Protein Phosphorylation and sample prep
宰后早期猪肉、牛肉和鸡肉中能量代谢及蛋白质磷酸化
宰后早期猪肉、牛肉和鸡肉中能量代谢及蛋白质磷酸化张爽1,2，张楠2,*，朱良齐2，李春保2（1.芜湖职业技术学院，安徽芜湖 241003；2.南京农业大学食品科技学院，江苏南京 210095）摘 要：目的：探讨畜禽肉宰后能量代谢的差异及其可能的原因。
方法：于宰后45 min测定鸡肉、灰白（pale soft exudative，PSE）猪肉、正常猪肉和牛肉中糖原、ATP/ADP/AMP含量及乳酸脱氢酶（lactate dehydrogenase，LDH）活性，并提取肌浆和肌原纤维蛋白进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate- polyacrylamide gel electrophoresis，SDS-PAGE）和液相色谱-串联质谱鉴定，分析各组肉的磷酸化水平差异。
结果：牛肉中糖原含量显著高于其他组（P＜0.05），正常猪肉组糖原和ATP含量显著高于PSE猪肉组和鸡肉组（P＜0.05），而PSE猪肉组的LDH活性显著高于其他组（P＜0.05）；肌浆蛋白SDS-PAGE ProQ染色表明，宰后45 min PSE猪肉组中6 个条带的磷酸化水平显著低于正常猪肉组（P＜0.05），而另有6 个条带的磷酸化水平显著高于正常猪肉组（P＜0.05）。
牛肉组和鸡肉组中分别有3 个和1 个条带的磷酸化水平偏低，1 个条带和8 个条带的磷酸化水平明显偏高。
而肌原纤维蛋白电泳表明，牛肉中大部分蛋白质的磷酸化水平处于最高值，而鸡肉组处于最低值，猪肉介于两者之间；PSE猪肉组中7 个条带的磷酸化水平显著低于正常猪肉（P＜0.05）；6 个条带的磷酸化水平显著高于正常猪肉（P＜0.05）。
结论：宰后异质肉的形成受宰后糖原和ATP消耗速率、LDH活性的影响，同时与肌肉中糖酵解酶的磷酸化水平有关。
关键词：异质肉；乳酸脱氢酶；糖酵解；能量代谢Energy Metabolism and Protein Phosphorylation of Pork, Beef and Chicken during the Early Postmortem PeriodZHANG Shuang1,2, ZHANG Nan2,*, ZHU Liangqi2, LI Chunbao2(1. Wuhu Institute of Technology, Wuhu 241003, China;2. College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China)Abstract: Objective: To explore the major cause for abnormal meat. Methods: At 45 min postmortem, chicken, pale soft exudative (PSE) pork, normal pork and beef were taken for the analysis of glycogen, ATP/ADP/AMP contents, lactate dehydrogenase (LDH) activity. At the same time, myofibrillar and sarcoplasmic proteins were extracted and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the bands of interest were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The difference in band intensity was evaluated. Results: Glycogen was higher in beef than three other meats (P < 0.05). Glycogen and ATP contents were higher in PSE pork than normal pork and chicken (P < 0.05). LDH activity in PSE pork was much higher than all other groups (P < 0.05).SDS-PAGE with ProQ staining indicated that, at 45 min postmortem, the phosphorylation levels of sarcoplasmic proteins from 6 bands were lower in PSE pork than those of normal pork (P < 0.05), while sarcoplasmic proteins from other6 bands had higher phosphorylation levels than normal pork (P < 0.05). In addition, 3 bands in beef and 1 bands inchicken had lower phsophorylation levels, whilst higher phsophorylation levels were observed for 1 bands in beef and 8 bands in chicken. For myofibrillar proteins, the majority of bands in beef had highest phosphorylation levels, the lowest phosphorylation levels were found for chicken (P < 0.05), and pork was in the middle. Similarly, 7 bands in PSE pork had lower phosphorylation levels than normal pork (P < 0.05), while 6 bands had higher phosphorylation levels than normal pork收稿日期：2016-11-25基金项目：“十二五”国家科技支撑计划项目（2014BAD19B01）；江苏省农业自主创新项目（CX(15)1006）；国家现代农业（生猪）产业技术体系建设专项（CARS36-11）；南京农业大学基本业务费专项（KYCYL201502）；江苏省优势学科项目作者简介：张爽（1975—），女，副教授，硕士，研究方向为食品加工与安全。
蛋白质组学技术流程
蛋白质组学技术流程Protein proteomics is a powerful technology used to study the complete set of proteins within an organism or a specific cell type. It involves the identification and quantification of proteins present in a sample, as well as the analysis of their functions, interactions, and modifications. This technology has revolutionized our understanding of cellular processes and disease mechanisms, making it an essential tool in biological research.蛋白质组学是一种强大的技术，用于研究生物体或特定细胞类型中的完整蛋白质组。
它涉及在样本中识别和定量存在的蛋白质，以及分析它们的功能、相互作用和修饰。
这项技术已经彻底改变了我们对细胞过程和疾病机制的理解，使其成为生物研究中必不可少的工具。
The workflow of a typical protein proteomics experiment involves several key steps, starting with sample preparation. This includes cell lysis to release the proteins, followed by protein extraction and purification to remove contaminants and concentrate the sample. The next step is protein digestion, where proteins are broken down into peptides using enzymes such as trypsin. These peptides are thenseparated using a technique such as liquid chromatography before being analyzed by mass spectrometry.典型蛋白质组学实验的工作流程包括几个关键步骤，从样本制备开始。
Overview of Protein Phosphorylation 蛋白磷酸化
UNIT 18.1 Overview of Protein PhosphorylationHISTORYPhosphorylation is the most common and important mechanism of acute and reversible regulation of protein function. Studies of mammalian cells metabolically labeled with [32P]orthophosphate suggest that as many as one-third of all cellular proteins are covalently modified by protein phosphorylation. Protein phosphorylation and dephosphorylation func-tion together in signal transduction pathways to induce rapid changes in response to hor-mones, growth factors, and neurotransmitters. Most polypeptide growth factors (platelet-de-rived growth factor and epidermal growth fac-tor are among the best studied; Heldin, 1995) and cytokines (e.g., interleukin 2, colony stimu-lating factor 1, and γ-interferon; Ihle et al., 1994) stimulate phosphorylation upon binding to their receptors. Induced phosphorylation in turn activates cytoplasmic protein kinases, such as raf, MEK, and MAP kinases (Marshall, 1995). Additionally, in all nucleated organisms, cell cycle progression is regulated at both the G1/S and the G2/M transitions by cyclin-de-pendent protein kinases (Doree and Galas, 1994).Differentiation and development are also controlled by phosphorylation. Development of the R7 cell in the Drosophila retina (Simon, 1994) and of the vulva in Caenorhabditis ele-gans(Eisenmann and Kim, 1994) are both dependent on the function of receptor and cy-toplasmic protein kinases. Finally, metabo-lism—in particular, the interconversion of glu-cose and glycogen and the transport of glu-cose—is regulated by phosphorylation (Cohen, 1985). Biologists of all stripes therefore find, often unexpectedly and occasionally reluc-tantly, that they must study protein phosphory-lation in order to understand the regulation and function of their favorite gene and its product.In the early 1940s, soon after their discovery that glycogen phosphorylase existed in two distinct forms in skeletal muscle, Carl and Gerty Cori identified a cellular activity that converted active phosphorylase a into a less active phosphorylase b. Initial studies of what they called the prosthetic-group removing (PR) enzyme suggested that the inactivation of phos-phorylase a was mediated via proteolysis (Cori and Cori, 1945). More than ten years later, this converting enzyme was shown to be a protein phosphatase. Thus, a protein phosphatase was studied nearly 20 years prior to the identifica-tion of the first protein kinase. The finding that phosphorylase phosphatases were more widelyexpressed in tissues than phosphorylase a ledto the current realization that these enzymeshave a broad substrate specificity and regulatemany physiological processes. The use of sub-strates other than phosphorylase a also identi-fied several other protein serine/threonine phosphatases.LABELING STUDIESProtein phosphorylation is usually studiedby biosynthetic labeling with 32P-labeled inor-ganic phosphate (32P i; UNIT 18.2). This is intrin-sically quite simple—the label is just added togrowth medium. It is this step of an experiment,however, that makes many investigators themost nervous, given the perceived danger ofradioactive exposure and the real danger of contamination of laboratory equipment with radioactivity. Neither problem is insurmount-able. With proper shielding and technique, ex-posure of the investigator can be limited to thehands and contamination of the laboratory canbe avoided. A general protocol for biosyntheticlabeling with 32P i that maximizes incorporationand minimizes radioactive exposure of workersin the lab and contamination of lab equipmentis described in UNIT 18.2. (For a general discus-sion of radiation safety consult Safe Use of Radioisotopes, APPENDIX 1F.)Many protein kinases can be assayed by thetransfer of radiolabel from [32P]ATP to surro-gate protein and peptide substrates. Syntheticpeptide substrates have been particularly usefulin establishing consensus motifs that define thesubstrate for protein kinases. By comparison,protein phosphatases show very low activityagainst synthetic phosphopeptides, greatly pre-ferring the phosphoprotein substrates (Shenoli-kar and Ingebritsen, 1984). Therefore, attemptsto define structural requirements for protein dephosphorylation using synthetic phos-phopeptides have failed to establish the mo-lecular basis for substrate specificity of themajor cellular protein phosphatases.The rate of 32P incorporation into proteinsin vivo, at either basal or hormone-stimulatedlevels, depends heavily on the rate of turnoverof phosphate in the protein. For example, lowphosphatase activity against specific sites orproteins will reduce the turnover of protein-bound phosphates and hinder their metaboliclabeling in the intact cell. Conversely, physi-Supplement 33Contributed by Bartholomew M. Sefton and Shirish Shenolikar Current Protocols in Molecular Biology (1996) 18.1.1-18.1.5Copyright © 2000 by John Wiley & Sons, Inc.18.1.1 Analysis of Protein Phosphorylationological stimuli that increase phosphatase ac-tivity may facilitate metabolic labeling of pro-teins, allowing rapid replacement of endo-genous “cold” phosphate with the radiolabel.Thus, 32P incorporation by itself gives no indi-cation about the extent of phosphorylation of a particular protein. As most phosphoproteins are phosphorylated on multiple sites, this also makes it difficult to correlate changes in protein function with 32P labeling of a given protein in the intact cell.SITES OF PHOSPHORYLATIONMost proteins are found to be phosphory-lated at serine or threonine residues, and many proteins involved in signal transduction are also phosphorylated at tyrosine residues. These three hydroxyphosphoamino acids exhibit suf-ficient chemical stability at acidic pH that they can be recovered after acid hydrolysis and iden-tified in a straightforward manner. Proteins that contain covalently bound phosphate at histid-ine, cysteine, and aspartic acid residues, either as phosphoenzyme intermediates or as stable modifications, have also been described. Each of these phosphoamino acids is chemically labile and impossible to study with the standard techniques used for the acid-stable phos-phoamino acids. Indeed, they are often identi-fied by inference or elimination. A technique for identifying phosphoserine, phosphothreon-ine, and phosphotyrosine by acid hydrolysis and two-dimensional thin-layer electrophore-sis is described in UNIT 18.3. Techniques for analyzing acid-labile forms of protein phos-phorylation are described in Ringer, 1991;Kamps, 1991; and Duclos et al., 1991.Phosphotyrosine is not an abundant phos-phoamino acid. Therefore, its detection in sam-ples labeled with 32P i is often difficult, espe-cially if the samples contain large quantities of proteins phosphorylated at serine residues or are contaminated with RNA. Detection of phos-photyrosine, as well as of phosphothreonine,can be enhanced considerably by incubation of gel-fractionated samples in alkali. This hydro-lyzes RNA and dephosphorylates phosphoser-ine, allowing visualization of minor tyrosine-and threonine-phosphorylated proteins. A sim-ple procedure for alkaline treatment is de-scribed in the alternate protocol of UNIT 18.3.DETECTION OF UNLABELED PHOSPHOAMINO ACIDSIf a protein is modified by phosphorylation,identification of the phosphoamino acid canoften be accomplished without resorting to bio-synthetic labeling. For example, tyrosine phos-phorylation can be studied because proteins containing this rare phosphoamino acid can be detected with great specificity and sensitivity by antibodies to phosphotyrosine (UNIT 18.4). By comparison, the quantitation of phosphoserine and phosphothreonine is more difficult and requires partial acid hydrolysis of the phospho-protein and subsequent separation of phos-phoamino acids by high-voltage electrophore-sis on thin-layer cellulose acetate plates. At-tempts to generate antibodies that recognize phosphoserine or phosphothreonine have failed to produce reagents with the required specific-ity and/or sufficient sensitivity to be useful.However, once the primary sequence around a phosphorylation site containing phosphoserine or phosphothreonine has been determined, it is possible to make antibodies against synthetic phosphopeptides modeled on these phosphory-lation sites (Czernik et al., 1991). Such anti-phosphopeptide antibodies have been very use-ful tools for monitoring the phosphorylation of the parent protein at specific sites. The phos-pho-specific antibodies have also been used to inhibit the dephosphorylation of individual sites and demonstrate their role in protein func-tion. Several recent studies have produced phospho-specific antibodies against phospho-rylation sites predicted solely by the primary sequence of a protein. These reagents have provided new insights into phosphorylations that control protein function.More generally, because phosphorylation often alters the mobility of a protein during SDS–polyacrylamide gel electrophoresis and almost always alters its isoelectric point, the presence of phosphorylated residues in an unlabeled protein can be deduced from al-tered gel mobility after incubation of the protein with a phosphatase. Most phosphory-lation sites are thought to reside near the surface of phosphoproteins. Therefore, they are equally accessible to phosphatases and proteases. In-deed, limited proteolysis often produces func-tional changes in phosphoproteins that closely mimic their in vitro dephosphorylation by phosphatase. As monitoring the release of ra-dioactivity from a 32P-labeled phosphoprotein does not distinguish between a phosphatase and a protease reaction, additional steps must to be taken to differentiate orthophosphate from small phosphopeptides. This is particularly im-portant when dephosphorylation reactions are carried out in crude extracts (UNIT 18.5).Supplement 33Current Protocols in Molecular Biology18.1.2Overview ofProteinPhosphorylationPROTEIN KINASESProtein kinases exhibit a strict specificity for phosphorylation of either serine/threonine or tyrosine residues. Yet these enzymes share structural homology in several domains such as the ATP-binding and catalytic sites, emphasiz-ing their common function as phosphotrans-ferases. A third group of protein kinases more closely resembles the serine/threonine kinases in their primary sequence but phosphorylate both serine/threonine and tyrosine residues.MEK, a MAP kinase activator (Crews and Erickson, 1992) and wee1, an inhibitor of cdc2kinase (Parker et al., 1992) are examples of such dual-specificity protein kinases that phospho-rylate threonine and tyrosine residues which are closely located in substrate proteins.Kinases have been used in a number of ways to analyze protein phosphorylation. Mutations that abrogate ATP-binding inactive protein ki-nases. Such inactive kinases retain the ability to recognize substrates and therefore act as dominant-negative reagents to analyze the physiological function of kinases in the intact cell (Thorburn et al., 1994; Alberola-Ila et al.,1995). Cell-permeable compounds modeled on ATP also inhibit selected kinases in the intact cell.Growth factors promote dimerization and activation of transmembrane receptor kinases.Mutant receptors that can dimerize in the ab-sence of the natural ligand offer a novel ap-proach to activate receptor kinases and initiate the cellular responses to growth factors (Spencer et al., 1993). Several cytoplasmic ki-nases are also activated by binding of intracel-lular second messengers. The allosteric activa-tion of these kinases results from conformation changes that eliminate internal autoinhibitory interactions which silence the kinases in the absence of ligand. Deletion of the autoinhibi-tory sequences generates a constitutively active kinase that provides important insights into its physiological role (Planas-Silva and Means,1992). Synthetic peptides modeled on the autoinhibitory sequences can also be used to selectively inhibit these kinases and reveal their role in controlling protein phosphorylation.Still other kinases, which are not directly regulated by ligand binding, are activated by phosphorylation. Some of these kinases partici-pate in cascades of sequential phosphorylations that amplify physiological signals. Substitution of the amino acids phosphorylated during the activation of these kinases with nonphosphory-lated residues generates dominant-negative en-zymes that can block the functions of suchkinases in intact cells. Alternately, acidic amino acids may be substituted for phosphoamino acids to produce a constitutively active kinase (Brunet et al., 1994). Such reagents have been used to validate the role of protein kinases in specific phosphorylation events.PROTEIN PHOSPHATASESMany protein phosphatases also show a strict specificity for either phosphoserine/phos-phothreonine or phosphotyrosine residues.However, unlike kinases, the serine/threonine and tyrosine phosphatases are not evolutionar-ily related and exhibit no primary sequence homology (Shenolikar and Nairn, 1991; Char-bonneau and Tonks, 1992). Acid and alkali phosphatases can also dephosphorylate phos-phoproteins in vitro but share no structural homology with protein phosphatases. A new family of phosphatases, such as cdc25 and CL100, are distantly related to the tyrosine phosphatases but dephosphorylate both phos-photyrosine and phosphothreonine in their tar-get substrates. Mutation of an essential cysteine in tyrosine phosphatases has produced domi-nant-negative enzymes that have been shown to shield their substrates from dephosphoryla-tion by endogenous phosphatases, thereby pro-longing the biological effects of protein phos-phorylation (Sun et al, 1993). Mutating several conserved amino acids can inactivate a ser-ine/threonine phosphatase (Zhou et al, 1994),but the ability of such mutant phosphatases to modulate serine/threonine phosphorylation in cells has not yet been demonstrated.A number of potent phosphatase inhibitors have been identified in recent years. Okadaic acid and several other toxins inhibit protein (serine/threonine) phosphatase 1 and 2A, and vanadate and phenylarsenoxide inhibit tyrosine phosphatase. These compounds have impli-cated reversible phosphorylation as a regula-tory mechanism in many physiological proc-esses (Cohen, 1989; Hardie et al., 1991; She-nolikar and Nairn, 1991; Shenolikar, 1994);under certain conditions, they may be the domi-nant regulators of these cellular processes.These phosphatase inhibitors can also be used to distinguish protein dephosphorylation from proteolysis in crude tissue extracts (Cohen,1991). However, by far the most important contribution of these reagents has been that they have allowed assessment of the role played by phosphorylation in cellular processes where neither the identity of the phosphoprotein in-volved nor that of the kinase(s) that regulates its function are known.Current Protocols in Molecular BiologySupplement 3318.1.3Analysis of ProteinPhosphorylationThe units in this chapter describe techniques that detect protein phosphorylation and identify amino acids that have been covalently modified (UNITS 18.2, 18.3, & 18.4). The next step is to use phosphatases to dephosphorylate the substrate protein in vitro and address the functional rele-vance of the covalent modification (UNIT 18.5).Once the regulatory role of phosphorylation has been clearly established, the more sophisticated approaches discussed above can be used to to identify the specific kinases and phosphatases involved and thereby begin to elucidate the physiological mechanism that regulates the substrate in the intact cell.LITERATURE CITEDAlberola-Ila, J., Forbush, K.A., Seger, R., Krebs,E.G., and Perlmutter, R.M. 1995. Selective re-quirement for MAP kinase activation in thymo-cyte differentiation. Nature 373:620-623.Brunet, A., Pages, G., and Poussegur, J. 1994. Con-stitutively active mutants of MAP (MEK1) in-duce growth factor-relaxation and oncogenicity when expressed in fibroblasts. Oncogene 9:3379- 3387.Charbonneau, H. and Tonks, N.K. 1992. 1002 pro-tein phosphatases? Annu. Rev. Cell Biol. 8:463-493.Cohen, P . 1985. The role of protein phosphorylation in the hormonal control of enzyme activity. Eur .J. Biochem. 15:439-448.Cohen, P. 1989. The structure and regulation of protein phosphatases. Annu. Rev. Biochem.58:453-508.Cohen, P. 1991. Classification of protein ser-ine/threonine phosphatases: Identification and quantitation in cell extracts. Methods Enzymol.201:389-398.Cori, G.T. and Cori, C.F. 1945. Enzymatic conver-sion of phosphorylase a to b . J. Biol. Chem.158:321-345.Crews, C.M. and Erickson, R.L. 1992. Purification of a murine protein-tyrosine/threonine kinase that phosphorylates and activates the erk-1 gene product: Relationship to the fission yeast byr1gene product. Proc. Natl. Acad. Sci. U.S.A.89:8205-8209.Czernik, A.J., Girault, J.A., Nairn, A.C., Chen, J.,Snyder, G., Kebabian, J., and Greengard, P . 1991.Production of phosphorylation state–specific an-tibodies. Methods Enzymol. 201:264-283.Doree, M. and Galas, S. 1994. The cyclin-dependent protein kinases and the control of cell division.F ASEB J. 85:1114-1121.Duclos, B., Marcandier, S., and Cozzone, A.J. 1991.Chemical properties and separation of phos-phoamino acids by thin-layer chromatography and/or electrophoresis. Methods Enzymol.201:10-21.Eisenmann, D.M. and Kim, S.K. 1994. Signal transduction and cell fate specification during Caenorhabditis elegans vulval development.Curr . Opin. Genet. Dev. 4:508-516.Hardie, D.G., Haystead, T.A.J., and Sim, A.T.R.1991. Use of okadaic acid to inhibit protein phosphatases in intact cells. Methods Enzymol.201:469-477.Heldin, C.-H. 1995. Dimerization of cell surface receptors in signal transduction. Cell 80:213-223.Ihle, J.N., Witthuhn, B.A., Quelle, F.W., Yamamoto,K., Thierfelder, W.E., Kreider, B., and Silven-noinen, O. 1994. Signalling by the cytokine re-ceptor superfamily: JAKS and STA TS. Trends Biochem. Sci. 19:222-227.Kamps, M.P . 1991. Determination of phosphoamino acid composition by acid hydrolysis of protein blotted to Immobilon. Methods Enzymol.201:21-27.Marshall, C.J. 1995. Specificity of receptor tyrosine kinase signalling: Transient versus sustained ex-tracellular signal-regulated kinase activation.Cell 80:179-185.Parker, L.L., Atherton-Fessler, S., and Piwinica-Worms, H. 1992. p107 wee1 is a dual-specificity kinase that phosphorylates p34cdc2 on tyrosine 15. Proc. Natl. Acad. Sci. U.S.A. 89:2917-2921.Planas-Silva, M.D. and Means, A.R. 1992. Expres-sion of a constitutive form of calcium/cal-modulin-dependent protein kinase II leads to arrest of the cell cycle in G2. EMBO J. 11:507-517.Ringer, D.P . 1991. Separation of phosphotyrosine,phosphoserine, and phosphothreonine by high-performance liquid chromatography. Methods Enzymol. 201:3-10.Shenolikar, S. 1994. Protein serine/threonine phos-phatases: New avenues for cell regulation. Annu.Rev. Cell Biol. 10:55-86.Shenolikar, S. and Ingebritsen, T.S. 1984. Protein (serine, threonine) phosphate phosphatases.Methods Enzymol. 107:102-130.Shenolikar, S. and Nairn, A.C. 1991. Protein phos-phatases: Recent progress. Adv. Second Messen-ger Phosphoprotein Res. 23:1-121.Simon, M. 1994. Signal transduction during the development of the Drosophila R7 photorecep-tor. Dev. Biol. 166:431-442.Spencer, D.M., Wandless, T.J., Schreiber, S.L., and Crabtree, G.R. 1993. Controlling signal transduction with synthetic ligands. Science 262:1019-1024.Sun, H., Charles, C.H., Lau, L.F., and Tonks, N.K.1993. MKP-1 (3CH134), an immediate early gene product, is a dual-specificity phosphatase that dephosphorylates MAP kinase in vivo. Cell 75:487- 493.Supplement 33Current Protocols in Molecular Biology18.1.4Overview ofProteinPhosphorylationThorburn, J., McMahon, M. and Thorburn, A. 1994.Raf-1 kinase activity is necessary and sufficient for gene expression changes but not sufficient for cellular morphology changes associated with cardiac myocyte hypertrophy. J. Biol. Chem.269: 30580-30586.Zhou, S., Clemens, J.C., Stone, R.L., and Dixon, J.E.1994. Mutational analysis of a ser/thr phos-phatase. Identification of residues important in phosphatase substrate binding and catalysis. J.Biol. Chem. 269:26234-26238.Contributed by Bartholomew M. Sefton The Salk Institute San Diego, CaliforniaShirish ShenolikarDuke University Medical Center Durham, North CarolinaCurrent Protocols in Molecular BiologySupplement 3318.1.5Analysis of ProteinPhosphorylation。
基于网络药理学探讨青娥丸治疗绝经后骨质疏松症的作用机制
364中国骨质疏松杂志 2021 年 3 月第 27 卷第 3 期 Chin J Osteoporos , March 2021 ,Vol 27 , No. 3Published doi ：10. 3969/j.issn.l006-7108. 2021. 03. 011基于网络药理学探讨青娥丸治疗绝经后骨质疏松症的 作用机制邸学士1陈昭2贾育松2李晋玉2郑晨颖2白春晓2张帆2刘楚吟2袁巧妹1龙水文1冉宇1 康晟乾1陈江2*基金项目：国家自然科学基金项目(81603638)；中国博士后科学基金面上项目(2019M662791)；北京中医药大学2019年度基础科研业务费项目(2019-JYB-JS-042)；北京中医药大学东直门医院青苗人才项目(DZMY-201702)。
* 通信作者：陈江,Email : cjdzmhp@ 1. 北京中医药大学，北京1000292. 北京中医药大学东直门医院，北京100700中图分类号：R589. 5, R285 文献标识码：A 文章编号：1006-7108( 2021) 03-0364-08摘要：目的 运用网络药理学的方法探讨青娥丸治疗PM0可能的分子作用机制。
方法 运用TCMSP 、ETCM 、SymMap 数据 库及文献挖掘获取青娥丸主要活性成分，通过SwissTargetPrediction 平台进行成分靶标预测；经GeneCards 、OMIM 和DisGeNET数据库获取PM0相关靶点，与成分靶点取交集，获得青娥丸治疗PM0的潜在作用靶点;应用Cytoscape 软件构建青娥丸治疗PM0的中药-活性成分-交集靶点网络图，使用String 数据库及Cytoscape 软件对交集靶点进行蛋白质相互作用网络分析，依据节点度值筛选关键靶点;通过DAVID 平台对关键靶点进行GO 和KEGG 富集分析，以探究青娥丸治疗PM0的分子作用机制。
结果筛选岀青娥丸活性成分40个,包括飞燕草素、槲皮素、山奈酚等核心成分;青娥丸治疗PM0的潜在作用靶点178个;对交集靶点进行蛋白质互相作用网络分析获取关键靶点68个，包括MAPK1、AKT1、PIK3CA 、JAK2等核心靶点;关键靶点基因的GO 及KEGG 富集分析显示:关键靶标主要在质膜、膜筏、细胞核等位置发挥作用，通过一氧化氮生物合成过程的正调控、信号转导、蛋白质磷酸化等生物过程，发挥激酶活性、与蛋白质结合、与蛋白激酶结合等功能。
蛋白质纯化手册【ProteinPurificationHandbook】
Protein PurificationHandbook18-1132-29Edition ABHiTrap, Sepharose, STREAMLINE, Sephadex, MonoBeads, Mono Q,Mono S, MiniBeads, RESOURCE, SOURCE, Superdex, Superose, HisTrap, HiLoad, HiPrep, INdEX, BPG, BioProcess, FineLINE, MabTrap, MAbAssistant, Multiphor, FPLC, PhastSystem and ÄKTA are trademarks of Amersham Pharmacia Biotech Limitedor its subsidiaries.Amersham is a trademark of Nycomed Amersham plcPharmacia and Drop Design are trademarks of Pharmacia & Upjohn Inc Coomassie is a trademark of ICI plcAll goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Pharmacia Biotech group which supplies them. A copy of these terms and conditions of sale is available on request.