This document provides an introduction to quantitative proteomics using stable isotope labeling of amino acids in culture (SILAC). SILAC involves growing cells in media containing "light" or "heavy" isotopic forms of amino acids, which get incorporated into proteins and allow distinction and quantitation of proteins from different samples after mixing and mass spectrometry analysis. The document discusses applications of SILAC, how mass spectrometers identify isotopic species, and complementary labeling strategies like ICAT and iTRAQ.

1. Quantitative Proteomics

2. Introduction of SILAC and its applications
• Stable Isotope Labeling of Amino acids in Culture
• Develop and promoted by Matthias Mann
• Two papers:
– Lipid Rafts (PNAS)
– Focal Adhesion precursors (Cell under review)

3. What does a mass spectrometer do?
Precise identification of mass
Can trap a single ion species and fragment to get sub fragments (sequence)

4. Overview of Biological and Chemical Isotope Labeling Strategies
Two complementary samples
Labeling
Analysis

5. SILAC vs. ICAT
• Culture system only
• De Novo Proteins (no
serum contamination)
• No optimization
• Simplifies MS/MS
• More complete peptide
coverage
• ALL protein samples
• Labels selected
moieties
• Need to optimize
labeling
• Large linker group.
• Reduces complexity

6. Stable isotopic
amino acids (MW)
Cambridge
Isotope Catalog
Sigma-Aldich
Catalog
L-Lysine 2HCl (223.13)
(4,4,5,5-D4)
DLM-2640 -
L-Lysine HCl (186.67)
(4,4,5,5-D4)
- 616192
L-Lysine 2HCl (225.1)
(13
C6)
CLM-2247 -
L-Lysine HCl (188.6)
(13
C6)
- 643459
L-Lysine 2HCl (227.1)
(13
C6, 15
N2)
CNLM-291 -
L-Lysine HCl (190.59)
(13
C6, 15
N2)
- 608041
L-Arginine HCl (214.64)
(15
N4)
NLM-396 600113
L-Arginine HCl (216.62)
(13
C6)
CLM-2265 643440
L-Arginine HCl (220.59)
(13
C6, 15
N4)
CNLM-539 608033
L-Arginine HCl (221.6)
(15
N4, D7)
DNLM-7543 -
L-Arginine HCl (227.6)
(13
C6, 15
N4, D7)
CDNLM-6801
SILAC Labels

7. “Light”
“Heavy”
“Light/heavy” mix
How can you distinguish between isotoped species?

8. How do you actually get quantitation?
1) Once you’ve identified the complementary peptides,
2) Backtrack and integrate the Xtracted Ion Chromatogram

9. I-TRAQ (Isobaric Tag for Relative and Absolute Quantitation

10. Experimental Strategy
Sample preparation
Labeling
Chromatographic separations
Mass Spectrometry
Analysis