title: Metrological traceability in mass spectrometry-based targeted protein quantitation: a proof-of-principle study for serum apolipoproteins A-I and B100.
abstract: In this study, we have followed up on previous liquid chromatography (LC) multiple reaction monitoring (MRM) mass spectrometry (MS) approaches for measurement of apolipoprotein (apo) A-I and apo B100 in serum aiming for implementation of a multiplexed assay in a clinical chemistry laboratory with full metrological traceability. Signature peptides were selected and detected by dynamic MRM, and stable isotope labeled (SIL)-peptides were used as internal standards. Five apo A-I and four apo B100 peptides were measured in serum digests with linearity (R(2)>0.992) in the physiologically relevant concentration ranges. Linearity with regard to protein concentration was ascertained at five concentration levels (R(2)>0.926 and R(2)>0.965, for the apo A-I and apo B100 peptides, respectively). Three native value-assigned sera were used as external calibrators for further method verification. Imprecision values on sample preparation and LC-MS/MS acquisition were below the established minimal specifications for apo A-I and apo B100 (5.0% and 5.3%, respectively). Correlation of LC-MS/MS results with immunoturbidimetric assay results, for normo- and hypertriglyceridemic samples, showed R(2)>0.944 for apo A-I and R(2)>0.964 for apo B100. This LC-MS/MS method has potential for clinical application in normo- and dyslipidemic patients.
  
  
  CONCLUSIONS
  Measurement of apo A-I and apo B100 may offer an alternative to high and low density lipoprotein cholesterol (HDL-c and LDL-c) methods for cardiovascular disease risk assessment in dyslipidemic patients [1]. An LC-MS/MS method for apo A-I and apo B100 has the advantage of antibody independent and specific detection of protein signature peptides. The introduction of an LC-MS/MS method for apo A-I and apo B100 can serve as an example for many existing and newly developed (multiplex) biomarker methods in quantitative clinical chemistry proteomics (qCCP). Such LC-MS/MS methods should meet basic clinical chemistry principles with regard to test evaluation [2]. Criteria for imprecision should be pre-defined, e.g., based on biological variation. The use of commutable and traceable serum-based calibrators will improve inter-laboratory reproducibility of LC-MS/MS methods and may contribute to a more rapid transition of biomarker discovery to clinical utility with benefit for the patient treatment and improvement of general health care.
authors: Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands. Electronic address: N.Smit@lumc.nl.
authors: Nico P M
authors: NP
authors: Smit
authors: Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands.
authors: Fred P H T M
authors: FP
authors: Romijn
authors: Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands.
authors: Irene
authors: I
authors: van den Broek
authors: Department of Immunohematology and Bloodtransfusion, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands.
authors: Jan W
authors: JW
authors: Drijfhout
authors: Agilent Technologies Netherlands B.V., Amstelveen, The Netherlands.
authors: Martin
authors: M
authors: Haex
authors: Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands.
authors: Arnoud
authors: A
authors: van der Laarse
authors: Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands.
authors: Yuri E M
authors: YE
authors: van der Burgt
authors: Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands. Electronic address: C.M.Cobbaert@lumc.nl.
authors: Christa M
authors: CM
authors: Cobbaert
mesh_terms: D016632:Apolipoprotein A-I / Q000097:blood*; D053299:Apolipoprotein B-100 / Q000097:blood*; D050171:Dyslipidemias / Q000097:blood*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods* / Q000592:standards*; D010455:Peptides / Q000097:blood*
chemical_list: D016632:Apolipoprotein A-I; D053299:Apolipoprotein B-100; D010455:Peptides
keywords: Apolipoproteins; Biomarker; Cardiovascular disease; Clinical chemistry; Metrological traceability; Multiple reaction monitoring