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Nuts and Bolts of Protein Quantification by Online Trypsin Digestion Coupled LC-MS/MS Analysis

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Functional Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1871))


Protein digestion coupled to liquid chromatography and tandem mass spectrometry (LC-MS/MS) detection enables multiplexed quantification of proteins in complex biological matrices. However, the reproducibility of enzymatic digestion of proteins to produce proteotypic target peptides is a major limiting factor of assay precision. Online digestion using immobilized trypsin addresses this problem through precise control of digestion conditions and time. Because online digestion is typically for a short time, the potential for peptide degradation, a major source of measurement bias, is significantly reduced. Online proteolysis requires minimal sample preparation and is easily coupled to LC-MS/MS systems, further reducing potential method variability. We describe herein a method optimized for the multiplexed quantification of several apolipoproteins in human serum using on-column digestion. We highlight key features of the method that enhance assay accuracy and precision. These include the use of value-assigned serum as calibrators and stable isotope-labeled (SIL) peptide analogs as internal standards. We also comment on practical aspects of column switching valve design, instrument maintenance, tandem mass spectrometry data acquisition, and data processing.

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References in this article to any specific commercial products, process, service, manufacturer, or company do not constitute an endorsement or a recommendation by the U.S. Government or the Centers for Disease Control and Prevention. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of CDC.

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Correspondence to Christopher A. Toth .

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Toth, C.A., Kuklenyik, Z., Barr, J.R. (2019). Nuts and Bolts of Protein Quantification by Online Trypsin Digestion Coupled LC-MS/MS Analysis. In: Wang, X., Kuruc, M. (eds) Functional Proteomics. Methods in Molecular Biology, vol 1871. Humana Press, New York, NY.

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8813-6

  • Online ISBN: 978-1-4939-8814-3

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