Abstract
The enrichment of trace proteins from human fluid samples is of great importance in diverse clinical and industrial applications. In clinical diagnostics, such enrichment may enable detection of trace proteins that serve as biomarkers of disease. Affinity-based approaches, such as immunoaffinity pulldown, are widely used to enrich trace proteins, but this strategy relies on the availability and performance of antibodies that act on all proteoforms in an unbiased manner. Our prior work to characterize MUC16 (the mucin protein that carries the ovarian cancer biomarker CA125) by mass spectrometry successfully overcame the reliance on affinity-based enrichment and was used to enrich this biomarker from ascites of individual ovarian cancer patients, however, this strategy was not demonstrated on clinically relevant volumes of serum, a biofluid that is more accessible than ascites. The present work developed a non-affinity-based chromatographic method to enrich MUC16 from serum. The enriched MUC16 sample was further processed using a Midi Top 14 abundant protein depletion column. Peptides identified using bottom-up proteomics yielded 1-8% coverage of MUC16. Additionally, MUC16 was detected in samples containing less than the clinical cut-off level of CA125 (35 U mL-1), suggesting that this strategy of enrichment and bottom-up proteomics can enable analysis of CA125 from the serum of individuals with early-stage ovarian cancer and those whose tumors express CA125 (MUC16) at low levels.