蛋白质组学技术的详细讲座(非常详细)
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100
Cell State 2 (All cysteines labeled with heavy ICAT)
Relative Abundance
Analyze by LCLC-MS/MS
0 200 400 600 800
Thiol-specific group = binds to Cysteins
Wisniewski JR et al., 2012
Interactomics
Schulze and Mann, 2004 Schulze WX et al., 2005
Signaling Pathways
Take home message
1. Anything is possible!
SILAC
Summary
Kolkman A et al., 2005
Label-free
Mobile phase
C18 column, 25cm long A 20 s B
Time
A = 5% organic solvent in water B = 95% organic solvent in water
ICAT
100
Cell State 1 (All cysteines labeled with light ICAT)
Combine Optional fractionation Proteolyze Affinity separation
Relative Abundance
0
Quantitate relative protein levels by measuring peak ratios Identify proteins by sequence information (MS/MS scan)
Metabolic VS Chemical Labeling
• Metabolic labeling - 15N labeling - SILAC Living cells Efficient labeling Simple!
• Chemical methods - many… but ICAT is prototype Isolated protein sample Depends on chemistry Multi-step protocols Require optimization
Ion Intensity = Ion abundance
MS measure m/z
Sample 1 Sample 2
Intensity
m/z
Isotopic Labeling
Unlabeled peptide: a) b) a) Labeled peptide: b)
Element
1
Stable Isotope
Quantitative Proteomics: Applications and Strategies
October 2013
A little history…
1985 – First use: up to a 3 kDa peptide could be ionized
1987 – Method to ionize intact proteins (up to 34 kDa) described Instruments have no sequence capability 1989 – ESI is used for biomolecules (peptides) Sequence capability, but low sensitivity 1994 – Term «Proteome» is coined 1995 – LC-MS/MS is implemented «Gold standard» of proteomic analysis
Quantitation at MS1 level
Intensity
m/z
Double sample complexity, i.e. instrument have more “features” to identify, i.e. decrease in identification rate
SILAC
Cells in normal culture media
Media with Normal AA () Media with Labelled AA (*)
Start SILAC labelling by growing cells in labelling media
m/z
m/z
(labelled AA / dialized serum)
Label-free
Strassberger V et al., 2010
Summary
Summary
Take home message
1. Quantitation can be done gel-free 2. Labeling can be performed at protein or peptide level, during normal cell growth or in vitro 3. Quantitation can be achieved at MS1 or MS2 level 4. Method choice depends on experimental design, costs, expertise etc 5. In my PERSONAL OPINION, chemical label should be avoided at all costs unless heavy multiplexing is required
iTRAQ (isobaric Tag for Relative and Absolute Quantitation)
Recognizes Arg or Lys
Total mass of label = 145 Da ALWAYS
Sample prep
iTRAQ
iTRAQ
Multiplexing
2DE-based approach
2DE-based approach
“I see 1000 spots, but identify 50 only.”
LC-MS
Column (75 mm)/spray tip (8 mm)
Reverse-phase C18 beads, 3 mm
No precolumn or split
Geiger T et al., 2012
Applications – Cell Biolo源自文库y
Applications – Immunology
Meissner et al, Science 2013
Clinical Proteomics
A. Amyloid tissue stained in Congo Red; B. After LMD.
Pr ot ei n A Pr ot ei n B Pr ot ei n C Pr ot ei n D Pr ot ei n E Pr ot ei n F
....
Time
=Protein A
NH2-EACDPLREACDPLR-COOH
m/z
ICAT
Thiol-specific group = binds to Cysteins
O N S N O XX N O XX O XX O XX O N I
Biotin tag
Linker (heavy or light)
Thiol specific reactive group
Gygi SP et al., 1999
ICAT (Isotope-Coded Affinity Tag)
X3
m/z
Grow SILAC labelled cells to desired number of cells for experiment
Ong SE et al., 2002
Chemical Labeling
ICAT Reagents: Heavy reagent: d8-ICAT (X=deuterium) Light reagent: d0-ICAT (X=hydrogen)
Control vs Tumor Cell?
Protein Identification and Quantitation by LC-MS
Control vs drug treated cell? Control vs knock-out cell?
