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Mol Cell Proteomics DOI:10.1074/mcp.M113.034660

Simplified and efficient quantification of low-abundance proteins at very high multiplex via targeted mass spectrometry.

Publication TypeJournal Article
Year of Publication2014
AuthorsBurgess, MW, Keshishian, H, Mani, DR, Gillette, MA, Carr, SA
JournalMol Cell Proteomics
Date Published2014 Apr
KeywordsAnimals, Blood Proteins, Cattle, Chromatography, Liquid, Isotope Labeling, Mass Spectrometry, Peptides, Reproducibility of Results

Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) of plasma that has been depleted of abundant proteins and fractionated at the peptide level into six to eight fractions is a proven method for quantifying proteins present at low nanogram-per-milliliter levels. A drawback of fraction-MRM is the increased analysis time due to the generation of multiple fractions per biological sample. We now report that the use of heated, long, fused silica columns (>30 cm) packed with 1.9 μm of packing material can reduce or eliminate the need for fractionation prior to LC-MRM-MS without a significant loss of sensitivity or precision relative to fraction-MRM. We empirically determined the optimal column length, temperature, gradient duration, and sample load for such assays and used these conditions to study detection sensitivity and assay precision. In addition to increased peak capacity, longer columns packed with smaller beads tolerated a 4- to 6-fold increase in analyte load without a loss of robustness or reproducibility. The longer columns also provided a 4-fold improvement in median limit-of-quantitation values with increased assay precision relative to the standard 12 cm columns packed with 3 μm material. Overall, the optimized chromatography provided an approximately 3-fold increase in analysis throughput with excellent robustness and less than a 2-fold reduction in quantitative sensitivity relative to fraction-MRM. The value of the system for increased multiplexing was demonstrated by the ability to configure an 800-plex MRM-MS assay, run in a single analysis, comprising 2400 transitions with retention time scheduling to monitor 400 unlabeled and heavy labeled peptide pairs.


Alternate JournalMol. Cell Proteomics
PubMed ID24522978
PubMed Central IDPMC3977191
Grant ListU24 CA160034 / CA / NCI NIH HHS / United States
R01HL096738 / HL / NHLBI NIH HHS / United States
R01 HL096738 / HL / NHLBI NIH HHS / United States
HHSN268201000033C / / PHS HHS / United States
HHSN268201000033C / HL / NHLBI NIH HHS / United States
U24CA160034 / CA / NCI NIH HHS / United States