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.