= Emerging. More than 5 years before clinical availability. (26.55%)
= Expected to be clinically available in 1 to 4 years. (39.66%)
= Clinically available now. (33.79%)
MSACL 2020 US : Owusu

MSACL 2020 US Abstract

Topic: Proteomics

Podium Presentation in Room 5 on Wednesday at 11:00 (Chair: Chris Shuford)

Clinical Validation of an LC-MS/MS Assay for Measurement of Serum C-peptide

Benjamin Owusu (Presenter)
University of Washington

Presenter Bio(s): Dr. Benjamin Owusu is a Senior Clinical Chemistry Fellow at the University of Washington School of Medicine. Benjamin received his PhD degree in Biochemistry and Molecular Genetics and went on to do a one-year postdoctoral research fellowship at the University of Alabama at Birmingham (UAB). He obtained his Bachelor of Science degree in Biochemistry at the University of Ghana, where he also served as a Teaching Assistant at the Department of Biochemistry upon completion of his undergraduate training. He later moved to Emory University School of Medicine in Atlanta, GA on a visiting research scholarship prior to starting his doctoral training at UAB. His research over the years span across cancer, cardiovascular, hematological and endocrinological diseases. In addition to clinical service and research, Benjamin is passionate about teaching and mentoring, particularly in STEM education.

Authors: Benjamin Yaw Owusu, Hannah Pflaum, Rusty Garner, Thomas Laha, and Andrew Hoofnagle
University of Washington, Seattle, WA


INTRODUCTION: C-peptide is a widely used marker of endogenous insulin secretion. It is used for assessing residual beta cell function and in the diagnostic workup of hypoglycemia. C-peptide is routinely measured in the clinical laboratory by immunoassays, which are sensitive but prone to limitations such as cross-reactivity, between-lot variability, and a lack of concordance across different platforms. LC-MS/MS methods are more specific and can be multiplexed. Previous LC-MS/MS methods developed for serum c-peptide measurement detected intact peptide. Unfortunately, intact peptide is poorly ionized, requiring immunoaffinity extraction or solid phase extraction with two-dimensional chromatography.

OBJECTIVE: The objective of this study was to develop and validate a novel enzyme (Glu-C) digestion-based LC-MS/MS assay for the quantification of serum c-peptide.

METHODS: The internal standard was isotopically labeled at two leucine residues: EAED*LQVGQVELGGGPGAGSLQP*LALEGSLQ. Serum proteins were precipitated using acetonitrile (ACN) and the precipitated proteins were resuspended in 10% ACN in 90mM ammonium bicarbonate (AMBIC). Solid phase extraction was performed, followed by reduction and alkylation using dithiothreitol (DTT) and indoacetamide (IAA) respectively. Glu-C was used to digest c-peptide into smaller peptides and sample injected into LCMS for quantitative analysis of c-peptide. Using a Waters Xevo TQ-MS tandem mass spectrometer coupled with Waters Acquity UPLC, two peptide fragments, EAEDLQVGQVE (EAE1 Peptide; +1 Precursor m/z 1216.5692) and LGGGPGAGSLQPLALE (LGG1 Peptide; +1 Precursor m/z 1436.69), were monitored using MRM in positive ion mode. One peptide (EAE1) was used for quantitation and the other (LGG1) as a confirmatory ion.

RESULTS: The assay was linear between 0.1 to 15 ng/mL using an eleven-point dilution series (0.1-15 ng/mL), which was prepared by diluting a high patient pool (spiked with c-peptide certified reference material) with horse serum (negative serum). Within-batch and between-batch imprecision was 4.6 %CV and 5.8 %CV, respectively, with a total error 7.4 %CV. The limit of quantification was determined to be 0.05 ng/mL. No significant carryover was observed up to 50 ng/mL. Spiking 5 ng/mL c-peptide into patient samples showed 99 % (± 8 %) mean recovery, with <15 % bias from expected mean results.

CONCLUSION: We validated a robust LC-MS/MS assay which employs solid phase extraction and enzyme (Glu-C)-mediated peptide digestion for the quantification of c-peptide in human serum.

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