Applications – Cell Biology
Ong SE et al., 2002
Importance of Dialyzed Serum
• non-dialzed serum contains free (unlabeled) amino acids!
No alterations to cell phenotype
C2C12 myoblast cell line
15 cm
Platin-wire 2.0 kV
Sample Loading:500 nl/min Gradient elution:200 nl/min
ESI
Fenn et al., Science 246:64-71, 1989.
MS-based quantitation
Inlet
LC
Ion Source
m/z
m/z
(labelled AA / dialized serum)
*
m/z m/z
Passage cells to allow incorporation of labelled AA
*
m/z m/z
By 5 cell doublings cells have incorporated
*
X3
m/z
2
H
H
12C 14
13C 15
N
N
16O
18O
Enzymatic Labeling
Metabolic Labeling
SILAC
Cells in normal culture media
Media with Normal AA () Media with Labelled AA (*)
Start SILAC labelling by growing cells in labelling media
MALDI ES
Mass Analyzer
Time-of-Flight Quadrupole Ion Trap Quadrupole-TOF
Detector
Peak intensities can vary up to 100x between duplicate runs.
Quatitative analysis MUST be carried on a single run.
• Labeling is guaranteed close to 99%. All identified proteins in principle are quantifiable
• Quantitation of proteins affected by different stimuli, disruption of genes, etc. • Quantitation of post-translational modifications (phosphorylation, etc.) • Identification and quantitation of interaction partners
*
m/z m/z
Passage cells to allow incorporation of labelled AA
*
m/z m/z
By 5 cell doublings cells have incorporated
*
X3
m/z
X3
m/z
Grow SILAC labelled cells to desired number of cells for experiment
Applications
State A State B Upregulated protein - Peptide ratio >1
Arg13C 6
Light Isotope
Heavy Isotope
Mix 1:1
Arg12C 6
Optional Protein Fractionation
m/z
Digest with Trypsin
Labeled cells behaved as expected under differentiation protocols
Why SILAC is convenient?
Why SILAC is convenient? • Convenient
- no extra step introduced to experiment, just special medium
Cell State 2 (All cysteines labeled with heavy ICAT)
Relative Abundance
Analyze by LCLC-MS/MS
0 200 400 600 800
Thiol-specific group = binds to Cysteins
Wisniewski JR et al., 2012
Interactomics
Schulze and Mann, 2004 Schulze WX et al., 2005
Signaling Pathways
Take home message
1. Anything is possible!
SILAC
Summary
Kolkman A et al., 2005
Label-free
Mobile phase
C18 column, 25cm long A 20 s B
Time
A = 5% organic solvent in water B = 95% organic solvent in water
ICAT
100
Cell State 1 (All cysteines labeled with light ICAT)
Combine Optional fractionation Proteolyze Affinity separation
Relative Abundance
0
Quantitate relative protein levels by measuring peak ratios Identify proteins by sequence information (MS/MS scan)
Metabolic VS Chemical Labeling
• Metabolic labeling - 15N labeling - SILAC Living cells Efficient labeling Simple!
• Chemical methods - many… but ICAT is prototype Isolated protein sample Depends on chemistry Multi-step protocols Require optimization
Ion Intensity = Ion abundance
MS measure m/z
Sample 1 Sample 2
Intensity
m/z
Isotopic Labeling
Unlabeled peptide: a) b) a) Labeled peptide: b)
Element
1
Stable Isotope
Quantitative Proteomics: Applications and Strategies
October 2013
A little history…
1985 – First use: up to a 3 kDa peptide could be ionized
1987 – Method to ionize intact proteins (up to 34 kDa) described Instruments have no sequence capability 1989 – ESI is used for biomolecules (peptides) Sequence capability, but low sensitivity 1994 – Term «Proteome» is coined 1995 – LC-MS/MS is implemented «Gold standard» of proteomic analysis
Quantitation at MS1 level
Intensity
m/z
Double sample complexity, i.e. instrument have more “features” to identify, i.e. decrease in identification rate
SILAC
Cells in normal culture media
Media with Normal AA () Media with Labelled AA (*)
Start SILAC labelling by growing cells in labelling media
m/z
m/z
(labelled AA / dialized serum)
Label-free
Strassberger V et al., 2010
Summary
Summary
Take home message
1. Quantitation can be done gel-free 2. Labeling can be performed at protein or peptide level, during normal cell growth or in vitro 3. Quantitation can be achieved at MS1 or MS2 level 4. Method choice depends on experimental design, costs, expertise etc 5. In my PERSONAL OPINION, chemical label should be avoided at all costs unless heavy multiplexing is required
iTRAQ (isobaric Tag for Relative and Absolute Quantitation)
Recognizes Arg or Lys
Total mass of label = 145 Da ALWAYS
Sample prep
iTRAQ
iTRAQ
Multiplexing
2DE-based approach
2DE-based approach
“I see 1000 spots, but identify 50 only.”
