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Differential Protein Expression Analysis

iTRAQ/TMT Based Analysis (Reporter Ion quantification)

Complete analysis consists of 4 steps:

Step 1: Sample processing/preparation for following TMT/iTRAQ labeling – service codes 113, 113a
Step 2: TMT/iTRAQ labeling - service codes 114-119
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. Using TMT/iTRAQ analysis, the relative abundance of proteins can be determined in up to 10 protein extracts at a time (per analysis). This is achieved by labeling of (for example) 10 different multi-protein extracts using 10 different mass-spectrally resolvable, charge-matched labels. TMT or iTRAQ Labeling is conducted using a commercial iTRAQ (Applied Biosystems, Foster City, CA) or TMT (Thermo Fisher Sci., Waltham, MA) kit according to the manufacturer’s protocol. Equal amounts (20-100 µg) of total protein from each sample are separately digested with trypsin and labeled with one of TMT/iTRAQ reagents (TMT or iTRAQ tags are covalently linked to the amino group of the lysine and the N-terminal residues of tryptic peptides). The labeled samples are pooled and fractionated into 8 high pH RP fractions. Each fraction is analyzed by LC/MS/MS for peptide/protein identification and quantitation to maximize number of analyzed peptides.  Identical peptides with different TMT/iTRAQ tags are resolved in the same chromatographic peak, the masses of eluted peptides are determined, then, the peptides are fragmented - the fragment masses are determined for peptide sequence identification and the intensities of the released TMT/iTRAQ tags are determined for corresponding peptide quantification. The ratios (relative abundance) of differently tagged identical 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 peptides with different tags 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