Absolute quantitation of proteins in human blood by multiplexed multiple reaction monitoring mass spectrometry

Andrew J. Percy, Andrew G. Chambers, Carol E. Parker, Christoph H. Borchers

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

33 Citations (Scopus)

Abstract

Multiple reaction monitoring (MRM)-mass spectrometry (MS) with stable isotope-labeled standards (SIS) has proven adept in rapidly, precisely, and accurately quantifying proteins in complex biological samples. The impetus behind the early use of multiplexed MRM in proteomics was to expedite the veri fi cation and validation stages of the protein biomarker pipeline for clinical utility, which involves the analysis of hundreds or even thousands of samples. Moreover, once a multiplexed assay has been developed, however, it can be turned around and used for biomarker discovery, as has been demonstrated for cancer biomarkers by our laboratory and by others. Overall, these MRM-based methods compare favorably with antibodybased techniques, such as ELISAs or protein arrays, in that MRM-based methods are less expensive and can be developed more rapidly. There are two MRM-based platforms that are currently being developed: A standard- fl ow and a nanofl ow LC/ESI-MRM-MS (liquid chromatography- electrospray ionization) platform. In this book chapter, we describe a recent study in which we evaluated these two platforms, both interfaced to the same mass spectrometer. This study demonstrated the enhanced performance metrics (in terms of sensitivity, dynamic range, and robustness) of the standard- fl ow ultra-high performance liquid chromatography (UHPLC) system compared to the nano- fl ow HPLC-Chip for the absolute quantitation of 48 plasma proteins. Using the standard- fl ow platform, we also developed two high-throughput assays for the analysis of a panel of 67 cardiovascular disease (CVD) biomarkers in non-depleted and non-enriched human plasma and a panel of 25 putative biomarkers in dried human blood spots (DBS). Since the nanoLC/MRM-MS platform has advantages under sample-limited conditions and for the analysis of certain speci fi c peptides, the protocols for both systems are described here.

Original languageEnglish
Title of host publicationVascular Proteomics
Subtitle of host publicationMethods and Protocols
EditorsFernando Vivanco
Pages167-189
Number of pages23
DOIs
Publication statusPublished - 2013
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume1000
ISSN (Print)1064-3745

Keywords

  • Dried blood spot (DBS)
  • Mass spectrometry (MS)
  • Multiple reaction monitoring (MRM)
  • Plasma
  • Protein
  • Quantitative proteomics
  • Stable isotope-labeled standards (SIS)

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