= Emerging. More than 5 years before clinical availability. (24.37%, 2023)
= Expected to be clinically available in 1 to 4 years. (39.50%, 2023)
= Clinically available now. (36.13%, 2023)
MSACL 2023 : Wang

MSACL 2023 Abstract

Self-Classified Topic Area(s): Proteomics

Podium Presentation in Steinbeck 1 on Wednesday at 14:20 (Chair: Zuzana Demianova / Xin Cong)

Lessons in Calibration: Peptide versus Protein Based Calibrators

Meng Wang (1,2), Junyan Shi (1,2), Yu Zi Zheng (1,2), Mari L. DeMarco (1,2,3)
(1) Department of Pathology and Laboratory Medicine, University of British Columbia, (2) Centre for Heart Lung Innovation, University of British Columbia, (3) Department of Pathology and Laboratory Medicine, St Paul’s Hospital, Providence Health Care

Meng Wang, PhD (Presenter)
Vancouver General Hospital

Presenter Bio: I am a Research & Development Scientist working at Vancouver General Hospital, specializing in mass spectrometry method development and quality improvement in the clinical laboratory.

I received my PhD in Chemistry from the University of British Columbia, with a research focus on discovering biologically active natural products for potential drug candidates. Following the completion of my doctorate, I transitioned to the role of Research Scientist at IntelliSyn Pharma, where I used mass spectrometry and NMR techniques to analyze and characterize drug-like small molecules across various drug discovery and medicinal chemistry projects. After working in the pharmaceutical industry for two years, I started my postdoctoral training at the University of British Columbia (St Paul's Hospital). During this period, my research focused on the development, optimization, and validation of targeted mass spectrometry protein assays.


BACKGROUND: In the design of a calibration scheme for a quantitative protein mass spectrometry assay, the use of calibrators matching the full sequence of the measurand is generally considered best practice. For proteolysis-aided workflows in particular, full-length protein standards are desired to control for the variability in the sample preparation process including proteolytic digestion. However, depending on the source, structure and composition of the protein calibrant, full-length protein calibrators do not always serve as reasonable surrogates for the endogenous protein measurand. An ill-matched full-length protein calibrant can result in inaccurate quantification of the measurand, for example, via modified digestion kinetics, and thus differential production of proteolytic peptides relative to the endogenous protein. This highlights the need for careful consideration and evaluation of calibration material, and its value assignment approach.

OBJECTIVE: Using peptides, full-length proteins standards, and value reassigned peptides, we aimed to investigate the best performing calibration schemes for quantitative proteolysis-aided HPLC-MS/MS assays for apolipoprotein A1 (apoA1), and independently, C-reactive protein (CRP).

METHODS: For both CRP and apoA1 methods, 10 μL of sample (i.e., QCs, calibrators, and plasma) was diluted with digestion buffer and internal standard solution added. The mixture was then denatured at 99 °C for 10 min with shaking, left to cool at room temperature before the addition of trypsin, followed by digestion at 37 °C for 20 min. A volume of 12 μL of the quenched digest solution was injected into the HPLC-MS/MS (Phenomenex Aeris Peptide 3.6-μm XB-C18 column, Shimadzu LC 20AD, and SCIEX Triple Quad 5500) and run on a 10-min gradient at a flow rate of 0.25 mL/min. We compared the peptide calibrators, protein calibrators, and a hybrid approach of value-reassigned peptide calibrators using plasma specimens measured by immunonephelometry. Method comparisons were performed against the following immunoassays (IA): Siemens BNII immunonephelometry assay (n = 59 human EDTA plasma samples) for apoA1 and the Siemens ADVIA immunoturbidimetry assay (n = 60 human heparin plasma samples) for CRP.

RESULTS: The method comparison for apoA1 revealed the following regressions: HPLC-MS/MS = 1.48 × IA – 0.2 (R2 = 0.83) using protein calibrators prepared from reference material BCR-393, HPLC-MS/MS = 0.63 × IA + 0.02 (R2 = 0.91) using peptide calibrators, and HPLC-MS/MS = 1.01 × IA - 0.01 (R2 = 0.91) using value-reassigned peptide calibrators. The method comparison for CRP revealed the following regressions: HPLC-MS/MS = 1.28 × IA + 1.62 (R2 = 0.99) using protein calibrators prepared from reference material SRM 2924, HPLC-MS/MS = 2.58 × IA + 1.78 (R2 = 0.99) using peptide calibrators, and HPLC-MS/MS = 0.99 × IA + 1.09 (R2 = 0.99) using value-reassigned peptide calibrators.

CONCLUSION: By developing (i) a reproducible and rapid digestion process, and (ii) a value assignment process whereby the peptide calibrators are anchored to immunoassay values for the protein measurand, peptide calibrators can outperform protein-based calibration schemes, while also offering a more cost-effective approach.

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