title: Range of protein detection by selected/multiple reaction monitoring mass spectrometry in an unfractionated human cell culture lysate.
abstract: 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 MS1-triggered MS2 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.
authors.affiliation: Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zürich, Zürich, Switzerland.
authors.forename: H Alexander
authors.initials: HA
authors.lastname: Ebhardt
authors.forename: Eduard
authors.initials: E
authors.lastname: Sabidó
authors.forename: Ruth
authors.initials: R
authors.lastname: Hüttenhain
authors.forename: Ben
authors.initials: B
authors.lastname: Collins
authors.forename: Ruedi
authors.initials: R
authors.lastname: Aebersold
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D002138:Calibration; D002457:Cell Extracts / Q000737:chemistry*; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D012015:Reference Standards; D012984:Software
chemical_list: D002457:Cell Extracts; D010455:Peptides