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Protein Identification and Mapping of Specified PTMs

Complete analysis consists of 3 (1 optional) steps:

Step 1: Sample processing/preparation for following LC/MS/MS analysis – service codes 111, 111a
Step 2: (Optional) High pH RP fractionation; 8 step fractions – service code 110
Step 3: LC/MS/MS analysis including DB search – service codes 401-404 

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
  • Modification(s) and modification site(s) found in identified peptide/proteins
  • Confidence level of peptide/protein identification – FDR (False Discovery Rate)

Short description of analysis

Proteins are identified through identification of the peptides unique to a protein. Peptides (with or without PTM), in turn, are identified through fragmentation and determination of the corresponding fragment masses. A protein or protein mixture is digested by trypsin to generate tryptic peptides. Tryptic peptides are analyzed by mass spectrometry in line with reverse phase HPLC. Peptides are separated on an HPLC column and eluted peptides are analyzed in a mass spectrometer: peptide masses (m/z, mass per charge) are determined (MS spectrum), then, each analyzed peptide is fragmented and the corresponding fragment masses are determined (MS/MS spectrum). The MS/MS spectrum of each analyzed peptide is submitted to database search for sequence identification. The peptide sequence and the residue bearing (if any) a specified PTM is identified by comparison (identity) of the experimental MS/MS spectrum to the MS/MS spectra generated by software for all tryptic peptides (of similar masses with or without specified modification) of all database proteins. A unique set of identified peptides identifies the corresponding parent protein. False Discovery Rate (FDR) is determined through decoy (reversed) DB search.

Last Published: Oct 31, 2017