LC-MS
Column (75 mm)/spray tip (8 mm)
Reverse-phase C18 beads, 3 mm
No precolumn or split
Geiger T et al., 2012
Applications – Cell Biolo源自文库y
Applications – Immunology
Meissner et al, Science 2013
Clinical Proteomics
A. Amyloid tissue stained in Congo Red; B. After LMD.
Pr ot ei n A Pr ot ei n B Pr ot ei n C Pr ot ei n D Pr ot ei n E Pr ot ei n F
....
Time
=Protein A
NH2-EACDPLREACDPLR-COOH
m/z
ICAT
Thiol-specific group = binds to Cysteins
O N S N O XX N O XX O XX O XX O N I
Biotin tag
Linker (heavy or light)
Thiol specific reactive group
Gygi SP et al., 1999
ICAT (Isotope-Coded Affinity Tag)
X3
m/z
Grow SILAC labelled cells to desired number of cells for experiment
Ong SE et al., 2002
Chemical Labeling
ICAT Reagents: Heavy reagent: d8-ICAT (X=deuterium) Light reagent: d0-ICAT (X=hydrogen)
Control vs Tumor Cell?
Protein Identification and Quantitation by LC-MS
Control vs drug treated cell? Control vs knock-out cell?
Applications – Cell Biology
Ong SE et al., 2002
Importance of Dialyzed Serum
• non-dialzed serum contains free (unlabeled) amino acids!
No alterations to cell phenotype
C2C12 myoblast cell line
15 cm
Platin-wire 2.0 kV
Sample Loading:500 nl/min Gradient elution:200 nl/min
ESI
Fenn et al., Science 246:64-71, 1989.
MS-based quantitation
Inlet
LC
Ion Source
m/z
m/z
(labelled AA / dialized serum)
*
m/z m/z
Passage cells to allow incorporation of labelled AA
*
m/z m/z
By 5 cell doublings cells have incorporated
*
X3
m/z
2
H
H
12C 14
13C 15
N
N
16O
18O
Enzymatic Labeling
Metabolic Labeling
SILAC
Cells in normal culture media
Media with Normal AA () Media with Labelled AA (*)
Start SILAC labelling by growing cells in labelling media
MALDI ES
Mass Analyzer
Time-of-Flight Quadrupole Ion Trap Quadrupole-TOF
Detector
Peak intensities can vary up to 100x between duplicate runs.
Quatitative analysis MUST be carried on a single run.
• Labeling is guaranteed close to 99%. All identified proteins in principle are quantifiable
• Quantitation of proteins affected by different stimuli, disruption of genes, etc. • Quantitation of post-translational modifications (phosphorylation, etc.) • Identification and quantitation of interaction partners
*
m/z m/z
Passage cells to allow incorporation of labelled AA
*
m/z m/z
By 5 cell doublings cells have incorporated
*
X3
m/z
X3
m/z
Grow SILAC labelled cells to desired number of cells for experiment
Applications
State A State B Upregulated protein - Peptide ratio >1
Arg13C 6
Light Isotope
Heavy Isotope
Mix 1:1
Arg12C 6
Optional Protein Fractionation
m/z
Digest with Trypsin
Labeled cells behaved as expected under differentiation protocols
Why SILAC is convenient?
Why SILAC is convenient? • Convenient
- no extra step introduced to experiment, just special medium