High sensitivity quantitative proteomics using automated multidimensional nano-flow chromatography and accumulated ion monitoring on quadrupole-orbitrap-linear ion trap mass spectrometer Journal Article


Authors: Cifani, P.; Kentsis, A.
Article Title: High sensitivity quantitative proteomics using automated multidimensional nano-flow chromatography and accumulated ion monitoring on quadrupole-orbitrap-linear ion trap mass spectrometer
Abstract: Quantitative proteomics using high-resolution and accuracy mass spectrometry promises to transform our understanding of biological systems and disease. Recent development of parallel reaction monitoring (PRM) using hybrid instruments substantially improved the specificity of targeted mass spectrometry. Combined with high-efficiency ion trapping, this approach also provided significant improvements in sensitivity. Here, we investigated the effects of ion isolation and accumulation on the sensitivity and quantitative accuracy of targeted proteomics using the recently developed hybrid quadrupole-Orbitraplinear ion trap mass spectrometer. We leveraged ultrahigh efficiency nano-electrospray ionization under optimized conditions to achieve yoctomolar sensitivity with more than seven orders of linear quantitative accuracy. To enable sensitive and specific targeted mass spectrometry, we implemented an automated, two-dimensional (2D) ion exchange-reversed phase nanoscale chromatography system. We found that automated 2D chromatography improved the sensitivity and accuracy of both PRM and an intact precursor scanning mass spectrometry method, termed accumulated ion monitoring (AIM), by more than 100-fold. Combined with automated 2D nano-scale chromatography, AIM achieved subattomolar limits of detection of endogenous proteins in complex biological proteomes. This allowed quantitation of absolute abundance of the human transcription factor MEF2C at ∼100 molecules/cell, and determination of its phosphorylation stoichiometry from as little as 1 μg of extracts isolated from 10,000 human cells. The combination of automated multidimensional nano-scale chromatography and targeted mass spectrometry should enable ultrasensitive high-accuracy quantitative proteomics of complex biological systems and diseases. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Keywords: protein phosphorylation; unclassified drug; human cell; sensitivity analysis; protein analysis; transcription factor; automation; proteomics; quantitative analysis; bioaccumulation; stoichiometry; synthetic peptide; limit of detection; electrospray; chromatography; quadrupole mass spectrometry; human; priority journal; article; transcription factor mef2c; ion exchange; accumulated ion monitoring; automated multidimensional nano flow chromatography; ion monitoring; ion trap mass spectrometry; oci-aml2 cell line; parallel reaction monitoring; quadrupole orbitrap linear ion trap mass spectrometry; two-dimensional imaging
Journal Title: Molecular & Cellular Proteomics
Volume: 16
Issue: 11
ISSN: 1535-9476
Publisher: Amer Soc Biochemistry Molecular Biology Inc  
Date Published: 2017-11-01
Start Page: 2006
End Page: 2016
Language: English
DOI: 10.1074/mcp.RA117.000023
PROVIDER: scopus
PMCID: PMC5672005
PUBMED: 28821601
DOI/URL:
Notes: Article -- Export Date: 4 December 2017 -- Source: Scopus
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MSK Authors
  1. Alex   Kentsis
    50 Kentsis
  2. Paolo   Cifani
    17 Cifani