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Differential Protein Expression Analysis

SILAC Analysis (Precursor Ion Quantification)

Complete analysis consists of 4 steps:

Step 1: Protein labeling in cell culture, SILAC – performed by the investigator
Step 2: Sample processing/preparation for following differential LC/MS/MS analysis – service codes 112, 112a
Step 3: High pH RP fractionation; 8 step fractions – service code 110
Step 4: LC/MS/MS analysis including DB search – service codes 501-503

Results summarized in a spreadsheet include (among others) the following columns:

  • List of identified proteins, description, accession number, sequence coverage
  • List of identified peptide sequences for each identified protein
  • Confidence level of peptide/protein identification – FDR (False Discovery Rate)
  • Abundances of identified peptides/proteins in analyzed samples
  • Average abundance of each identified peptide/protein in a group of samples (e.g. control) and corresponding standard error (SE)
  • Relative abundances (molar ratios) of identical proteins in compared sample types/groups

Short description of analysis

Differential protein expression analysis determines the relative abundances (molar ratios) of identical proteins in two or more samples (multi-protein mixtures) representing different conditions (groups) – e.g. control vs patient, treated vs untreated, etc. An optimal number of biological and/or technical replicates must be analyzed per condition (group) for statistical validation of results. SILAC - stable isotope labeling by amino acids in cell culture is used for in vivo labeling of proteins for MS-based quantitative proteomics;  proteins are labeled through metabolic incorporation of ‘light’ or ‘heavy’ form of a given amino acid (usually L-Lysine, L-Arginine). ‘light’ and  ‘heavy’ forms of an amino acid  are chemically identical but differ in content of stable isotopes (usually 13C, 15N), i.e. SILAC labels are mass spectrally resolvable. Two cell populations are grown in media with different labels and labeled protein samples are isolated. Equal amounts of protein of 2 samples are pooled, digested by trypsin, and fractionated into 8 high pH RP fractions. Each fraction is analyzed by LC/MS/MS for peptide sequence identification and quantitation:  identical ‘light’ and ‘heavy’ peptides derived from different samples are resolved in the same chromatographic peak, the masses and the intensities of eluted peptides are determined for quantification, then, the peptides are fragmented and the corresponding fragment masses are determined for sequence identification. The ratios (relative abundance) of identical ‘light’ and ‘heavy’ peptides are preserved even if losses of peptides occur during analysis,. The acquired MS data is submitted to database search: the sequence of each analyzed peptide is identified and the abundances of identical ‘light’ and ‘heavy’ peptides are determined. False Discovery Rate (FDR) for sequence identification is determined through decoy (reversed) DB search. A unique set of Identified and quantified peptides determine the identity and the abundance of the corresponding parent protein.

May 26, 2022