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.

雄性新西兰肥胖（NZO）小鼠的病理生理进程与肥胖相关2型糖尿病（T2D）患者的发病进程高度相似。当前的核心挑战在于如何从微量血浆样本中建立定量蛋白质组学分析方法。本研究搭建了一套标准化分析流程，可实现高丰度蛋白的可重复去除，具备优异的高通量适用性，且能够通过液相色谱-选择反应监测质谱（LC-SRM-MS）对小鼠血浆样本中的蛋白质进行绝对定量。本研究针对微量样本体量对ProteoMiner蛋白均衡技术进行了适配优化，并通过外源性加标蛋白对蛋白质鉴定的重现性进行监控。基于对一组NZO小鼠测试队列的去高丰度蛋白血浆样本开展无标记相对定量分析，本研究针对潜在候选蛋白设计了靶向检测方法，以搭建选择反应监测（SRM）靶向定量平台并实现绝对定量。通过绝对定量肽段（AQUA peptides）分析，我们证实：与非糖尿病对照组相比，载脂蛋白E（Apoe）、甘露糖结合凝集素2（Mbl2）及腮腺分泌蛋白（Psp）在糖尿病NZO小鼠的去高丰度蛋白血浆中的含量存在显著差异。本研究采用酶联免疫吸附测定（ELISA）技术，对非去高丰度蛋白血浆样本中的Mbl2定量结果进行了验证。综上，该蛋白去除技术可适用于微量受限样本，且可用于鉴定血浆中的T2D特征标志物。