Chemistry Department , University of Manitoba , Winnipeg , Manitoba R3T 2N2 , Canada.
Department of Internal Medicine , University of Manitoba , Winnipeg , Manitoba R3A 1R9 , Canada.
Anal Chem. 2019 Nov 5;91(21):13360-13366. doi: 10.1021/acs.analchem.9b02584. Epub 2019 Oct 14.
The sequence-specific retention calculator algorithm (SSRCalc) [ Krokhin , O. V. 2006 , 78 , 7785 ] was adapted for the prediction of retention times of -glycopeptides separated by reversed-phase high performance liquid chromatography (RPLC). The retention time shifts (dHI = HI - HI, where HI is the hydrophobicity index, measured in percent acetonitrile units) used for modeling were measured for 602 glycopeptides versus 123 of their deglycosylated analogues. Our method used a tryptic digest of 12 purified glycoproteins, glycopeptide enrichment, deglycosylation with PNGaseF, and RPLC-MS/MS analysis of combined (deglycosylated and intact) peptide mixtures. On average, glycosylation yields a 0.79% acetonitrile unit decrease in retention, compared with the hydrophobicity indices of their deglycosylated analogues. These values, however, are drastically different for asialo (-1.37% acetonitrile units), monosialylated (-0.47% acetonitrile units), disialylated (+0.61% acetonitrile units), and trisialylated (+1.94% acetonitrile units) glycans. Peptide retention time shifts upon glycosylation (dHI) vary depending on the number of monosaccharide units, the presence or absence of sialic acid, peptide hydrophobicity, and the number of position-dependent features. These features are mostly driven by competing effects of acidic residues (aspartic acid and sialic acid) on ion-pair formation and by nearest-neighbor effects of hydrophilic glycans. The accuracy of the modified prediction model for glycopeptides approaches that of the prediction for nonmodified species ( = 0.97 vs 0.98). However, retention time prediction based on the experimental retention values of deglycosylated analogues (HI = HI + dHI, = 0.995) is much more accurate, thus providing a solid support for glycopeptide identification in complex samples based on mass and retention time.
SSRCalc 算法 [ Krokhin, O. V. 2006, 78, 7785 ] 被改编用于预测反相高效液相色谱 (RPLC) 分离的 -糖肽的保留时间。用于建模的保留时间偏移量 (dHI = HI - HI，其中 HI 是疏水性指数，以乙腈单位的百分比表示) 是针对 602 个糖肽与其 123 个去糖基化类似物测量得到的。我们的方法使用了 12 种纯化糖蛋白的胰蛋白酶消化物、糖肽富集、PNGaseF 去糖基化以及组合 (去糖基化和完整) 肽混合物的 RPLC-MS/MS 分析。与去糖基化类似物的疏水性指数相比，糖基化平均导致保留时间减少 0.79%的乙腈单位。然而，对于无唾液酸 (-1.37%乙腈单位)、单唾液酸化 (-0.47%乙腈单位)、二唾液酸化 (+0.61%乙腈单位)和三唾液酸化 (+1.94%乙腈单位)糖，这些值差异很大。糖基化后肽的保留时间偏移量 (dHI) 取决于单糖单位的数量、唾液酸的存在与否、肽的疏水性以及位置相关特征的数量。这些特征主要受酸性残基 (天冬氨酸和唾液酸) 对离子对形成的竞争影响以及亲水糖的近邻效应的驱动。经修正的糖肽预测模型的准确性接近非修饰种的预测准确性 ( = 0.97 对 0.98)。然而，基于去糖基化类似物的实验保留值 (HI = HI + dHI， = 0.995) 的保留时间预测更为准确，从而为基于质量和保留时间在复杂样品中鉴定糖肽提供了坚实的支持。