© Amersham Pharmacia Biotech AB 1999-All rights reserved.Amersham Pharmacia Biotech ABSE-751 84 Uppsala SwedenAmersham Pharmacia Biotech UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Pharmacia Biotech Inc800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USAProtein Purification HandbookContents Introduction (7)Chapter 1Purification Strategies - A Simple Approach (9)Preparation (10)Three Phase Purification Strategy (10)General Guidelines for Protein Purification (12)Chapter 2 Preparation (13)Before You Start (13)Sample Extraction and Clarification (16)Chapter 3Three Phase Purification Strategy (19)Principles (19)Selection and Combination of Purification Techniques (20)Sample Conditioning (26)Chapter 4Capture (29)Chapter 5Intermediate Purification (37)Chapter 6Polishing (40)Chapter 7Examples of Protein Purification Strategies (45)Three step purification of a recombinant enzyme (45)Three step purification of a recombinant antigen binding fragment (49)Two step purification of a monoclonal antibody (54)One step purification of an integral membrane protein (57)Chapter 8Storage Conditions (61)Extraction and Clarification Procedures (62)Chapter 9Principles and Standard Conditions for Purification Techniques (73)Ion exchange (IEX) (73)Hydrophobic interaction (HIC) (79)Affinity (AC) (85)Gel filtration (GF) (88)Reversed phase (RPC) (92)Expanded bed adsorption (EBA) (95)IntroductionThe development of techniques and methods for protein purification has been an essential pre-requisite for many of the advancements made in biotechnology. This booklet provides advice and examples for a smooth path to protein purification. Protein purification varies from simple one-step precipitation procedures to large scale validated production processes. Often more than one purification step is necessary to reach the desired purity. The key to successful and efficient protein purification is to select the most appropriate techniques, optimise their performance to suit the requirements and combine them in a logical way to maximise yield and minimise the number of steps required.Most purification schemes involve some form of chromatography. As a result chromatography has become an essential tool in every laboratory where protein purification is needed. The availability of different chromatography techniques with different selectivities provides a powerful combination for the purification of any biomolecule.Recombinant DNA developments over the past decade have revolutionised the production of proteins in large quantities. Proteins can even be produced in forms which facilitate their subsequent chromatographic purification. However, this has not removed all challenges. Host contaminants are still present and problems related to solubility, structural integrity and biological activity can still exist. Although there may appear to be a great number of parameters to consider, with a few simple guidelines and application of the Three Phase Purification Strategy the process can be planned and performed simply and easily, with only a basic knowledge of the details of chromatography techniques.78Chapter 1Purification Strategies- a simple approachApply a systematic approach to development of a purification strategy. The first step is to describe the basic scenario for the purification. General considerations answer questions such as: What is the intended use of the product? What kind of starting material is available and how should it be handled? What are the purity issues in relation to the source material and intended use of the final product? What has to be removed? What must be removed completely? What will be the final scale of purification? If there is a need for scale-up, what consequences will this have on the chosen purification techniques? What are the economical constraints and what resources and equipment are available?Most purification protocols require more than one step to achieve the desired level of product purity. This includes any conditioning steps necessary to transfer the product from one technique into conditions suitable to perform the next technique. Each step in the process will cause some loss of product. For example, if a yield of 80% in each step is assumed, this will be reduced to only 20% overall yield after 8 processing steps as shown in Figure 1. Consequently, to reach the targets for yield and purity with the minimum number of steps and the simplest possible design, it is not efficient to add one step to another until purity requirements have been fulfilled. Occasionally when a sample is readily available purity can be achieved by simply adding or repeating steps. However, experience shows that, even for the most challenging applications, high purity and yield can be achieved efficiently in fewer than four well-chosen and optimised purification steps. Techniques should be organised in a logical sequence to avoid the need for conditioning steps and the chromatographic techniques selected appropriately to use as few purification steps as possible.Limit the number of steps in a purification procedure910Fig.1.Yields from multi-step purifications.PreparationThe need to obtain a protein, efficiently, economically and in sufficient purity and quantity, applies to every purification. It is important to set objectives for purity,quantity and maintenance of biological activity and to define the economical and time framework for the work. All information concerning properties of the target protein and contaminants will help during purification development. Some simple experiments to characterise the sample and target molecule are an excellent investment. Development of fast and reliable analytical assays is essential to follow the progress of the purification and assess its effectiveness. Sample preparation and extraction procedures should be developed prior to the first chromatographic purification step.With background information, assays and sample preparation procedures in place the Three Phase Purification Strategy can be considered.Three Phase Purification Strategy Imagine the purification has three phases Capture, IntermediatePurification and Polishing.In the Three Phase Strategy specific objectives are assigned to each step within the process:In the capture phase the objectives are to isolate, concentrate and stabilise the target product.During the intermediate purification phase the objective is to remove most of the bulk impurities such as other proteins and nucleic acids, endotoxins and viruses.In the polishing phase the objective is to achieve high purity by removing any remaining trace impurities or closely related substances.The selection and optimum combination of purification techniques for Capture,Intermediate Purification and Polishing is crucial to ensure fast method development, a shorter time to pure product and good economy.108060402012345678Number of steps 95% / step90% / step 85% / step 80% / step 75% / stepYield (%)The final purification process should ideally consist of sample preparation, including extraction and clarification when required, followed by three major purification steps, as shown in Figure 2. The number of steps used will always depend upon the purity requirements and intended use for the protein.Fig. 2.Preparation and the Three Phase Purification Strategy11Guidelines for Protein PurificationThe guidelines for protein purification shown here can be applied to any purification process and are a suggestion as to how a systematic approach can be applied to the development of an effective purification strategy. As a reminder these guidelines will be highlighted where appropriate throughout the following chapters.Define objectivesfor purity, activity and quantity required of final product to avoid over or under developing a methodDefine properties of target protein and critical impuritiesto simplify technique selection and optimisationDevelop analytical assaysfor fast detection of protein activity/recovery and to work efficientlyMinimise sample handling at every stageto avoid lengthy procedures which risk losing activity/reducing recovery Minimise use of additivesadditives may need to be removed in an extra purification step or may interfere with activity assaysRemove damaging contaminants earlyfor example, proteasesUse a different technique at each stepto take advantage of sample characteristics which can be used for separation (size, charge, hydrophobicity, ligand specificity)Minimise number of stepsextra steps reduce yield and increase time, combine steps logicallyKEEP IT SIMPLE!12Chapter 2PreparationBefore You StartThe need to obtain a protein, efficiently, economically and in sufficient purity and quantity, applies to any purification, from preparation of an enriched protein extract for biochemical characterisation to large scale production of a therapeutic recombinant protein. It is important to set objectives for purity and quantity, maintenance of biological activity and economy in terms of money and time. Purity requirements must take into consideration the nature of the source material, the intended use of the final product and any special safety issues. For example, it is important to differentiate between contaminants which must be removed and those which can be tolerated. Other factors can also influence the prioritisation of objectives. High yields are usually a key objective, but may be less crucial in cases where a sample is readily available or product is required only in small quantities. Extensive method development may be impossible without resources such as an ÄKTA™design chromatography system. Similarly, time pressure combined with a slow assay turnaround will steer towards less extensive scouting and optimisation. All information concerning properties of the target protein and contaminants will help during purification development, allowing faster and easier technique selection and optimisation, and avoiding conditions which may inactivate the target protein.Development of fast and reliable analytical assays is essential to follow the progress of the purification and assess effectiveness (yield, biological activity, recovery).Define objectivesGoal:To set minimum objectives for purity and quantity, maintenance of biological activity and economy in terms of money and time.Define purity requirements according to the final use of the product. Purity requirement examples are shown below.Extremely high > 99%Therapeutic use, in vivo studiesHigh 95- 99 %X-ray crystallography and most physico-chemicalcharacterisation methodsModerate < 95 %Antigen for antibody productionN-terminal sequencing13Identify 'key' contaminantsIdentify the nature of possible remaining contaminants as soon aspossible.The statement that a protein is >95% pure (i.e. target protein constitutes 95% of total protein) is far from a guarantee that the purity is sufficient for an intended application. The same is true for the common statement "the protein was homogenous by Coomassie™ stained SDS-PAGE". Purity of 95% may be acceptable if the remaining 5% consists of harmless impurities. However, even minor impurities which may be biologically active could cause significant problems in both research and therapeutic applications. It is therefore important to differentiate between contaminants which must be removed completely and those which can be reduced to acceptable levels. Since different types of starting material will contain different contaminant profiles they will present different contamination problems.It is better to over-purify than to under-purify.Although the number of purification steps should be minimised, thequality of the end product should not be compromised. Subsequent results might be questioned if sample purity is low and contaminants are unknown.Contaminants which degrade or inactivate the protein or interfere withanalyses should be removed as early as possible.The need to maintain biological activity must be considered at every stage during purification development. It is especially beneficial if proteases are removed and target protein transferred into a friendly environment during the first step.Economy is a very complex issue. In commercial production the time to market can override issues such as optimisation for recovery, capacity or speed. Robustness and reliability are also of great concern since a batch failure can have major consequences.It may be necessary to use analytical techniques targetted towards specific conta-minants in order to demonstrate that they have been removed to acceptable levels. 14Define properties of target protein and critical impurities Goal:To determine a 'stability window' for the target protein for easier selection and optimisation of techniques and to avoid protein inactivation during purification.Check target protein stability window for at least pH and ionic strength. All information concerning the target protein and contaminant properties will help to guide the choice of separation techniques and experimental conditions for purification. Database information for the target, or related proteins, may give size, isoelectric point (pI) and hydrophobicity or solubility data. Native one and two dimensional PAGE can indicate sample complexity and the properties of the target protein and major contaminants. Particularly important is a knowledge of the stability window of the protein so that irreversible inactivation is avoided. Itis advisable to check the target protein stability window for at least pH and ionic strength. Table 1 shows how different target protein properties can affect a purification strategy.Table 1.Protein properties and their effect on development of purification strategies. Sample and target protein properties Influence on purification strategyTemperature stability Need to work rapidly at lowered temperaturepH stability Selection of buffers for extraction and purificationSelection of conditions for ion exchange, affinity orreversed phase chromatographyOrganic solvents stability Selection of conditions for reversed phasechromatographyDetergent requirement Consider effects on chromatographic steps and the needfor detergent removal. Consider choice of detergent.Salt (ionic strength)Selection of conditions for precipitation techniques andhydrophobic interaction chromatographyCo-factors for stability or activity Selection of additives, pH, salts, buffersProtease sensitivity Need for fast removal of proteases or addition ofinhibitorsSensitivity to metal ions Need to add EDTA or EGTA in buffersRedox sensitivity Need to add reducing agentsMolecular weight Selection of gel filtration mediaCharge properties Selection of ion exchange conditionsBiospecific affinity Selection of ligand for affinity mediumPost translational modifications Selection of group specific affinity medium Hydrophobicity Selection of medium for hydrophobic interactionchromatography15Develop analytical assaysGoal:To follow the progress of a purification, to assess effectiveness (yield, biological activity, recovery) and to help during optimisation.Select assays which are fast and reliable.To progress efficiently during method development the effectiveness of each step should be assessed. The laboratory should have access to the following assays:• A rapid, reliable assay for the target protein• Purity determination• Total protein determination• Assays