title: High precision quantification of human plasma proteins using the automated SISCAPA Immuno-MS workflow.
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.
authors.affiliation: SISCAPA Assay Technologies, Inc., Box 53309, Washington, DC 20009, USA. Electronic address: mrazavi@siscapa.com.
authors.forename: Morteza
authors.initials: M
authors.lastname: Razavi
authors.affiliation: SISCAPA Assay Technologies, Inc., Box 53309, Washington, DC 20009, USA.
authors.forename: N
authors.initials: N
authors.lastname: Leigh Anderson
authors.affiliation: SISCAPA Assay Technologies, Inc., Box 53309, Washington, DC 20009, USA.
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Pope
authors.affiliation: SISCAPA Assay Technologies, Inc., Box 53309, Washington, DC 20009, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Yip
authors.affiliation: SISCAPA Assay Technologies, Inc., Box 53309, Washington, DC 20009, USA; Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada.
authors.forename: Terry W
authors.initials: TW
authors.lastname: Pearson
mesh_terms: D001331:Automation; D015415:Biomarkers / Q000032:analysis; D001709:Biotechnology; D001798:Blood Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D006801:Humans; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D012371:Robotics; D053719:Tandem Mass Spectrometry / Q000379:methods; D014357:Trypsin; D057188:Workflow
chemical_list: D015415:Biomarkers; D001798:Blood Proteins; D010455:Peptides; D014357:Trypsin