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Abstract
Efficient robotic workflows for trypsin digestion of human plasma and subsequent antibody-mediated
peptide enrichment (the SISCAPA method) were developed with the goal of improving
assay precision and throughput for multiplexed protein biomarker quantification. First,
an 'addition only' tryptic digestion protocol was simplified from classical methods,
eliminating the need for sample cleanup, while improving reproducibility, scalability
and cost. Second, methods were developed to allow multiplexed enrichment and quantification
of peptide surrogates of protein biomarkers representing a very broad range of concentrations
and widely different molecular masses in human plasma. The total workflow coefficients
of variation (including the 3 sequential steps of digestion, SISCAPA peptide enrichment
and mass spectrometric analysis) for 5 proteotypic peptides measured in 6 replicates
of each of 6 different samples repeated over 6 days averaged 3.4% within-run and 4.3%
across all runs. An experiment to identify sources of variation in the workflow demonstrated
that MRM measurement and tryptic digestion steps each had average CVs of ∼2.7%. Because
of the high purity of the peptide analytes enriched by antibody capture, the liquid
chromatography step is minimized and in some cases eliminated altogether, enabling
throughput levels consistent with requirements of large biomarker and clinical studies.