for impurities which must be removedThe importance of a reliable assay for the target protein cannot be over- emphasised. When testing chromatographic fractions ensure that the buffers used for separation do not interfere with the assay. Purity of the target protein is most often estimated by SDS-PAGE, capillary electrophoresis, reversed phase chromatography or mass spectrometry. Lowry or Bradford assays are used most frequently to determine the total protein.The Bradford assay is particularly suited to samples where there is a high lipid content which may interfere with the Lowry assay.For large scale protein purification the need to assay for target proteins and critical impurities is often essential. In practice, when a protein is purified for research purposes, it is too time consuming to identify and set up specific assays for harmful contaminants. A practical approach is to purify the protein to a certain level, and then perform SDS-PAGE after a storage period to check for protease cleavage. Suitable control experiments, included within assays forbio-activity, will help to indicate if impurities are interfering with results.Sample Extraction and Clarification Minimise sample handlingMinimise use of additivesRemove damaging contaminants earlyDefinition:Primary isolation of target protein from source material.Goal:Preparation of a clarified sample for further purification. Removal of particulate matter or other contaminants which are not compatible with chromatography.16The need for sample preparation prior to the first chromatographic step is dependent upon sample type. In some situations samples may be taken directly to the first capture step. For example cell culture supernatant can be applied directly to a suitable chromatographic matrix such as Sepharose™ Fast Flow and may require only a minor adjustment of the pH or ionic strength. However, it is most often essential to perform some form of sample extraction and clarification procedure.If sample extraction is required the chosen technique must be robust and suitable for all scales of purification likely to be used. It should be noted that a technique such as ammonium sulphate precipitation, commonly used in small scale, may be unsuitable for very large scale preparation. Choice of buffers and additives must be carefully considered if a purification is to be scaled up. In these cases inexpensive buffers, such as acetate or citrate, are preferable to the more complex compositions used in the laboratory. It should also be noted that dialysis and other common methods used for adjustment of sample conditions are unsuitable for very large or very small samples.For repeated purification, use an extraction and clarification techniquethat is robust and able to handle sample variability. This ensures areproducible product for the next purification step despite variability instarting material.Use additives only if essential for stabilisation of product or improvedextraction. Select those which are easily removed. Additives may need tobe removed in an extra purification step.Use pre-packed columns of Sephadex™ G-25 gel filtration media, forrapid sample clean-up at laboratory scale, as shown in Table 2.Table 2.Pre-packed columns for sample clean-up.Pre-packed column Sample volume Sample volume Code No.loading per run recovery per runHiPrep™Desalting 26/10 2.5 -15 ml7.5 - 20 ml17-5087-01HiTrap Desalting0.25 - 1.5 ml 1.0 - 2.0 ml17-1408-01Fast Desalting PC 3.2/100.05 - 0.2 ml0.2 - 0.3 ml17-0774-01PD-10 Desalting 1.5 - 2.5 ml 2.5 - 3.5 ml17-0851-01 Sephadex G-25 gel filtration media are used at laboratory and production scale for sample preparation and clarification of proteins >5000. Sample volumes of up to 30%, or in some cases, 40% of the total column volume are loaded. In a single step, the sample is desalted, exchanged into a new buffer, and low molecular weight materials are removed. The high volume capacity, relative insensitivity to sample concentration, and speed of this step enable very large sample volumes to be processed rapidly and efficiently. Using a high sample volume load results in a separation with minimal sample dilution (approximately 1:1.4). Chapter 8 contains further details on sample storage, extraction and clarification procedures.17Sephadex G-25 is also used for sample conditioning i.e. rapid adjustment of pH, buffer exchange and desalting between purification steps.Sephadex G-25 gel filtrationFor fast group separations between high and low molecular weight substances Typical flow velocity 60 cm/h (Sephadex G-25 SuperFine, Sephadex G-25 Fine), 150 cm/h (Sephadex G-25 Medium).If large sample volumes will be handled or the method scaled-up in the future, consider using STREAMLINE™ expanded bed adsorption. This technique is particularly suited for large scale recombinant protein and monoclonal antibody purification. The crude sample containing particles can be applied to the expanded bed without filtration or centrifugation. STREAMLINE adsorbents are specially designed for use in STREAMLINE columns. Together they enable the high flow rates needed for high productivity in industrial applications of fluidised beds. The technique requires no sample clean up and so combines sample preparation and capture in a single step. Crude sample is applied to an expanded bed STREAMLINE media. Target proteins are captured whilst cell debris, cells, particulate matter, whole cells, and contaminants pass through. Flow is reversed and the target proteins are desorbed in the elution buffer.STREAMLINE (IEX, AC, HIC)For sample clean-up and capture direct from crude sample.STREAMLINE adsorbents are designed to handle feed directly from both fermentation homogenate and crude feedstock from cell culture/fermentation at flow velocities of 200 - 500 cm/h, according to type and application.Particle size: 200 µmNote:cm/h: flow velocity (linear flow rate) = volumetric flow rate/cross sectional area of column.18Chapter 3Three Phase Purification StrategyPrinciplesWith background information, assays, and sample preparation and extraction procedures in place the Three Phase Purification Strategy can be applied (Figure 3). This strategy is used as an aid to the development of purification processes for therapeutic proteins in the pharmaceutical industry and is equally efficient as an aid when developing purification schemes in the research laboratory.Fig. 3.Preparation and the Three Phase Purification Strategy.Assign a specific objective to each step within the purification process.In the Three Phase Strategy a specific objective is assigned to each step. The purification problem associated with a particular step will depend greatly upon the properties of the starting material. Thus, the objective of a purification step will vary according to its position in the process i.e. at the beginning for isolation of product from crude sample, in the middle for further purification of partially purified sample, or at the end for final clean up of an almost pure product.The Three Phase Strategy ensures faster method development, a shorter time to pure product and good economy.In the capture phase the objectives are to isolate, concentrate and stabilise the target product. The product should be concentrated and transferred to an environment which will conserve potency/activity. At best, significant removal of other critical contaminants can also be achieved.19During the intermediate purification phase the objectives are to remove most of the bulk impurities,such as other proteins and nucleic acids, endotoxins and viruses.In the polishing phase most impurities have already been removed except for trace amounts or closely related substances. The objective is to achieve final purity.It should be noted that this Three Phase Strategy does not mean that all strategies must have three purification steps. For example, capture and intermediate purification may be achievable in a single step, as may intermediate purification and polishing. Similarly, purity demands may be so low that a rapid capture step is sufficient to achieve the desired result, or the purity of the starting material may be so high that only a polishing step is needed. For purification of therapeutic proteins a fourth or fifth purification step may be required to fulfil the highest purity and safety demands.The optimum selection and combination of purification techniques for Capture, Intermediate Purification and Polishing is crucial for an efficient purification process.Selection and Combination ofPurification TechniquesMinimise sample handlingMinimise number of stepsUse different techniques at each stepGoal:Fastest route to a product of required purity.For any chromatographic separation each different technique will offer different performance with respect to recovery, resolution, speed and capacity. A technique can be optimised to focus on one of these parameters, for example resolution, or to achieve the best balance between two parameters, such as speed and capacity.A separation optimised for one of these parameters will produce results quite different in appearance from those produced using the same technique, but focussed on an alternative parameter. See, for example, the results shown on page 49 where ion exchange is used for a capture and for a polishing step.20Select a technique to meet the objectives for the purification step. Capacity,in the simple model shown, refers to the amount of target protein loaded during purification. In some cases the amount of sample which can be loaded may be limited by volume (as in gel filtration) or by large amounts of contaminants rather than the amount of the target protein.Speed is of the highest importance at the beginning of a purification where contaminants such as proteases must be removed as quickly as possible. Recovery becomes increasingly important as the purification proceeds because of the increased value of the purified product. Recovery is influenced by destructive processes in the sample and unfavourable conditions on the column. Resolution is achieved by the selectivity of the technique and the efficiency of the chromatographic matrix to produce narrow peaks. In general, resolution is most difficult to achieve in the final stages of purification when impurities and target protein are likely to have very similar properties.Every technique offers a balance between resolution, speed, capacity and recovery and should be selected to meet the objectives for each purification step. In general, optimisation of any one of these four parameters can only be achieved at the expense of the others and a purification step will be a compromise. The importance of each parameter will vary depending on whether a purification step is used for capture, intermediate purification or polishing. This will steer the optimisation of the critical parameters, as well as the selection of the most suitable media for the step.Proteins are purified using chromatographic purification techniques which separate according to differences in specific properties, as shown in Table 3. Table 3.Protein properties used during purification.Protein property TechniqueCharge Ion exchange (IEX)Size Gel filtration (GF)Hydrophobicity Hydrophobic interaction (HIC),reversed phase (RPC)Biorecognition (ligand specificity)Affinity (AC)Charge, ligand specificity or hydrophobicity Expanded bed adsorption (EBA) follows theprinciples of AC, IEX or HIC21。
蛋白质组学复习重点
蛋白质组学复习重点1.名词解释(掌握名词的中英文)1、蛋白质组（proteome)是指一个基因组、一种细胞或组织表达的所有蛋白质。
2、蛋白质组学 Proteomic蛋白质组学是通过大规模研究蛋白的表达水平变化、翻译后修饰、蛋白质与蛋白质之间的相互作用，以获取蛋白质水平上疾病变化、细胞进程及蛋白质网络相互作用的整体综合信息的科学研究，是生命科学研究的热点领域之一。
3、电喷雾电离（Electrospray Ionization，ESI）电喷雾离子化是在质谱系统离子源毛细管的出口处施加一高电压，所产生的高电场使从毛细管流出的液体雾化成细小的带电液滴，随着溶剂蒸发，液滴表面的电荷强度逐渐增大，最后液滴崩解为大量带一个或多个电荷的离子，致使分析物以单电荷或多电荷离子的形式进入气相。
4、噬菌体展示技术 (phage display technology)一种将外源蛋白或多肽的DNA序列插入到噬菌体外壳蛋白结构基因的适当位置，使外源基因随外壳蛋白的表达而表达，同时，外源蛋白随噬菌体的重新组装而展示到噬菌体表面的生物技术。
5、双向电泳（two-dimensional electrophoresis,2-DE）指的是按照蛋白质的两个性质即“等电点”和“分子量”进行二维电泳分离。
过程主要是先进行等电聚焦电泳，按照等电点分离，然后再进行SDS-PAGE，按照分子大小分离，经染色得到的电泳图是个二维分布的蛋白质图。
6、等电点（isoelectric point）在某一pH的溶液中，氨基酸或蛋白质解离成阳离子和阴离子的趋势或程度相等，成为兼性离子，呈电中性，此时溶液的pH 称为该氨基酸或蛋白质的等电点。
7、质谱分析（mass spectrometry，MS）MS是在高真空系统中测定样品的分子离子及碎片离子质量，以确定样品相对分子质量及分子结构的方法。
8、生物信息学（bioinformatics）生物信息学是综合运用数学、计算机科学和生物学的各种工具，来阐明和理解大量数据所包含的生物学意义的新兴交叉学科，包含了生物信息的获取、处理、存储、发布、分析和解释等在内的所有方面。
SNP rs6265 Regulates Protein Phosphorylation and Osteoblast Differentiation
