A data reduction workflow for mass spectrometry proteoform testing in the clinical laboratory: from antithrombin peaks to diagnosis

Objectives Proteoforms affect biological and clinical processes and are anticipated to play a major role in establishing precision medicine. However, mass spectrometry-based proteoform tests generate complex data which needs to be processed thoroughly prior to reporting to clinical chemists and clinicians. We developed a workflow for handling (clinical) data, based on our in-house developed test for the molecular characterization of antithrombin proteoforms by LC-MRM-MS, which serves as a proof-of-concept for laboratories aiming to develop MS-based proteoform tests. Methods Using previously collected study data investigating the diagnostic value of an MS-based antithrombin proteoform test, a workflow was developed based on three layers. Quantitative information assessed deficiencies, qualitative information assessed mutations or PTMs, and a last confirmatory step verified mutations. All steps were (semi-)automated by a custom R script. Results The workflow comprised of quantitation through verification of a quantifying peptide and interpretation to previously established reference intervals. Qualitative information was based on the ratio between qualitative peptides and the established concentration, for which deviations >20 % indicated the presence of variant proteoforms, subsequently confirmed by variant peptide transitions. The R script analyzed and condensed the data per sample to a bi-level diagnosis report providing concentration and molecular proteoforms for easy integration in a LIS. Conclusions MS-based proteoform analysis is a feasible and robust application for the clinical laboratory when combined with automated data analysis and we here provide guidance for establishing such analyses. MS-based proteoform tests will aid the transition to personalized medicine, placing the clinical chemist at the forefront of precision diagnostics.