Implementation of a data repository-driven approach for targeted proteomics experiments by multiple reaction monitoring

Abstract

Multiple Reaction Monitoring (MRM), commonly employed for the mass spectrometric detection of small molecules, is rapidly gaining ground in proteomics. Its high sensitivity and specificity makes this targeted approach particularly useful when sample throughput or proteome coverage limits global studies. Existing tools to design MRM assays rely exclusively on theoretical predictions, or combine them with previous observations on the same type of sample. The additional mass spectrometric experimentation this requires can pose significant demands on time and material. To overcome these challenges, a new MRM worksheet was introduced into The Global Proteome Machine database (GPMDB) that provided all of the information needed to design MRM transitions based solely on archived observations made by other researchers in previous experiments. This required replacing the precursor ion intensity by the number of peptide observations, which proved to be an adequate substitute if peptides did not occur in multiple forms. While the absence of collision energy information proved largely inconsequential, successful prediction of unique transitions depended on the type of fragment ion involved. The design of MRM assays for iTRAQ-labeled tryptic peptides obtained from human platelet proteins demonstrated the usefulness of the MRM worksheet also for quantitative applications. This workflow, which relies exclusively on experimental observations stored in data repositories, therefore represents an attractive alternative for the prediction of MRM transitions prior to experimental validation and optimization.

Mass spectrometry of peptides and proteins from human blood

“…An SQL database containing more than 4,300 blood proteins, the columns that best identify them, and a redundant set of more than 200,000 peptide ions, charge states and observed masses from sera has recently been published (Tucholska et al, 2009). The compilation of ion databases will facilitate the absolute quantification of proteins by triple quadrupole or other internal standard methods (Walsh et al, 2009). The working embodiment of proteomics may include a survey with a large number of fractionation techniques to achieve sensitivity and determine analytes of interest with regard to physiological states and thus create a local protein, peptide, and ion, database.…”

An extensive library of surrogate peptides for all human proteins

“…In our previous paper [4], we have reviewed a few of these, including ESP predictor [15], Peptide Sieve [16], and PepFly [17,18], PeptideAtlas [8,19] PABST (Peptide Atlas Best SRM Transition Tool) [7,13,8,20], Automated and Targeted Analysis with Quantitative SRM-ATAQS [21], MRMaid, [22], GPMDB MRM Worksheet [23], SRM/MRMAtlas [24,25], TIQAM [26,27] (used in ATAQS workflow), Skyline [28,29], and TPP-MaRiMba [30,31].…”

The Effect of Diabetes and Poor Left Ventricular Function on Bone Marrow Cell-Induced Myocardial Protection

“…For SRM analysis, the mrm worksheets tool [12] of the gpm database (gpmdb.thegpm.org) was used to find suitable peptides in conjunction with a manual analysis of the raw data. Five proteins were chosen for SRM analysis, defensin a 1 (DEFA1, ENSP00000 372126), calgranulin A (S100-A8, ENSP00000357721), calgranulin B (S100-A9, ENSP00000357727), cytoplasmic actin 2 (b actin, ACTIN, ENSP00000331514), and hemoglobin delta chain (HEMOGLOBIN SUBUNIT DELTA, ENSP00000369654).…”