O r i g i n a l A r t i c l eSNP rs6265 Regulates Protein Phosphorylation and Osteoblast Differentiation and Influences BMD in Humans†Fei-Yan Deng 1-3, Li-Jun Tan 2-4, Hui Shen 2, 3, Yong-Jun Liu 2, 3, Yao-Zhong Liu 2, 3, Jian Li 2, 3, Xue-Zhen Zhu 5, Xiang-Ding Chen 4, Qing Tian 2, 3, Ming Zhao 2, 3, andHong-Wen Deng 2-5*1. Center of Genetic Epidemiology and Genomics, School of Public Health, SoochowUniversity, Suzhou, Jiangsu 215123, P. R. China2. Center for Bioinformatics and Genomics, Tulane University School of PublicHealth and Tropical Medicine, New Orleans, LA 70112, USA3. Department of Biostatistics and Bioinformatics, Tulane University School of PublicHealth and Tropical Medicine, New Orleans, LA 70112, USA4. Laboratory of Molecular and Statistical Genetics, College of Life Sciences, HunanNormal University, Changsha, Hunan 410081, P. R. China5. Center for Systematic Biomedical Research, Shanghai University of Science and Technology, Shanghai 200093, P. R. ChinaRunning title: phosSNP and human BMD*Corresponding author: Hong-Wen Deng, Ph. D.Center for Bioinformatics and Genomics Tulane University School of Public Health and Tropical Medicine 1440 Canal Street, Suite 2001, New Orleans, LA 70112, U.S.A. Phone: (001) 504 988-1310Email: hdeng2@All authors state that they have no conflicts of interestAdditional Supporting Information may be found in the online version of this article.†This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/jbmr.1997]Initial Date Submitted March 17, 2013; Date Revision Submitted April 17, 2013; Date Final Disposition Set May 17, 2013Journal of Bone and Mineral Research© 2013 American Society for Bone and Mineral ResearchDOI 10.1002/jbmr.1997Bone Mineral Density (BMD) is major index for diagnosing osteoporosis. PhosSNPs are non-synonymous SNPs that affect protein phosphorylation. The relevance and significance of phosSNPs to BMD and osteoporosis is unknown. This study aims to identify and characterize phosSNPs significant for BMD in humans. We conducted a pilot genome-wide phosSNP association study for BMD in three independent population samples, involving ~5,000 unrelated individuals. We identified and replicated three phosSNPs associated with both spine BMD and hip BMD in Caucasians. Association with hip BMD for one of these phosSNPs, i.e., rs6265 (major/minor allele: G/A) in BDNF gene, was also suggested in Chinese. Consistently in both ethnicities, individuals carrying AA genotype have significant lower hip BMD than carriers of GA and GG genotype s. Through in vitro molecular and cellular studies, we found that compared to osteoblastic cells transfected with wild-type BDNF-Val66 (encoded with allele G at rs6265), transfection of variant BDNF-Met66 (encoded with allele A at rs6265) significantly decreased BDNF protein phosphorylation (at amino acid residue T62), expression of osteoblastic genes (OPN, BMP2, and ALP), and osteoblastic activity. The findings are consistent with and explain our prior observations in general human populations. We conclude that phosSNP rs6265, via regulating BDNF protein phosphorylation and osteoblast differentiation, influence hip BMD in humans. This study represents our first endeavor to dissect the functions of phosSNPs in bone, which might stimulate extended large-scale studies on bone or similar studies on other human complex traits and diseases.Key words: BMD, SNP, protein phosphorylation, BDNF, osteoblastIntroductionGenetics of BMD and Osteoporosis: Bone mineral density (BMD), as a measurable and powerful index predicting osteoporosis and OF risks, is the current “golden standard” for diagnosing osteoporosis. BMD is a complex trait largely determined by genetic factors, with heritability larger than 0.6(1). In the past decades, extensive studies, including candidate gene association studies, genome-wide linkage and association studies, have been launched to search for quantitative trait loci underlying BMD variation in humans. However, currently identified quantitative trait loci alone can only account for a small proportion of BMD variation, leaving the majority of genetic variation to be explained by novel factors to be identified. In addition, most of the genes identified in one population have yet to be tested for their significance in independent sample(s). So far, an increasing number of genome-wide association studies (GWAS) systemically explore the relationship between single nucleotide polymorphisms (SNPs) and phenotypic variation, and also established some associations. However, the mechanism(s) underlying a majority of these associations are largely unclear. Identification of significant SNPs influencing BMD and characterization of their cellular and molecular functions will contribute to elucidating pathophysiological mechanism of osteoporosis.Proteins, Protein Phosphorylation, and Diseases: Proteins, as direct executors of genes’ function, participate in a wide variety of biological processes. As such, proteome-wide protein expression studies have been utilized to systemically search for causal proteins or protein markers which are differentially expressed in diseased vs. healthy subjects. However, such expression proteomics studies generally focused on quantitative change(s) of protein(s) expression levels and cast little attention to amino acid residue change(s) and potential impact of such sequence changes on phenotypes of interest.As the most widely studied post-translational modification, protein phosphorylation is involved in various signaling pathways and plays fundamental roles in regulating almostall kinds of normal cellular functions and biological processes. Thus, abnormal regulationof protein phosphorylation has been found associated with pathogenesis of a serial of diseases, such as cancer (2), Alzheimer’s disease (3), osteoporosis (4), etc. In the bone field, more than a dozen of review articles highlight the importance of protein phosphorylation-regulated signaling events for osteoporosis, such as IL-6 signaling (5), TNF signaling (6), and events in regulation of CSF-1 induced osteoclast spreading (7), integrin-mediated osteoclast adhesion and activation (8), etc. However, few studies have systemically explored the relationship between protein phosphorylation and bone metabolism and/or assess the significance of phosphorylation sites in influencing BMD in humans.SNP and Protein Phosphorylation: Protein phosphorylation is a reversible post-translational modification mainly targeting at amino acid residues serine (S), threonine (T), and tyrosine (Y). It is catalyzed by protein kinases. Ren et al. reported that ~70% of non-synonymous SNP (nsSNP) in human genome are potential phosphorylation-related SNPs, i.e., so called phosSNPs (9). They reported a total of 64,035 phosSNPs, among which 2,004 are experimentally validated. PhosSNPs could affect protein phosphorylation by either changing protein phosphorylation amino acid site(s) or changing protein kinase(s) which catalyze phosphorylation of specific amino acid site(s). Despite their potential significance, so far, whether phosSNPs are associated with variation in complex traits, such as human BMD, is unknown.Study Purpose: Through a pilot genome-wide phosSNP association study and replication studies in three independent human populations, the present work is attempted to identify significant phosSNPs influencing BMD. Furthermore, through follow-up in vitro studies on top significant phosSNP identified, we aim to characterize their biological functions, hence to illustrate pathophysiological mechanism of osteoporosis in humans.Materials and MethodsHuman SubjectsThe study was approved by Institutional Review Boards of involved institutes. Signed informed-consent documents were obtained from all study participants before enrollment in the study.Caucasian Sample 1 (CAU1): This sample contains 1,000 unrelated subjects (age: 50.3 ± 18.3 years) selected from our established and expanding genetic database currently containing more than 7,000 subjects and largely recruited in Midwestern U.S. in Omaha, Nebraska. All the subjects were U.S. Caucasians of European origin. CAU1 serves as a discovery cohort in this study.Caucasian Sample 2 (CAU2): This sample contains 2,286 unrelated subjects (age: 51.4 ± 13.8 years) recruited in Midwestern U.S. in Kansas City, Missouri and Omaha, Nebraska. All the subjects were U.S. Caucasians of European origin. This sample, independent of CAU1, serves as a replication cohort to validate findings in CAU1.Chinese Sample (CHN): This sample consists of 1,627 unrelated subjects (age: 34.5 ± 13.2 years) recruited from central south region of China. All the subjects were Han Chinese. This sample serves as a replication cohort for across-ethnicity validation to test ethnic- general or specific effects of phosSNPs identified and/or validated in Caucasians.For subject recruitment, strict exclusion criteria (10) were adopted to minimize any known or potential confounding effects on variation of bone phenotype. Briefly, patients with chronic diseases/conditions that may potentially affect bone mass were excluded. These diseases/conditions included chronic disorders involving vital organs (heart, lung, liver, kidney, brain), serious metabolic diseases (diabetes, hypo- or hyperparathyroidism, hyperthyroidism), other skeletal diseases (Paget’s disease, osteogenesis imperfecta, rheumatoid arthritis), chronic use of drugs affecting bone metabolism (corticosteroidtherapy, anticonvulsant drugs), and malnutrition conditions (chronic diarrhea, chroniculcerative colitis). Bone mineral density (g/cm2) at lumbar spine (L1_4) and total hip were measured using daily calibrated dual energy X-ray absorptiometry (DXA) machines (Hologic Inc., Bedford, MA, USA).PhosSNP GenotypingOut of the total 64,035 phosSNPs in the phosSNP 1.0 database (9), those covered by Affymetrix SNP Arrays (Affymetrix, Inc., Santa Clara, CA, USA) were studied herein. Specifically, genomic DNA was extracted from leukocytes using Puregene DNA Isolation Kit (Gentra systems, Minneapolis, MN, USA). For the CAU1 sample, SNP genotyping with the Affymetrix Mapping 250 k Nsp and 250 k Sty arrays was performed in Vanderbilt Microarray Shared Resources (VMSR) (/) using the standard protocol recommended by Affymetrix. For CAU2 and CHN samples, SNP genotyping with Affymetrix Genome-Wide Human SNP Array 6.0 was performed using the standard protocol recommended by the manufacturer. Fluorescence intensities were quantified using an Affymetrix array scanner 30007G. Data management and analyses were performed using the Affymetrix® GeneChip® Command Console® Software. Contrast quality control (QC) threshold was set at the default value of greater than 0.4 for data QC.After excluding SNPs with minor allele frequency (MAF) less than 0.01 and/or SNPs deviating from Hardy-Weinberg Equilibrium (HWE test, p <0.01) in individual population sample, a total of 2,474 phosSNPs retained in the three study samples, including 660, 1,797, and 1,662 phosSNPs in the CAU1, CAU2, and CHN samples, respectively. The discrepancy between the numbers of phosSNPs “retained” in CAU1 and the other two samples mainly reflects the difference in SNP coverage between the genotyping arrays used. The relatively small difference in the numbers of phosSNPs between CAU2 and CHN primarily reflects the difference in genetic background between the two ethnicities of Caucasian and Chinese. Among those retained phosSNPs, 653 phosSNPs were overlapped in all the three samples.Age, gender, height, and weight were used as covariates to adjust the raw BMD measurements. PLINK (11) was used to perform genotypic association analyses between phosSNPs and adjusted BMD. Specifically, the analyses compared the difference of mean BMD values among carriers of the three different genotypes for each phosSNP. PhosSNPs, discovered to be significant to both spine BMD and hip BMD in the CAU1 sample, were analyzed in the CAU2 sample for within-ethnicity validation purpose, and further analyzed in the CHN sample for across-ethnicity validation. Miscellaneous statistical analyses were performed using the software packages SAS (SAS Institute Inc., Cary, NC) and Minitab (Minitab Inc., State College, PA).Predicting and Validating Impact of PhosSNP on Protein PhosphorylationGroup-based Prediction System (GPS 2.0) (12) was applied to predict the impact of phosSNPs on protein phosphorylation, i.e., to predict the resultant changes of protein phosphorylation sites and/or corresponding changes of protein kinases (12). For a specific phosSNP rs6265 that we identified as important for hip BMD in general human populations, we conducted the following studies to validate its predicted impact on protein phosphorylation (detailed in the section of Results). To be noted, rs6265 (major/minor allele: G/A) is located in BDNF gene (Entrez Gene database accession number: 627), which encodes brain-derived neurotrophic factor.Firstly, to ascertain the substrate-kinase relationship between the protein BDNF and the predicted protein kinase CHEK2, we tested their protein-protein interaction in bone cells. Briefly, we co-transfected the human fetal osteoblastic 1.19 cell line (hFOB, ATCC, Cat CRL-11372) with expression vector pCMV6-AC-GFP-CHEK2, together with wild-type pCDNA3.1-BDNF-V66T62, variant pCDNA3.1-BDNF-M66T62, or mutant pCDNA3.1-BDNF-V66A62, respectively. Herein, pCDNA3.1-BDNF-V66A62 was to replace asuspect target site of phosphorylation (i.e., Threonine at amino acid residue 62) with anunphosphorylatable residue (i.e., Alanine, A) at the encoded BDNF protein product. After48 hours, we collected hFOB cell lysate and quantified total proteins. We then employed Co-Immunoprecipitation (Co-IP) procedures to pull down BDNF protein from the cell lysate and to probe CHEK2 protein in the Co-IP product through Western Blotting (WB) procedures.Secondly, for testing whether rs6265 affect BDNF protein phosphorylation, we co-transfected hFOB cells with expression vector pCMV6-AC-GFP-CHEK2, together with wild-type pCDNA3.1-BDNF-V66T62, variant pCDNA3.1-BDNF-M66T62, or mutant pCDNA3.1-BDNF-V66A62, respectively. After 48 hours, we collected hFOB cell lysate, quantified total proteins, and purified BDNF from equal amount of total proteins. Then, we conducted phosphoprotein phosphate estimation assay to quantify overall BDNF phosphoyrlation levels under different conditions.Thirdly, to validate the prediction that BDNF-T62 is the target site of phosphorylation regulated by rs6265, we co-transfected human osteoblast-like cell line MG63 (ATCC, Cat CRL-1427) with expression vector pCMV6-AC-GFP-CHEK2, together with wild-type pCDNA3.1-BDNF-V66T62, variant pCDNA3.1-BDNF-M66T62, or mutant pCDNA3.1-BDNF-V66A62, respectively. After 48 hours, we collected MG63 cell lysate and quantified total proteins. We then analyzed BDNF-T62 site-specific protein phosphorylation levels by Western Blotting method using anti-BDNF-pT62 antibody.Detailed procedures of the above experiments are described as follows.Plasmid ConstructsThe pCDNA3.1-BDNF-V66T62 and pCDNA3.1-BDNF-M66T62 constructs were kind gifts from Dr. Francis Lee’s lab (13). Specifically, the human BDNF cDNA was subcloned into pCDNA3.1 hygro expression vector (Invitrogen, Cat V870-20) at Hind III and Xho I restriction endonuclease sites. The