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Differential Protein Expression Analysis

Label Free Analysis (Precursor ion Peak Area Detection)

Complete analysis consists of 3 steps:

Step 1: Sample processing/preparation for following differential LC/MS/MS analysis – service codes 112, 112a
Step 2: High pH RP fractionation; 8 step fractions – service code 110
Step 3: 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. Each sample is analyzed separately. Proteins are digested by trypsin and generated peptides are analyzed by LC/MS/MS. Complex samples are usually fractionated into 8 high pH RP fractions, each fraction is analyzed by LC/MS/MS, and the data collected for all fractions of a sample are combined. Raw MS data is submitted to database search for peptide/protein identification and quantification. Peptides sequences are identified using acquired experimental MS/MS spectra. False Discovery Rate (FDR) for sequence identification is determined through decoy (reversed) DB search. Identified peptides are quantified using experimental high resolution accurate mass (HRAM) spectra – individual peptide chromatogram is extracted and the peak area equivalent to its abundance is determined. A unique set of Identified and quantified peptides determine the identity and the abundance of the corresponding parent protein.

May 26, 2022