Evaluating the effects of preanalytical variables on the stability of the human plasma proteome.

High quality clinical biospecimens are vital for biomarker discovery, verification, and validation. Variations in blood processing and handling can affect protein abundances and assay reliability. Using an untargeted LC-MS approach, we systematically measured the impact of preanalytical variables on the plasma proteome. Time prior to processing was the only variable that affected the plasma protein levels. LC-MS quantification showed that preprocessing times <6h had minimal effects on the immunodepleted plasma proteome, but by 4 days significant changes were apparent. Elevated levels of many proteins were observed, suggesting that in addition to proteolytic degradation during the preanalytical phase, changes in protein structure are also important considerations for protocols using antibody depletion. As to processing variables, a comparison of single- vs double-spun plasma showed minimal differences. After processing, the impact ⩽3 freeze-thaw cycles was negligible regardless of whether freshly collected samples were processed in short succession or the cycles occurred during 14-17 years of frozen storage (-80 °C). Thus, clinical workflows that necessitate modest delays in blood processing times or employ different centrifugation steps can yield valuable samples for biomarker discovery and verification studies.

Mass spectrometry-based analyses showing the effects of secretor and blood group status on salivary N-glycosylation.

BACKGROUND: The carbohydrate portions of salivary glycoproteins play important roles, including mediating bacterial and leukocyte adhesion. Salivary glycosylation is complex. Many of its glycoproteins present ABO and Lewis blood group determinants. An individual's genetic complement and secretor status govern the expression of blood group antigens. We queried the extent to which salivary glycosylation varies according to blood group and secretor status. First, we screened submandibular/sublingual and parotid salivas collected as ductal secretions for reactivity with a panel of 16 lectins. We selected three lectins that reacted with the largest number of glycoproteins and one that recognized uncommon lactosamine-containing structures. Ductal salivas representing a secretor with complex blood group expression and a nonsecretor with a simple pattern were separated by SDS-PAGE. Gel slices were trypsin digested and the glycopeptides were individually separated on each of the four lectins. The bound fractions were de-N-glycosylated. LC-MS/MS identified the original glycosylation sites, the peptide sequences, and the parent proteins. RESULTS: The results revealed novel salivary N-glycosites and glycoproteins not previously reported. As compared to the secretor, nonsecretor saliva had higher levels of N-glycosylation albeit with simpler structures. CONCLUSIONS: Together, the results suggested a molecular basis for inter-individual variations in salivary protein glycosylation with functional implications for oral health.