pCDNA3.1-BDNF-V66T62 construct wasmutated to pCDNA3.1-BDNF-V66A62 (i.e., ACT->GCT at codon 62, for amino acidsubstitution: T62->A62) through PCR-based method by using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, Cat 200524). Primers used were 5'-ttgacatcattggctgacgctttcgaacacgtgatag-3' and 5'-ctatcacgtgttcgaaagcgtcagccaatgatgtcaa-3'. All the constructs were confirmed by Sanger sequencing to exclude potential PCR-introduced mutations. The pCMV6-AC-GFP-CHEK2 construct was purchased from Origene (Rockville, MD, USA, Cat RG201278).Osteoblastic Cell CultureHuman fetal osteoblastic 1.19 cell line (hFOB) was obtained from American Type Culture Collection (ATCC, Cat CRL-11372). The hFOB has the ability to differentiate into mature osteoblasts expressing normal osteoblast phenotype (14). In this study, hFOB cells were maintained at 37°C in complete medium consisting of 1:1 Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 medium (DMEM/F-12) without phenol red (Hyclone, Cat SH3027201), supplemented with 10% fetal bovine serum (Hyclone, Cat SH30070.03) and 0.3 mg/mL G418/geneticin (Invitrogen, Cat 10131-035). For osteoblastic differentiation, confluent cultures of hFOB cells were maintained at 37°C in complete medium with the addition of differentiation cocktail: 50 µg/µL ascorbic acid, 10-8 M dexamethasone, and 8mM β-glycerolphosphate (all from Sigma, St Louis, MO).Human osteoblast-like cell line MG63 (ATCC, Cat CRL-1427) was cultured in Eagle's Minimum Essential Medium (EMEM) (ATCC, Cat 30-2003) containing 10% fetal bovine serum and 1% penicillin and streptomycin (Sigma-Aldrich, Cat P4333) at 37°C in an atmosphere of 5% CO2.Transient and Stable TransfectionFor transient transfection, the hFOB cells or MG63 cells were seeded at 6×105 cells/well in polystyrene-coated 6-well plates. After 24 hours, cells were transfected with 1.0 µg of plasmids, 6.0 µl of Lipofectamine PLUS reagent (Invitrogen, Cat 11514-015), plus 4.0 µlof Lipofectamine™ reagent per well (Invitrogen, Cat 18324-020). The pCMV6-AC-GFP-CHEK2 was co-transfected with pCDNA3.1-BDNF-V66T62, pCDNA3.1-BDNF-M66T62, and pCDNA3.1-BDNF-V66A62, respectively. After 1-3 days, hFOB cells were harvested and lysed for downstream assays. All transfection experiments were performed three times (duplicates for each condition).For stable transfection, hFOB cells were transfected with pCDNA3.1-BDNF-V66T62, pCDNA3.1-BDNF-M66T62, and pCDNA3.1-BDNF-V66A62, respectively. After 24 hours, 50 µg/ml selective antibiotic hygromycin B was added to the culture medium. After one week selection, 25 µg/ml hygromycin B was maintained in the culture.BDNF-CHEK2 Protein Interaction AssayCo-immunoprecipitation (Co-IP) and Western Blotting (WB) procedures were employed to test protein-protein interaction between protein BDNF and kinase CHEK2. Briefly, 48 hours after transient transfection, hFOB cells were lysed and clarified by centrifugation. Total proteins in supernatants were quantified using a BCA Protein Assay Kit (Pierce Chemical Co., Cat 23225). A Co-immunoprecipitation Kit (Pierce, Cat 26149) was used to pull down BDNF from hFOB total proteins. Specifically, mouse anti-human BDNF antibody (Abcam, Cat ab10505) was coupled to an amine-reactive gel. Then, the supernatants of cell lysate (50 µg total protein) were incubated with the antibody-coupled gel in spin-columns for 2.0 hours at 4 °C. After washing, proteins retained in the spin columns were eluted. The eluates were separated by electrophoresis on a 12% Tris-HCl gel and transferred to a PVDF membrane. The membrane was incubated with mouse anti-human CHEK2 monoclonal antibody (Abcam, Cat ab3292) or an IgG control antibody, and then incubated with goat anti-mouse HRP-conjugated secondary antibody (Abnova, Cat PAB0096). Protein bands were visualized using chemiluminescent detection reagents (Bio-Rad, Cat 170-5070) and imaged by using VesDoc MP 4000 system (Bio-Rad, Hercules, CA, USA).BDNF Protein Phosphorylation Assaya) BDNF total protein phosphorylation assayBDNF protein was purified from transiently transfected hFOB cell lysate through IP procedure by using the Co-IP kit (Pierce, Cat 26149) and mouse anti-human BDNF antibody (Abcam, Cat ab10505). Then, BDNF protein phosphorylation level was quantitated using a Phosphoprotein Phosphate Estimation Assay Kit (Thermo Scientific, Cat 23270) according to manufacturer’s instruction. The assay is based on the alkaline hydrolysis of phosphate from seryl and threonyl residues in phosphoprotein, followed by colorimetric quantification of the released phosphate by use of malachite green and ammonium molybdate. Herein, BioTek Synergy HT Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA) and BioTek Gen5 Data Analyses Software were used to collect and analyze the data.b) BDNF T62 site-specific phosphorylation assayTransiently transfected MG63 were lysed in 1X cell lysis buffer (62.5 mM Tris-HCl pH 6.8, 10% glycerol, and 2% SDS) and homogenized. Equal amount of cell lysate (25 µg total protein) was subjected to electrophoresis on a 12% Tris-HCl gel under reducing conditions, and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). Membrane was blocked with 5% non-fat milk in phosphate buffered saline (0.1% Tween). The membrane was incubated with rabbit anti-human BDNF-pT62 polyclonal antibody (Custom-made, ProSci Incorporated, Poway, CA, USA), and a goat anti-rabbit HRP-conjugated secondary antibody (Sigma-aldrich, Cat A0545). In a parallel blot, total BDNF was incubated with mouse anti-human BDNF primary antibody (Abcam, Cat ab10505) and goat anti-mouse HRP-conjugated secondary antibody (Abnova, Cat PAB0096). Both the BDNF-pT62 and total BDNF proteins were visualized using chemiluminescence detection kit (Bio-Rad, Cat 170-5070) and imaged with VesDoc MP 4000 system (Bio-Rad, Hercules, CA, USA). Then, both blots were stripped and incubated with goat anti-rabbit actin antibody (Sigma, Cat A5060) and imaged again to detect house-keeping protein beta-actin (internal control).CA, USA). For each sample, the intensity of the BDNF-pT62 and total BDNF proteinbands were normalized against that of beta-actin band on the two parallel blots,respectively. The ratio of the normalized BDNF-pT62 intensity to the normalized totalBDNF intensity was used to represent BDNF phosphorylation level at site T62. Westernblot experiments were performed twice.Testing Effect of PhosSNP rs6265 on Osteoblastogenesis in VitroOsteopontin (OPN) and alkaline phosphatase (ALP) are known to be regulated duringosteoblastic differentiation and are commonly used as "osteoblast markers" (15-17). Bonemorphogenetic protein 2 (BMP2) is a potent osteoblastic factor stimulatingosteoblastogenesis. To assess the effect of rs6265 on osteoblastogenesis, we comparedmRNA expression levels (OPN and BMP2) and enzyme activity (ALP) of theseosteoblastic maker genes in hFOB cells that had been transfected with wild-type vs.variant BDNF gene. Related experimental procedures are detailed as follows.mRNA Expression Assay of Osteoblastic Genes (OPN and BMP2) by Quantitative realtime PCR24 hours after transfection of pCDNA3.1-BDNF-V66T62, pCDNA3.1-BDNF-M66T62,pCDNA3.1-BDNF-V66A62 constructs, and pCDNA3.1 empty vector individually, hFOBcells were lysed and reverse transcribed using the Power SYBR Green Cells-to-CT Kit(Applied Biosciences, Cat 4402954) according to the manufacturer’s instructions. OPNand BMP2 mRNA levels were quantified by quantitative PCR using a MiQ qPCR cyclerand MiQ software (Bio-Rad). Both gene expression levels were normalized against thehouse-keeping gene Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH, internalcontrol). The PCR primers used are shown in Supplemental Table 1. The quantitativeRT-PCR experiments were performed in triplicates for each condition and repeated twice.Osteoblastic ALP Enzyme Activity Assay by ALP StainingALP staining was performed to test ALP enzyme activity by using TRACP & ALP Double-Stain Kit (Takara, Cat MK300) following the manufacturer’s instructions. 5×104 stably transfected hFOB cells were plated on 48-well plates the day before staining. The experiments were performed in triplicates for each condition and repeated twice.ResultsIdentification of PhosSNPs Significant for BMD in HumansBasic characteristics of the three study population samples were summarized in Table 1. In the CAU1 sample, we identified nine phosSNPs associated with both spine BMD and hip BMD (p<0.05) (Table 2). Among the nine phosSNPs, three phosSNPs (rs16861032, rs2657879, and rs6265) were replicated in the CAU2 sample (p=0.05). Notably, association of phosSNP rs6265 (major/minor allele: G/A) with hip BMD was also suggested in the CHN sample (p=0.09), suggesting that rs6265 has ethnic-general effect on hip BMD. Interestingly, the direction of genotypic effect is consistent among the three studied population samples. Specifically, homozygous AA carriers have significantly decreased hip BMD than allele G carriers (GA or GG) (Figure 1).Predictive Impact of PhosSNP on Protein PhosphorylationThe potential impacts of the above three significant phosSNPs (rs16861032, rs2657879, and rs6265) on protein phosphorylation were predicted and summarized in Supplemental Table 2. Generally speaking, the allele-specific gene sequences encode different protein isoforms, which either change (create or remove) phosphorylation site(s) of the encoded protein or change the kinases that catalyze phosphorylation of target site(s). To take phosSNP rs6265 as an example, the BDNF gene harboring major allele G and minor allele A encodes BDNF protein isoforms with amino acid residue Val (V) and Met (M) at position 66 (abbreviated as BDNF-V66 and BDNF-M66), respectively. Bioinformatics analyses predicted that compared with BDNF-M66, BDNF-V66 creates a new phosphorylation site at residue T62, which can be targeted and catalyzed by proteinkinase CHEK2. CHEK2 is the only one kinase that is predicated to act on this specificphosphorylation site of BDNF. However, the substrate-kinase relationship between BDNF and CHEK2 and the effect of rs6265 on BDNF-T62 phosphorylation have not been experimentally validated previously.Experimental Validation of rs6265 on Protein PhosphorylationWe found that CHEK2 protein kinase in hFOB cell lysates can be pulled down together with BDNF protein by anti-BDNF antibody during co-IP process, as visualized on Western blot using anti-CHEK2 antibody (Figure 2). The finding supports and validates substrate-kinase interaction between BDNF and CHEK2 proteins in bone cells. Furthermore, comparison of CHEK2 protein band intensity on Western blot, after beta-actin normalization, showed that more CHEK2 protein was pulled down with BDNF-V66T62 than BDNF-M66T62 or BDNF-V66A62, suggesting weakened interaction between protein kinase CHEK2 and the variant/mutant BDNF protein isoforms.We also found that BDNF total protein phosphorylation level was significantly reduced in hFOB cells transfected with variant BDNF-M66T62, compared with that transfected with wild-type BDNF-V66T62 (Figure 3). The finding suggests that BDNF total protein phosphorylation is regulated by the amino acid substitution Val66Met, resulting from the nucleotide variation at rs6265.Furthermore, site-specific protein phosphorylation analyses showed that normalized BDNF-pT62 protein band intensities level on Western blot was significantly decreased in MG63 cells transfected with variant BDNF-M66T62, compared with that transfected with wild-type BDNF-V66T62(Figure 4). The finding suggests that T62 is the target phosphorylation site regulated by amino acid residue at position 66, and that substitution of amino acid residue V66 to M66 attenuates BDNF phosphorylation at site T62. Consistently, when T62 is substituted with unphosphorylatable A62, BDNF total phosphorylation level was significantly decreased (Figure 3, V66A62 vs. V66T62), suggesting that T62 is a primary target site of phosphorylation in BDNF protein.。
荧光探针在蛋白磷酸化和糖基化检测中的应用
第43 卷第 1 期2024 年1 月Vol.43 No.1157~165分析测试学报FENXI CESHI XUEBAO（Journal of Instrumental Analysis）荧光探针在蛋白磷酸化和糖基化检测中的应用常永新1，李军荣1，2，邵娟1，杨新迪1，卿光焱1*（1．中国科学院大连化学物理研究所中国科学院分离分析重点实验室，辽宁大连116023；2．大连理工大学精细化工国家重点实验室，辽宁大连116023）摘要：蛋白质翻译后修饰在细胞内起着关键的调控作用，对细胞的功能和代谢具有重要影响。
近年来，荧光探针在研究蛋白质翻译后修饰中的应用不断发展。
该文重点介绍了荧光探针在蛋白磷酸化和糖基化等修饰中的应用，包括其原理、设计策略以及在细胞和生物体内的应用。
荧光探针的发展为研究蛋白质翻译后修饰提供了强有力的工具，有望深化对这些修饰过程的理解，并为药物研发和疾病诊断提供新的途径。
关键词：荧光探针；蛋白质；翻译后修饰；磷酸化；糖基化中图分类号：O657.3；O629.7文献标识码：A 文章编号：1004-4957（2024）01-0157-09Application of Fluorescent Probes in Protein Phosphorylation andGlycosylation DetectionCHANG Yong-xin1，LI Jun-rong1，2，SHAO Juan1，YANG Xin-di1，QING Guang-yan1*（1．CAS Key Laboratory of Separation Science for Analytical Chemistry，Dalian Institute of Chemical Physics，Chinese Academy of Sciences，Dalian 116023，China；2．State Key Laboratory of Fine Chemicals，Dalian University of Technology，Dalian 116023，China）Abstract：Post-translational modifications of proteins play a crucial regulatory role within cells，ex⁃erting significant impacts on cellular functions and metabolism. In recent years，the application of fluorescent probes in the study of protein post-translational modifications has been continuously evolv⁃ing. This review will primarily focus on the applications of fluorescent probes in modifications such as protein phosphorylation and glycosylation，encompassing their principles，design strategies，as well as their utilization within cells and organisms. The development of small molecule fluorescent probes provides a robust tool for investigating protein post-translational modifications，holding the potential to deepen our understanding of these processes and offering novel avenues for drug development and disease diagnosis.Key words：fluorescent probe；protein；post-translational modification；phosphorylation；glyco⁃sylation蛋白质翻译后修饰（PTM）调节几乎所有的细胞过程，是细胞内调控生物功能和信号传导的重要机制之一［1］。
蛋白质修饰
