Center for Translational Biomedical Research, University of North Carolina at Greensboro , North Carolina Research Campus, Kannapolis, North Carolina 28081, United States.
Department of Chemistry & Biochemistry, University of North Carolina at Greensboro , Greensboro, North Carolina 27412, United States.
Anal Chem. 2018 Jan 16;90(2):1081-1086. doi: 10.1021/acs.analchem.7b03342. Epub 2017 Dec 27.
Glycated proteins are emerging as good indicators for diabetes and age related diseases. However, the platform for analysis of glycated proteome has been relatively less well established. We here introduce an online 2D-LC-HCD-MS/MS platform for comprehensive glycated peptide quantification. This platform includes a boronate affinity column in the first dimension for enrichment, reversed phase nanoLC column in the second dimension for separation, a benchtop Orbitrap mass spectrometer with HCD-MS/MS for peptide sequencing, and MaxQuant bioinformatics tool for identification and quantification of glycated peptides. This online 2D-LC-HCD-MS/MS platform has high enrichment efficiency with 85% of identified peptides in the enriched fraction as glycated, high sensitivity for detection of glycated peptides with LOD and LOQ at 1.2 and 2.4 pg, respectively, and high reproducibility with interday CVs < 20% for 80% of the glycated peptides. The number of glycated peptides quantified in in vitro glycated human plasma increased more than 3-fold using this platform in comparison to that obtained using 1D-LC-HCD-MS/MS platform without boronate affinity enrichment. Application of this online platform to human plasma identified 376 glycated peptides from 10 μg of protein digests. This highly sensitive and reproducible online 2D platform is promising for glycated protein analysis of complex clinical samples.
糖化蛋白正逐渐成为糖尿病和与年龄相关疾病的良好指标。然而，糖化蛋白质组学的分析平台相对来说还不够完善。我们在这里介绍了一种用于全面糖化肽定量的在线二维液相色谱-高分辨串联质谱（2D-LC-HCD-MS/MS）平台。该平台在第一维包含硼酸盐亲和柱进行富集，在第二维包含反相纳升液相色谱柱进行分离，采用台式 Orbitrap 质谱仪进行 HCD-MS/MS 肽测序，以及 MaxQuant 生物信息学工具进行糖化肽的鉴定和定量。这种在线二维液相色谱-高分辨串联质谱（2D-LC-HCD-MS/MS）平台具有高的富集效率，在富集部分鉴定出的肽中有 85%是糖化的；对糖化肽具有高的检测灵敏度，LOD 和 LOQ 分别为 1.2 和 2.4 pg；80%的糖化肽的日内 CVs 小于 20%，具有高的重现性。与没有硼酸盐亲和富集的一维液相色谱-高分辨串联质谱（1D-LC-HCD-MS/MS）平台相比，该平台在体外糖化人血浆中的糖化肽定量数量增加了 3 倍以上。将该在线平台应用于人血浆，从 10 μg 蛋白消化物中鉴定出 376 个糖化肽。这种高灵敏度和重现性的在线二维平台有望用于复杂临床样本中的糖化蛋白分析。
