Evaluation of interspecimen trypsin digestion efficiency prior to multiple reaction monitoring-based absolute protein quantification with native protein calibrators

J Proteome Res. 2013 Dec 6;12(12):5760-74. doi: 10.1021/pr400763d. Epub 2013 Nov 13.


Implementation of quantitative clinical chemistry proteomics (qCCP) requires targeted proteomics approaches, usually involving bottom-up multiple reaction monitoring-mass spectrometry (MRM-MS) with stable-isotope labeled standard (SIS) peptides, to move toward more accurate measurements. Two aspects of qCCP that deserve special attention are (1) proper calibration and (2) the assurance of consistent digestion. Here, we describe the evaluation of tryptic digestion efficiency by monitoring various signature peptides, missed cleavages, and modifications during proteolysis of apolipoprotein A-I and B in normo- and hypertriglyceridemic specimens. Absolute quantification of apolipoprotein A-I and B was performed by LC-MRM-MS with SIS peptide internal standards at two time points (4 and 20 h), using three native protein calibrators. Comparison with an immunoturbidimetric assay revealed recoveries of 99.4 ± 6.5% for apolipoprotein A-I and 102.6 ± 7.2% for apolipoprotein B after 4 h of trypsin digestion. Protein recoveries after 20 h trypsin incubation equaled 95.9 ± 6.9% and 106.0 ± 10.0% for apolipoproteins A-I and B, respectively. In conclusion, the use of metrologically traceable, native protein calibrators looks promising for accurate quantification of apolipoprotein A-I and B. Selection of rapidly formed peptides, that is, with no or minor missed cleavages, and the use of short trypsin incubation times for these efficiently cleaved peptides are likely to further reduce the variability introduced by trypsin digestion and to improve the traceability of test results to reach the desirable analytical performance for clinical chemistry application.

MeSH terms

  • Amino Acid Sequence
  • Apolipoprotein A-I / blood*
  • Apolipoprotein A-I / chemistry
  • Apolipoproteins B / blood*
  • Apolipoproteins B / chemistry
  • Calibration
  • Case-Control Studies
  • Chromatography, Liquid / standards*
  • Humans
  • Hypertriglyceridemia / blood*
  • Mass Spectrometry / standards*
  • Molecular Sequence Data
  • Nephelometry and Turbidimetry
  • Peptide Fragments / chemistry
  • Peptide Fragments / isolation & purification*
  • Proteolysis
  • Reference Standards
  • Reproducibility of Results
  • Trypsin / chemistry


  • Apolipoprotein A-I
  • Apolipoproteins B
  • Peptide Fragments
  • Trypsin