• posttranslational modifications: alter their interaction with DNA and nuclear proteins. H3 & H4: long tails; can be modified at several places, including methylation, acetylation, phosphorylation, ubiquitination, sumoylation, citrullination and ADPribosylation. The core of the histones H2A and H3 can also be modified. • Histone Code: hypothesized to be a code consisting of covalent histone tail modifications → epigenetic code
– modification site may be a targeting signal – modification may be a membrane anchor
• Degradation
– identify the protein for degradation
• ……
For more information, see /wiki/Posttranslational_modification
Non-histone Acetylation/Deacetylation
a significant post-translational regulatory mechanism
生物化学名词英汉互译
碳水化合物( carbohydrate )单糖( monosaccharide )寡糖( oligosaccharide )多糖( polysaccharide )醛糖(aldose )酮糖( ketose )蔗糖(sucrose )乳糖(lactose ) 麦芽糖(maltose )纤维二糖(cellobiose )多糖(polysaccharides )淀粉(starch ) 直链淀粉( amylose)支链淀粉( amylopectin )纤维素( cellulose )半纤维素( hemicellulose )糖原(glycogen )几丁质(chitin )糖胺聚糖( glycosaminolgycan ) 脂类( lipids )脂肪酸( fatty acid )甘油三酯( glycerol triester )亲水脂类 ( amphipathic lipids )蜡( wax)磷酸甘油脂( phosphoglyceride )甘油磷脂 ( glycerophospholipid )磷脂酰胆碱( phosphatidylcholine )磷脂酰乙醇胺 ( phosphatidylethanolamine )磷脂酰丝氨酸( phoshatidylserine )磷脂酰肌醇 ( phosphatidylinositol, PI )肌醇三磷酸 ( inositol-1,4,5-trisphosphate ，IP3) 二脂酰甘油( diacylglycerol ，DAG)磷脂酸( phosphatidic acid ，PA)磷脂酶A2 ( phospholipase A2，PLA2)磷脂酶C( phospholipase C，PLC)磷脂酶D(phospholipase D，PLD)溶血磷脂(1ysophospholipid) 鞘磷脂(sphingomyelin )神经酰胺( ceramide ) 类固醇(steroids )萜类(terpenes )胆固醇(cholesterol )麦角固醇(ergosterol ) 蛋白质protein 简单蛋白质simple protein 氨基酸amino acid 结合蛋白质conjugated protein 多肽polypeptide 肽peptide 肽键peptide bond 介电常数dielectric constant 范德华力van der waals force 层析法chromatography吸附层析法adsorption chromatography 分配系数partition or distribution confficient 活性肽active peptide 二硫键disulfide bond 兼性离子zwitterion 一级结构primary structure 疏水效应hydrophobic effectSDS- 聚丙烯酰胺凝胶电泳SDS-PAGE毛细管电泳( capillary eletrophoresis, CE)离子交换层析ion exchange chromatography 同源蛋白homologous protein 构象conformation 构象角conformatiomal angle 糖脂( glycolipid ) 糖基甘油酯( glycosylglyceride )鞘糖脂( glycosphingolipid ) 脑苷脂( cerebroside )N-乙酰神经氨酸( N-acetylneuraminic acid ) 神经节苷脂( ganglioside )硫酸脑苷脂( cerebroside sulfate ) 糖蛋白( glycoproteins )蛋白聚糖( proteoglycans )生物膜( biomembrane) 膜脂( membrane lipids )膜蛋白( membrane proteins )脂质双层分子( lipid bilayers )外周蛋白( peripheral protein )外源性( extrinsic protein )内在蛋白( integral protein )内源性蛋白( intrinsic protein ) 跨膜蛋白( transmembrane proteins )流动镶嵌模型( fluid mosaic model ) 简单扩散( simple diffusion )协助扩散( facilitated diffusion ) 被动运输( passive transport )主动运输( active transport ) 介导性运输( mediated transport )非介导性运输( nonmediated transport ) 载体蛋白( carrier protein )通道蛋白( channel protein ) 离子通道( ionic channel )离子载体( ionophore )内吞作用( endocytosis ) 胞饮作用”(pinocytosis )外排作用(exocytosis )基团转移( group translocation ) 脂蛋白( lipoprotein )染色体( chromosome)染色质( chromatin ) 组蛋白( histone )核小体( nucleosome)病毒( virus ) 噬菌体( bacteriophage 或简称phage)变性denaturation 沉降系数( S)Svedberg(S) 抗体antibody 亲和层析法affinity chromatography盐溶salting in 盐析salting out 二级结构secondary structure三级结构tertiary structure - 螺旋-helix超二级结构super-secondaery structure 结构域structure domain 氢键hydrogen bend 疏水相互作用hydrophoblic interaction 肌红蛋白myoglobin 寡聚蛋白质oligomeric protein 无规则卷曲randon coil 复性renaturation 镰刀状细胞贫血病sickle-cell anermia 酶( enzyme)酶的专一性( specificity )单体酶(monomeric enzyme)寡聚酶(oligomeric enzyme) 多酶复合体系( multienzyme system )酶活性中心( active center of enzyme ) 催化基团( catalytic site )酶原(zymogeno r proenzyme)诱导契合(induced-fit theory )抗体酶( abzyme)酸碱催化( acid-base catalysis ) 共价催化(covalent catalysis) 激活剂( activator )抑制剂( inhibitor ) 可逆抑制( reversible inhibition ) 竞争性抑制作用( competitive inhibition )非竞争性抑制作用( noncompetitive inhibition ) 调节酶( modulator ) 别构酶( allosteric enzyme ) 同配位效应( isosteric effect ) 变构效应( allosteric effect ) 变构激活( allosteric activation ) 正协同效应( positive cooperative effect )负协同效应( negative cooperative effect )效应物( effector ) 维生素( vitamin ) 维生素缺少症( avitaminosis ) 调节中心( regulatory center ) 催化亚基( catalytic subunit ) 调节亚基( regulatory subunit ) 诱导酶( induced enzyme ) 结构酶( structural enzyme ) 核酶( ribozyme ) 辅酶( coenzyme) 比活力( specific activity )脱氧核酶( deoxyribozyme ) 酶工程( enzyme engineering ) 酶纯度( purity of enzyme ) 酶活力( enzyme activity )- 淀粉酶( -amylase ) - 淀粉酶( -amylase ) 脱支酶( debranching enzyme ) 淀粉的磷酸化酶( amylophosphorylase )糖酵解( glycolysis ) 三羧酸循环( tricarboxylic acid cycle ，TCA)磷酸戊糖途径( pentose phosphate pathway ，PPP) 生物氧化( biological oxidation )烟酰胺脱氢酶类( nicotinamide dehydrogenase ) 黄素脱氢酶类( flavin dehydrogenase ) 铁硫蛋白类( iron-sulfur protein ) 泛醌( ubiquinone ) 细胞色素类( cytochromes ) 细胞色素氧化酶( cytochromeoxidase ) 鱼藤酮( rotenone ) 安密妥( amytal ) 杀粉蝶菌素( piericidine ) 抗霉素A( antimycin A ) 底物水平磷酸化( substrate-level phosphorylation ) 氧化磷酸化( oxidative phosphorylation ) 化学渗透假说( chemiosmotic coupling hypothesis ) 化学偶联假说( chemical coupling hypothesis ) 构象偶联假说( conformational coupling hypothesis )甘油-磷酸穿梭途径( glycerophosphate shuttle ) 苹果酸- 天冬氨酸穿梭途径( malate- aspartate shuttle )异柠檬酸穿梭途径( isocitrate shuttle )能荷( energy charge ) 肉碱(肉毒碱，carnitine ) 乙醛酸体(乙醛酸循环体，glyoxysome ) 乙醛酸循环( glyoxylate cycle ) 酮体( ketone bodies ) 饱和脂肪酸的从头合成( de novo synthesis ) 谷氨酸脱氢酶( glutamate dehydrogenase, GDH ) 转氨基作用( transamination ) 转氨酶( transaminase ) 磷酸吡哆醛( pyridoxal phosphate ，PLP) 谷丙转氨酶( glutamic pyruvic transaminase，GPT或alanine transaminase ALT) 谷草转氨酶( glutamic oxaloacetic transaminase ，GOT或aspartate transaminase ，AST) γ-谷氨酰-半胱氨酸合成酶( γ-glutamyl systeine synthetase ，γ-ECS)谷胱甘肽( glutathione ) 谷胱甘肽合成酶( glutathione synthetase ) 生物固氮( biological nitrogen fixation )固氮酶( nitrogenase )自身固氮微生物( diazatrophs ) 共生固氮微生物( symbiotic microorganism ) 硝酸还原酶( nitrate reductase ，NR) 亚硝酸还原酶( nitrite reductase ，NiR)谷氨酸合酶( glutamate: oxo-glutarate aminotransferase ，GOGA)T谷氨酰胺合成酶( glutamine synthetase ，GS) 腺苷-5'- 磷酸硫酸酐( adenosine-5'-phosphosulfate ，APS) 3'- 磷酸腺酐-5'- 磷酰硫酸( 3'-phosphoadenosine-5'-phosphosulfate ，PAPS) 5- 磷酸核糖焦磷酸( phosphoribosyl pyrophosphaet ，PRPP) 天冬氨酸转氨甲酰酶( aspartate trsnscarbamoy lase ) 腺嘌呤磷酸核糖转移酶( adenine phosphoribosyl fransferase ，APRT)黄嘌呤- 鸟嘌呤磷酸核糖转移酶( hypoxanthineguanine phosphoribosyl transferase ，HGPR)T 谷胱甘肽还原酶( glutathione reductase ，GR) 谷氧还蛋白( glutaredoxin ) 谷氧还蛋白还原酶( glutaredoxin reductase ) 胸腺嘧啶核苷酸合酶( thymidylate synthase )DNA复制( DNA replication ) 中心法则( central dogma ) 冈崎片段( Okazaki fragement ) 前导链( leading strand ) 滞后链( lagging strand ) 引物( primer ) 复制叉( replication fork ) 半保留式复制( semiconservative replication ) 模板( template ) 反转录( reverse transcription )转换( transition ) 颠换( transversion ) 错配修复( mismatch repair ) 核苷酸切除修复( nucleotide excision repair )碱基切除修复( base excision repair ) 同源重组( homologous recombination ) 特异性重组( site-specific recombination ) 转座子( transposon ) 启动子( promoter ) 限制性内切酶( restriction endonuclease ) 修饰( modification ) 单链结合蛋白( single stranded binding proteins, SSB ) 遗传密码( genetic code ) 读码框架( reading frame )移码突变( frame-shift mutation ) 简并性( degeneracy ) 同义密码子( synonymous codon) 起始密码子( initiatlon codon ) 终止密码子( termination codon ) 摆动假说( wobble hypothesis) 同功受体tRNA( isoaccepting tRNA ) 反密码子( anticodon ) 多核糖体( polyribisome ) 氨酰-tRNA合成酶( aminoacyl-tRNA synthetase ) Shine –Dalgarno 序列( Shine –Dalgarno sequence ) 起始因子( initiation factor ) 延伸因子( elongation factor ) 释放因子( release factor ) 转肽( transpeptidation ) 移位( translocation ) 分子伴侣( molecular chapeones ) 共翻译转移( co-translational translocation ) 翻译后转移( post-translational translocation ) 信号肽( signal sequence ) 信号识别颗粒( signal recognition particle SPR ) 代谢 (metabolism ) 代谢调节 ( metabolic regulation ) 共价修饰 ( covalent modification ) 反馈抑制 ( feedback inhibition ) 操纵子模型 (operon model ) 衰减作用 ( attenuation ) 级联放大作用 (amplification cascade ) 变(别)构效应 (allosteric effect )诱导和阻遏 (induction and repression ) 蛋白激酶C (protein kinase C ，PKC)第二信使 (second messenger )受体 ( receptor )G 蛋白 (guanosine triphosphate-binding protein )信号转导 ( signal transductionphospholipase C ，PLC) 钙调素（calmodulin ，CaM）磷酯酶。
蛋白质磷酸化修饰的研究进展_姜铮
doi:10．3969／j．issn．1009－0002．2009．02．025综述蛋白质磷酸化修饰的研究进展姜铮，王芳，何湘，刘大伟，陈宣男，赵红庆，黄留玉，袁静中国人民解放军疾病预防控制研究所，北京100071［摘要］蛋白质磷酸化是最常见、最重要的一种蛋白质翻译后修饰方式，它参与和调控生物体内的许多生命活动。
通过蛋白质的磷酸化与去磷酸化，调控信号转导、基因表达、细胞周期等诸多细胞过程。
随着蛋白质组学技术的发展和应用，蛋白质磷酸化的研究越来越受到广泛的重视。
我们介绍了蛋白质磷酸化修饰的主要类型与功能、磷酸化蛋白质分析样品的富集及制备、磷酸化蛋白的鉴定及磷酸化位点的预测、蛋白分离后磷酸化蛋白的检测，及蛋白质磷酸化的分子机制，并综述了近年来国内外的主要相关研究进展。
［关键词］磷酸化修饰；磷酸化蛋白鉴定；磷酸化位点检测［中图分类号］Q52［文献标识码］A［文章编号］1009－0002(2009)02－0233－05Progress on Protein／Peptide PhosphorylationJIANG Zheng,WANG Fang,HE Xiang,LIU Da－Wei,CHEN Xuan－Nan,ZHAO Hong－Qing,HUANG Liu－Yu,YUAN JingInstitute of Disease Control and Prevention,Academy of Military Medical Sciences,100071Beijing,China［Abstract］Phosphorylation is one of the most important post－translational modifications of proteins,which is related to many activities of life．By reversible protein phosphorylation eukaryotes control many cellular processes including signal transduction,gene expression,and the cell cycle etc．As the development and application of the proteomics,the studies of the protein phosphorylation have become more important．This article has introduced the main types and functions of the protein phosphorylation,the enrichment and preparation of phosphoproteins and phosphopeptides,the identification of the phosphopeptides,the determination and prediction of the specific－phosphorylation－site,the phosphorelated modifications of the proteins,and the progress on studies above as well．［Key words］phosphorelated modifications;identification of the phosphopeptides;determination of the specific－phosphory-lation－site几乎所有的蛋白质在合成过程中或合成后都要经过某些形式的翻译后修饰，一些不合适的修饰常常与疾病相关，某些特定的翻译后修饰还被作为疾病的生物标志或治疗的靶标。
解释载体蛋白磷酸化过程
解释载体蛋白磷酸化过程载体蛋白磷酸化是一种重要的信号转导机制，它参与细胞内多种生物学过程，如细胞分化、生长、再生、信号传导和发育等，其过程对于实现细胞的正常功能至关重要。
载体蛋白磷酸化是一个化学反应，它通过载体蛋白上蛋白质磷酸化（Protein Phosphorylation），把磷酸形式的小分子（例如磷酸化乙酰腺苷）和磷酸化脂质（例如磷酰胆碱）转换为磷酸盐（例如磷酸乙酰腺苷），进而改变载体蛋白活性，并发挥对应的生物学功能。
载体蛋白磷酸化过程通常分为两个步骤：磷酸化和脱磷酸化。
在磷酸化过程中，ATP通过激酶（Kinase）的催化转化成ADP，同时将一个磷酸基从ATP转移到载体蛋白上，从而改变其活性。
在脱磷酸化过程中，一个磷酸基从载体蛋白转移到一种磷酸酶（Phosphatase）中，从而改变载体蛋白的活性。
载体蛋白磷酸化过程是一种动态的过程，可以促进细胞内许多生物学过程。
载体蛋白磷酸化促进了细胞信号传导，并参与调节许多细胞应激反应、细胞凋亡和干细胞分化过程。
此外，载体蛋白磷酸化也可以参与调节DNA激活和复制，从而影响蛋白质表达。
载体蛋白磷酸化过程中参与的过程复杂，与许多其他细胞过程关联密切，十分显著的是它的影响不仅限于活性蛋白质的合成，而且也可以影响蛋白质的后处理，例如乙酰化（acetylation）、糖基化（glycosylation）和磷酸化（phosphorylation）。
载体蛋白磷酸化过程是一个细胞内复杂的过程，它可以变换蛋白质的活性，从而影响许多细胞过程。
它的功能可以通过激酶（Kinases）和磷酸酶（Phosphatases）的活动调控，其中激酶通过把磷酸从ATP 转移到蛋白质上，而磷酸酶通过把磷酸从蛋白质上转移出来，从而变换蛋白质的活性，实现许多细胞功能。
因此，载体蛋白磷酸化过程对于细胞功能的调节至关重要。
蛋白磷酸化后条带的变化
蛋白磷酸化后条带的变化英文回答：Protein phosphorylation is a post-translational modification that plays a crucial role in regulating various cellular processes. It involves the addition of a phosphate group to specific amino acid residues in a protein, typically serine, threonine, or tyrosine. This modification can have significant effects on the structure, function, and localization of the protein.When a protein is phosphorylated, it can undergo several changes in its electrophoretic mobility, which can be visualized as shifts in the protein bands on a gel. These changes in the protein's mobility are primarily due to alterations in its charge and size. Phosphorylation introduces negatively charged phosphate groups, which can increase the overall negative charge of the protein and affect its migration during gel electrophoresis.In addition to changes in charge, protein phosphorylation can also lead to changes in protein conformation. Phosphorylation can induce conformational changes that expose or hide certain protein domains, affecting protein-protein interactions or enzymatic activity. For example, phosphorylation of the regulatory subunit of protein kinase A (PKA) causes a conformational change that releases the catalytic subunit, enabling it to phosphorylate target proteins.Furthermore, phosphorylation can also influence protein localization within the cell. Phosphorylation events can trigger the recruitment or release of proteins fromspecific cellular compartments or organelles. For instance, phosphorylation of the nuclear localization signal (NLS) can mask it, preventing nuclear import and leading to the retention of the protein in the cytoplasm.To illustrate these changes, let's consider the example of the tumor suppressor protein p53. Phosphorylation of p53 at specific sites can lead to its stabilization and activation, promoting cell cycle arrest or apoptosis. Thisphosphorylation event causes a shift in the protein bandson a gel due to changes in charge and size. The modifiedp53 protein can then translocate to the nucleus, where it regulates the expression of target genes involved in cell cycle control and DNA repair.中文回答：蛋白磷酸化是一种后转录修饰，对调节细胞过程起着重要作用。
棕榈酰化蛋白组学 实验流程
棕榈酰化蛋白组学实验流程（中英文版）Title: Palmitylation Proteomics Experimental ProcessObjective: This document outlines the step-by-step procedure for the palmitylation proteomics experiment.实验目的：本文件详细介绍了棕榈酰化蛋白质组学实验的每一步骤。
1.Sample Preparation: Cell cultures or tissue samples are harvested and lysed to release the proteins.样本准备：收获细胞或组织样本，并裂解以释放蛋白质。
2.Protein Extraction: Proteins are extracted using a suitable buffer and sonicated to achieve a uniform particle size.蛋白质提取：使用适宜的缓冲液提取蛋白质，并通过超声波处理以获得均匀的粒度。
3.Protein Digestion: Extracted proteins are digested with trypsin or other suitable enzymes to generate peptide fragments.蛋白质消化：提取的蛋白质与胰蛋白酶或其他适宜的酶一起消化，以产生肽片段。
4.Palmitylation Analysis: Peptide fragments are analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect palmitylated sites.棕榈酰化分析：使用液相色谱-串联质谱法（LC-MS/MS）分析肽片段，以检测棕榈酰化位点。
高级分子实验技术定量方法Affinity Enrichment Based Strategy for
Schematic overview of recent work
Methods development
Immobilized trypsin
Anal. Chem. 2012
Anal. Chem. 2014
In vivo glycan
imaging and differential analysis
35
Lab Chip 2010, 10, 3397
9
Microchannel
41
Lab Chip 2010, 10, 2887
Immobilized trypsin (1)
MS spectra of BSA digests by PHMN-Trypsin
MS spectrum of BSA digests obtained by PHMN-Trypsin digestion of BSA
Amino acid sequence coverage: 93% (PHMNTrypsin) VS ~70% (free trypsin digestion)
HILIC materials Combined Anal.
