Apoe, Mbl2, and Psp Plasma Protein Levels Correlate with Diabetic Phenotype in NZO Mice—An Optimized Rapid Workflow for SRM-Based Quantification

Abstract

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.

Peptide serum markers in islet autoantibody-positive children

“…Sample preparation for MS Plasma samples were depleted from highly abundant proteins and proteolysed with trypsin as previously described [27]. All samples were randomly distributed into one of three batches for processing, and the experimenters were blinded to the sample-group allocation during the experiment.…”

MASP1, THBS1, GPLD1 and ApoA-IV are novel biomarkers associated with prediabetes: the KORA F4 study

“…Plasma depletion and on-bead digestions using ProteoMiner beads Depletion and tryptic digestion of plasma samples which had been stored at −80°C were performed as previously described in six processing batches [12]. For quality control of depletion, digestion and MS measurements, a final concentration of 50 fmol ribulose-1,5-bisphosphate carboxylase oxygenase protein (RuBisCO; Sigma-Aldrich, Taufkirchen, Germany) was spiked into each 10 μl plasma sample as an internal standard (see ESM methods section 'Quality control of sample preparation and MSmeasurement').…”

“…Candidate protein selection Candidate proteins were obtained from a previous pilot mouse study [12] and unpublished mouse data (von Toerne and Hauck, data not shown). These were complemented by results from shotgun measurements of human plasma samples from different sources (data not shown).…”

“…LC-tandem MS (MSMS) analysis was performed on a Tempo nano MDLC system (Eksigent Technologies, Dublin, CA, USA) coupled online to a QTrap4000 (ABSCIEX, Framingham, MT, USA) mass spectrometer by a nano spray ion source, as previously described [12] with the following changes. Peptides were separated on a 90 min non-linear gradient of 2% acetonitrile and 3% DMSO in 0.1% formic acid (solution A) to 95% acetonitrile and 3% DMSO in 0.1% formic acid (solution B) at a flow rate of 250 nl/min.…”

“…We used a nano-liquid chromatography (LC) MS approach in SRM mode (SRM-MS) to quantify the relative levels of 23 proteins in 439 plasma samples from the population-based Cooperative Health Research in the Region of Augsburg (KORA) F4 study (see electronic supplementary material [ESM] Table 1). The candidate proteins were selected based on previous results from a mouse model for type 2 diabetes [12], literature mining and unpublished shotgun discovery proteomic analyses (von Toerne and Hauck, data not shown).…”