title: Quantification of serum apolipoproteins A-I and B-100 in clinical samples using an automated SISCAPA-MALDI-TOF-MS workflow.
abstract: A fully automated workflow was developed and validated for simultaneous quantification of the cardiovascular disease risk markers apolipoproteins A-I (apoA-I) and B-100 (apoB-100) in clinical sera. By coupling of stable-isotope standards and capture by anti-peptide antibodies (SISCAPA) for enrichment of proteotypic peptides from serum digests to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS detection, the standardized platform enabled rapid, liquid chromatography-free quantification at a relatively high throughput of 96 samples in 12h. The average imprecision in normo- and triglyceridemic serum pools was 3.8% for apoA-I and 4.2% for apoB-100 (4 replicates over 5 days). If stored properly, the MALDI target containing enriched apoA-1 and apoB-100 peptides could be re-analyzed without any effect on bias or imprecision for at least 7 days after initial analysis. Validation of the workflow revealed excellent linearity for daily calibration with external, serum-based calibrators (R(2) of 0.984 for apoA-I and 0.976 for apoB-100 as average over five days), and absence of matrix effects or interference from triglycerides, protein content, hemolysates, or bilirubins. Quantification of apoA-I in 93 normo- and hypertriglyceridemic clinical sera showed good agreement with immunoturbidimetric analysis (slope = 1.01, R(2) = 0.95, mean bias = 4.0%). Measurement of apoB-100 in the same clinical sera using both methods, however, revealed several outliers in SISCAPA-MALDI-TOF-MS measurements, possibly as a result of the lower MALDI-TOF-MS signal intensity (slope = 1.09, R(2) = 0.91, mean bias = 2.0%). The combination of analytical performance, rapid cycle time and automation potential validate the SISCAPA-MALDI-TOF-MS platform as a valuable approach for standardized and high-throughput quantification of apoA-I and apoB-100 in large sample cohorts.
authors.affiliation: Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA Leiden, The Netherlands. Electronic address: i.van_den_broek@lumc.nl.
authors.forename: Irene
authors.initials: I
authors.lastname: van den Broek
authors.affiliation: Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
authors.forename: Jan
authors.initials: J
authors.lastname: Nouta
authors.affiliation: SISCAPA Assay Technologies Inc., Box 53309, Washington, DC 20009, USA.
authors.forename: Morteza
authors.initials: M
authors.lastname: Razavi
authors.affiliation: SISCAPA Assay Technologies Inc., Box 53309, Washington, DC 20009, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Yip
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
authors.forename: Marco R
authors.initials: MR
authors.lastname: Bladergroen
authors.affiliation: Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
authors.forename: Fred P H T M
authors.initials: FP
authors.lastname: Romijn
authors.affiliation: Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
authors.forename: Nico P M
authors.initials: NP
authors.lastname: Smit
authors.affiliation: Bruker Daltonics GmbH, Fahrenheitstraße 4, 28359 Bremen, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Drews
authors.affiliation: Bruker Daltonics GmbH, Fahrenheitstraße 4, 28359 Bremen, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Paape
authors.affiliation: Bruker Daltonics GmbH, Fahrenheitstraße 4, 28359 Bremen, Germany.
authors.forename: Detlev
authors.initials: D
authors.lastname: Suckau
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
authors.forename: André M
authors.initials: AM
authors.lastname: Deelder
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
authors.forename: Yuri E M
authors.initials: YE
authors.lastname: van der Burgt
authors.affiliation: SISCAPA Assay Technologies Inc., Box 53309, Washington, DC 20009, USA.
authors.forename: Terry W
authors.initials: TW
authors.lastname: Pearson
authors.affiliation: SISCAPA Assay Technologies Inc., Box 53309, Washington, DC 20009, USA.
authors.forename: N Leigh
authors.initials: NL
authors.lastname: Anderson
authors.affiliation: Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
authors.forename: Christa M
authors.initials: CM
authors.lastname: Cobbaert
mesh_terms: D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I / Q000097:blood* / Q000276:immunology; D053299:Apolipoprotein B-100 / Q000097:blood* / Q000276:immunology; D015415:Biomarkers / Q000097:blood; D002138:Calibration; D006801:Humans; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D057188:Workflow
chemical_list: C505693:APOA1 protein, human; C569471:APOB protein, human; D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I; D053299:Apolipoprotein B-100; D015415:Biomarkers
keywords: Analytical method validation; Cardiovascular disease risk assessment; Clinical proteomics; Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF); Stable-isotope standards and capture by anti-peptide antibodies (SISCAPA)