Chem. 2014
Application
Phosphorylation analysis of DNABinding-Protein
Core fucoslated glycoprotein based biomarker screening and verification Anal. Chem. 2011
1
Brush polymer modified column
Anal. Chem. 2014
and TiO2 based phosphopeptide
protein A磁珠免疫沉淀DynabeadsProteinA_man
• For sensitive proteins and phosphorylation studies, the isolation protocol
Catalog nos. 10001D, 10002D, 10008D
Store at 2˚C to 8˚C
including elution may be performed at 4°C, to avoid protein complex
Product Description
Dynabeads® Protein A are designed for immunoprecipitation of proteins, protein complexes, protein-nucleic acid complexes, and other antigens.
Figure 1: Principle of immunoprecipitation of antigen using Dynabeads® Protein A.
Required Materials
• Magnet (DynaMag™). See /magnets for recommendations.
Rev. Date: October 2011 (Rev. 007)
dissociation and minimize enzymatic a
Cat. no. 10001D 10002D 10008D
Volume 1 mL 5 mL 50 mL
Dynabeads® Protein A contains 30 mg Dynabeads®/mL in phosphate buffered saline (PBS), pH 7.4, with 0.01% Tween®-20 and 0.09% sodium azide as a preservative.
载体蛋白的磷酸化
载体蛋白的磷酸化载体蛋白的磷酸化（Protein Phosphorylation）是一种重要的信号传导机制，它可以活化或抑制蛋白质功能和活性。
磷酸化反应是ATP通过蛋白激酶将磷酸基团转移到蛋白质上的一种反应，由于磷酸基团在蛋白质中具有负电荷，它可以改变蛋白质的表面电荷，从而影响蛋白质的结构和功能。
磷酸化蛋白质是影响和调节生物体内各种代谢过程、细胞凋亡和细胞周期调控过程、细胞内信号传导等的重要机制。
磷酸化蛋白质通过两种主要方式来调节蛋白质的活性：一种是修饰蛋白质的结构，改变蛋白质的三维结构，使其失去其原来的活性；另一种是改变蛋白质的活性，使其能够识别新的结合伙伴，从而改变其活性。
磷酸化蛋白质的活性可以通过多种不同的方式来调节，例如蛋白质激酶磷酸化、磷酸酶磷酸化、磷酸二酯酶磷酸化等。
磷酸化蛋白质是一种重要的信号传导机制，它可以调节细胞内外的信号传导和调控，并且在植物和动物的生殖发育、胚胎发育、细胞凋亡和细胞周期调控等研究中发挥着重要作用。
磷酸化蛋白质的研究也可以帮助我们理解疾病的发生机制以及药物的作用机制，并且可以用于开发新的药物。
磷酸化蛋白质可以用多种方法检测，包括生物化学方法、分子生物学方法和免疫学方法。
生物化学方法是测定蛋白质激酶磷酸化水平的常用方法，它可以检测蛋白质激酶的活性，从而反映磷酸化水平。
分子生物学方法可以检测磷酸化蛋白质的表达水平，例如免疫印迹、RT-PCR等，而免疫学方法，如Western Blotting，可以用于直接检测蛋白质磷酸化水平。
磷酸化蛋白质的研究已经取得了很大的进展，但仍然存在许多未解的问题，例如磷酸化蛋白质的分子机制仍然不太清楚，以及如何有效地利用磷酸化蛋白质的基础研究成果来开发治疗疾病的药物。
研究人员正在努力探索磷酸化蛋白质的分子机制和规律，以及如何利用磷酸化蛋白质的基础研究成果开发新药。
随着研究的不断深入，人们将有望利用磷酸化蛋白质的研究成果开发出更有效的治疗药物，为人类提供更好的治疗方案。
环磷酸腺苷天然提取项目简介(中英文)
环磷酸腺苷（cAMP）的营养保健功能成分实验研究刘孟军河北农业大学科技处中国枣研究中心一．认识cAMPcAMP是一种蛋白激酶制活剂，它的中文名字叫环磷酸腺苷，它与环磷酸鸟苷（cGMP）并称为环核苷酸，二者的比例约为50：1，是受神经内分泌系统控制的下属单位，是中枢神经细胞的“第二信使”，作为肽类激素、儿茶酚胺和前列腺等激素的第二信使发挥生物效应，对中枢神经系统活动起重要调节作用；环核苷酸可以调节基因的活动，促进mRNA基因的转录，影响酶的合成，起到代谢调节的作用，与人体的生命活动、疾病的发生、发展和康复、病理生理的变化都息息相关。
二．cAMP对人体的重要性cAMP可以促进人体的三大物质代谢，在细胞水平的调节上有非常重要的作用。
它可以促进脑细胞的新陈代谢及功能修复，缓解脑细胞疲劳，延缓脑细胞的衰老，对增强记忆力有非常良好的作用。
在2000年，有一位叫做坎德尔的美国科学家发现记忆的形成机制而获得诺贝尔生理及医学奖，他发现无论是短时记忆还是长期记忆都与cAMP密切相关，蛋白质磷酸化对记忆形成中分子机制的作用，在坎德尔获奖的理论中是极为重要的一环。
短期记忆由较弱的刺激形成。
神经递质促进cAMP制造→cAMP再活化蛋白质活化酶A （PKA）→PKA再使特定离子管道（如钾离子管道）蛋白质磷酸化→导致神经递质在突出的释放增加可，就形成短期的记忆。
较强和较久的刺激即形成长期记忆。
因此我们可以看出cAMP在整个记忆形成过程中所起到的重要作用，它的缺乏与不足将严重影响后续过程，导致记忆力低下！医学研究证明至少有40多种疾病与环核苷酸的代谢有关，人体内缺乏环磷酸腺苷（cAMP）即cAMP/cGMP值下降，会导致恶性肿瘤、癌症、失眠健忘、贫血、心血管病等众多疾病的发生。
三．cAMP研究与发展1957年，美国科学家萨瑟蓝德发现并报道了cAMP这种物质的存在及其相应的作用机理，他也因此荣膺1971年的诺贝尔生理及医学奖。
自从cAMP被发现以后，全世界上千所实验室都在对它进行研究。
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Protein PhosphorylationandSample PrepJohan ÖhmanSenior ScientistOutlineIntroductionCase Study 1pervanadate treated CHO cells Case Study 2phosphoproteomicsCase Study 3drug treatment of cancer cells Summary1000 / 100 / 1•localization and dynamicsSample prep - phosphoproteinsImportant considerations • • • • • Phosphatase inhibitors • Reduce phosphatase activity Obscured (hidden) PO4 groups Mapping protein interactions Multi-protease approach may be needed (LC-MS/MS) Reduce ”background” by using good controls • Low abundanceAmount material needed: 100-200 fmol peptide in LC-MS/MS 10-200 million cells, 1-20 ml cell extract11MagTM SepharoseAttractive sample preparation made easy12Products for enrichment of proteins and phosphopeptidesMagnetic beads Additional materialSpecific Ligands, e.g.: Antibodies Other Binders NHS Mag Sepharose™ Protein A Mag Sepharose Protein G Mag Sepharose TiO2 Mag Sepharose MagRack 6 Low µg scale Variable amount of beads Variable sample volume Enzyme inhibitors, e.g.: Protease inhibitors Phosphatase inhibitors Extraction and immunoprecipitation buffersYour Sample – Cells or tissue13Enrichment of phosphoproteins and peptides Case study 1: Finding low abundant tyrosine phosphoproteins in CHO cells14Key sample preparation issues for phosphoprotein and phosphopeptide analysisCoverage of the phosphoproteome: Selective enrichment of phosphoproteins and –peptides Alteration of the phosphoproteome: Stop in vitro dephosphorylation15Sample prep - enrichment techniquesSample mixture Immunoprecipitation Affinity matrixIMAC Fe3+ Ga3+ Al3+16Chemical modificationpTyrantibodypSer/pThrantibodyMOAC TiO2Zr4+Sample prep – workflow phosphoproteins17Changes in tyrosine phoshorylation in CHO cells upon pervanadate treatmentBiological system CHO cells Treatment with pervanadate (H2O2 + vanadate), known to introduce a decrease in tyrosine phosphorylation Tools and methods Phospo tyrosine specific antibody, PY20 Protein G Mag Sepharose™ beads Trypsin Mass spectrometry, LC-MS/MS18Enrichment of tyrosine phosphorylated proteins in CHO cells™Trypsin digestion19Changes in tyrosine phoshorylation in CHO cells upon pervanadate treatment• 76 potential tyrosine phosphoproteins identified Single analysis , simple MS method.. • Enrichment with Protein G Mag Sepharose™ immobilized with an anti-pTyr-antibody offers a sensitive and efficient capture of the low abundant pTyr proteinsOxidative stress indicated !20Sample prep –workflow phosphoproteinsChallenges for low abundant proteins Low abundant proteins are often biologically important but difficult to detectRegulatory proteins are often present in low amounts and only for a short time.Labeling or staining will result in very weak or no signal due to limits in detection or sensitivityof label or stainUse more sensitive stains or labels Enrich the protein or group of proteinsUse antibody based detection and amplify the signal Perform selective labeling Remove high abundant proteins Over express your protein of interestHow can we improve the possibility fordetection of low abundant proteins?(pg)Biological systemK562 chronic myeloid leukemia cellsTreatment with imatinib (Gleevec), known to introduce a decrease in tyrosine phosphorylationQuestionsCan we detect and analyze changes in the very low abundant tyrosine phosphorylated proteins?Can we identify the proteins that are differentially regulated?Collaboration with Uppsala University, Uppsala, Sweden: Dr. Sara Lind et al, Rudbeck laboratoryChanges in tyrosine phoshorylation in cancer cells upon drug treatmentUse more sensitive stains or labels Enrich the protein or group of proteinsUse antibody based detection and amplify the signal Perform selective labeling Remove high abundant proteins Over express your protein of interestHow can we improve the possibility fordetection of low abundant proteins?Enrichment of proteins and peptides using Mag Sepharose™beadsNHS Mag SepharoseProtein A Mag SepharoseProtein G Mag SepharoseTiO2Mag Sepharose(peptides)Analysis of low abundant phosphoproteinsTools and methodsPhospho tyrosine specific antibody, 4G10Protein G Mag Sepharose™beads2-D fluorescence differential gel electrophoresis (2-D DIGE) DeCyder™2-D differential analysis softwareMass spectrometryWork flow enrichment of pTyrproteinsPhenyl phosphate (phospho tyrosine analogue)Overnight incubation with K562 cell lysate at +4ºCDesalting, concentration andexchange into DIGE labeling bufferVivaspin ultracentrifugation columnsAnti phospho tyrosine antibody, 4G10Protein G MagSepharose™Non-enriched EnrichedMany tyrosine phosphoproteins are down regulated upon drug treatmentControl Imatinib treated overlayDeCyder™ 2-D differential analysis software41Large down regulationControl Imatinib treatedFold change-5.14-11.52-11.1342Protein Identification resultsRatio control/treated Spot no 1 2 3 4 Protein CRKL n/a n/a v-crk sarcoma virus CT10 oncogene homolog (avian)-like n/a Protein full name gi 4885153 Protein accession1 Gel 12 -2.68 -2.93 -2.64 -2.93 Gel 22 -2.07 -1.94 -2.47 -1.955 6 7 8 9 10TPM3 14-3-3 ε 14-3-3 γ GRB2Tropomyosin 3 isoform 5 14-3-3 protein epsilon 14-3-3 protein gamma n/a n/a Growth factor receptor-bound protein 2 isoform 1gi 114155148 gi 5803225 P61981 gi 4504111-3.09 -5.14 -11.62 -3.14 -2.65 -11.13-1.48 -9.98 -11.56 -8.81 -2.39 -2.33432-D Western blotting showed decrease in tyrosine phosphorylationTotal protein pre-labeled with Cy™3 Unlabeled total protein 2-D electrophoresis Transfer to membraneControlCy 3Drug treatedAntibody probingCy 5Anti phospho tyrosine primary, 4G10 ECL Plex™ Cy 5 secondaryPMembrane image Cy 3/Cy 5 overlay44Conclusions, case study 3Enrichment enabled the detection of differences in very low abundant proteins Tyrosine phosphoproteins were decreased in response to drug treatment Results from analysis of CyDye™ labeled enriched proteins and Western blotting were in good agreement It was necessary to enrich the proteins to get protein identity with MS45SummaryEnrichment of low abundant phosphoproteins is necessary for detection and identification Good sample prep methods are necessary for high quality results46Thank You for your attention!47Questions!48GE, imagination at work, and GE monogram are trademarks of General Electric Company. 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A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. /sampleprep GE Healthcare Bio-Sciences AB Björkgatan 30 SE-751 84 Uppsala Sweden49Recently Launched Sample Prep Products。