A highly-reproducible automated protein sample preparation workflow for quantitative mass spectrometry in plasma or blood

Recorded On: 02/05/2018

Sample preparation for protein quantification by mass spectrometry requires multiple processing steps including denaturation, reduction, alkylation, protease digestion, and peptide cleanup. Scaling these procedures for the analysis of numerous complex biological samples, such as plasma, can be tedious and time-consuming, as there are many liquid transfer steps and timed reactions where technical variations can be introduced and propagated. Therefore, we have automated this digestion workflow and adapted it to include the preparation of dried blood obtained from remote sampling devices, allowing high throughput analysis of both archived and “real-time” sampling of our pathological surveillance biomarkers.  Our pathological surveillance biomarker assay is composed of 72 plasma proteins that screen for 8 pathological signatures. METHODS. We established an automated sample preparation workflow with a total processing time for 96 plasma or blood samples of 5 hours, including a 2-hour incubation with trypsin. Peptide cleanup is accomplished by online diversion during the LC/MS/MS analysis.  RESULTS. In a selected reaction monitoring (SRM) assay targeting 6 plasma biomarkers and spiked β-galactosidase, mean intra-day CVs for 5 samples ranged from 5.5%-8.9% for serum and 3.9%-7.2% for plasma, and mean inter-day CVs over 5 days ranged from 5.8%-10.6% for serum and 3.9%-6.0% for plasma. As well for the highly multiplex surveillance biomarker assay, 90% of the transitions from 6 plasma samples repeated on 3 separate days had total CVs below 20%. Similar results were obtained when the workflow was transferred to a second site: 93% of peptides had CVs below 20%. In an analysis of plasma samples from 48 individuals (disease and healthy), the average CVs for spiked β-galactosidase was < 15%. The workflow was adapted for the direct processing of remote blood sampling devices (Neoteryx) and achieved equivalent high performance for spiked β-galactosidase when part of a 10 and 72 protein SRM assays. 

Jennifer Van Eyk

Cedar Sinai Medical Center

PhD in Biochemistry

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