Kozlik Petr, Goldman Radoslav, Sanda Miloslav
Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA.
Department of Analytical Chemistry, Faculty of Science, Charles University, Czech Republic.
Electrophoresis. 2017 Sep;38(17):2193-2199. doi: 10.1002/elps.201600547. Epub 2017 May 17.
Analysis of glycosylation is challenging due to micro- and macro-heterogeneity of the protein attachment. A combination of LC with MS/MS is one of the most powerful tools for glycopeptide analysis. In this work, we show the effect of various monosaccharide units on the retention time of glycopeptides. Retention behavior of several glycoforms of six peptides obtained from tryptic digest of haptoglobin, hemopexin, and sex hormone-binding globulin was studied on a reversed phase chromatographic column. We observed reduction of the retention time with increasing number of monosaccharide units of glycans attached to the same peptide backbone. Fucosylation of larger glycans provides less significant retention time shift than for smaller ones. Retention times of glycopeptides were expressed as relative retention times. These relative retention times were used for calculation of upper and lower limits of glycopeptide retention time windows under the reversed phase conditions. We then demonstrated on the case of a glycopeptide of haptoglobin that the predicted retention time window boosts confidence of identification and minimizes false-positive identification. Relative retention time, as a qualitative parameter, is expected to improve LC-MS/MS characterization of glycopeptides.
由于蛋白质连接存在微观和宏观异质性，糖基化分析具有挑战性。液相色谱与串联质谱联用是糖肽分析最强大的工具之一。在这项工作中，我们展示了各种单糖单元对糖肽保留时间的影响。在反相色谱柱上研究了从触珠蛋白、血红素结合蛋白和性激素结合球蛋白的胰蛋白酶消化产物中获得的六种肽的几种糖型的保留行为。我们观察到，连接在同一肽主链上的聚糖单糖单元数量增加时，保留时间会缩短。较大聚糖的岩藻糖基化导致的保留时间变化比小聚糖的要小。糖肽的保留时间以相对保留时间表示。这些相对保留时间用于计算反相条件下糖肽保留时间窗口的上限和下限。然后，我们以触珠蛋白的一种糖肽为例证明，预测的保留时间窗口提高了鉴定的可信度，并将假阳性鉴定降至最低。相对保留时间作为一个定性参数，有望改善糖肽的液相色谱 - 串联质谱表征。
