Apoe, Mbl2, and Psp plasma protein levels correlate with diabetic phenotype in NZO mice--an optimized rapid workflow for SRM-based quantification.

Male New Zealand Obese (NZO) mice progress through pathophysiological stages similar to humans developing obesity-associated type 2 diabetes (T2D). The current challenge is to establish quantitative proteomics from small plasma sample amounts. We established an analytical workflow that facilitates a reproducible depletion of high-abundance proteins, has high throughput applicability, and allows absolute quantification of proteins from mouse plasma samples by LC-SRM-MS. The ProteoMiner equalizing technology was adjusted to the small sample amount, and reproducibility of the identifications was monitored by spike proteins. Based on the label free relative quantification of proteins in depleted plasma of a test set of NZO mice, assays for potential candidates were designed for the setup of a targeted selected reaction monitoring (SRM) approach and absolute quantification. We could demonstrate that apolipoprotein E (Apoe), mannose-binding lectin 2 (Mbl2), and parotid secretory protein (Psp) are present at significantly different quantities in depleted plasma of diabetic NZO mice compared to non-diabetic controls using AQUA peptides. Quantification was validated for Mbl2 using the ELISA technology on non-depleted plasma. We conclude that the depletion technique is applicable to restricted sample amounts and suitable for the identification of T2D signatures in plasma.