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Range of protein detection by selected/multiple reaction monitoring mass spectrometry in an unfractionated human cell culture lysate
Author(s) -
Ebhardt H. Alexander,
Sabidó Eduard,
Hüttenhain Ruth,
Collins Ben,
Aebersold Ruedi
Publication year - 2012
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201100543
Subject(s) - selected reaction monitoring , chemistry , mass spectrometry , chromatography , lysis , liquid chromatography–mass spectrometry , analytical chemistry (journal) , tandem mass spectrometry , biochemistry
Selected or multiple reaction monitoring is a targeted mass spectrometry method ( S / MRM ‐ MS ), in which many peptides are simultaneously and consistently analyzed during a single liquid chromatography‐mass spectrometry ( LC ‐ S / MRM ‐ MS ) measurement. These capabilities make S / MRM ‐ MS an attractive method to monitor a consistent set of proteins over various experimental conditions. To increase throughput for S / MRM ‐ MS it is advantageous to use scheduled methods and unfractionated protein extracts. Here, we established the practically measurable dynamic range of proteins reliably detectable and quantifiable in an unfractionated protein extract from a human cell line using LC ‐ S / MRM ‐ MS . Initially, we analyzed S / MRM transition peak groups in terms of interfering signals and compared S / MRM transition peak groups to MS 1‐triggered MS 2 spectra using dot‐product analysis. Finally, using unfractionated protein extract from human cell lysate, we quantified the upper boundary of copies per cell to be 35 million copies per cell, while 7500 copies per cell represents a lower boundary using a single 35 min linear gradient LC ‐ S / MRM ‐ MS measurement on a current, standard commercial instrument.