title: Quantifying protein measurands by peptide measurements: where do errors arise?
abstract: Clinically actionable quantification of protein biomarkers by mass spectrometry (MS) requires analytical performance in concordance with quality specifications for diagnostic tests. Laboratory-developed tests should, therefore, be validated in accordance with EN ISO 15189:2012 guidelines for medical laboratories to demonstrate competence and traceability along the entire workflow, including the selected standardization strategy and the phases before, during, and after proteolysis. In this study, bias and imprecision of a previously developed MS method for quantification of serum apolipoproteins A-I (Apo A-I) and B (Apo B) were thoroughly validated according to Clinical and Laboratory Standards Institute (CLSI) guidelines EP15-A2 and EP09-A3, using 100 patient sera and either stable-isotope labeled (SIL) peptides or SIL-Apo A-I as internal standard. The systematic overview of error components assigned sample preparation before the first 4 h of proteolysis as major source (∼85%) of within-sample imprecision without external calibration. No improvement in imprecision was observed with the use of SIL-Apo A-I instead of SIL-peptides. On the contrary, when the use of SIL-Apo A-I was combined with external calibration, imprecision improved significantly (from ∼9% to ∼6%) as a result of the normalization for matrix effects on linearity. A between-sample validation of bias in 100 patient sera further supported the presence of matrix effects on digestion completeness and additionally demonstrated specimen-specific biases associated with modified peptide sequences or alterations in protease activity. In conclusion, the presented overview of bias and imprecision components contributes to a better understanding of the sources of errors in MS-based protein quantification and provides valuable recommendations to assess and control analytical quality in concordance with the requirements for clinical use.
authors.affiliation: Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center (LUMC) , Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
authors.forename: Irene
authors.initials: I
authors.lastname: van den Broek
authors.forename: Fred P H T M
authors.initials: FP
authors.lastname: Romijn
authors.forename: Nico P M
authors.initials: NP
authors.lastname: Smit
authors.forename: Arnoud
authors.initials: A
authors.lastname: van der Laarse
authors.forename: Jan W
authors.initials: JW
authors.lastname: Drijfhout
authors.forename: Yuri E M
authors.initials: YE
authors.lastname: van der Burgt
authors.forename: Christa M
authors.initials: CM
authors.lastname: Cobbaert
mesh_terms: D001798:Blood Proteins / Q000032:analysis / Q000737:chemistry; D002853:Chromatography, Liquid; D006801:Humans; D007553:Isotope Labeling; D010446:Peptide Fragments / Q000032:analysis* / Q000097:blood / Q000737:chemistry / Q000592:standards*; D040901:Proteomics / Q000379:methods* / Q000592:standards*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry; D014357:Trypsin
chemical_list: D001798:Blood Proteins; D010446:Peptide Fragments; D014357:Trypsin
keywords: Apo A-I mutations; analytical method validation; matrix effects; quantitative Clinical Chemistry Proteomics (qCCP); selected or multiple reaction monitoring (SRM/MRM); stable-isotope labeled peptide or protein; trypsin digestion efficiency