Rush R S, Boss H J, Katta V, Rohde M F
Department of Protein Structure, Amgen Inc., Thousand Oaks, CA 91320-1789, USA.
Electrophoresis. 1997 May;18(5):751-6. doi: 10.1002/elps.1150180515.
High performance capillary electrophoresis (CE) of glycoprotein digests frequently reveals extensive microheterogeneity associated with specific protein glycosylation sites. The choice of the sample matrix can influence the electrophoretic migration time, peak shape and resolution, as well as the physical stability of the product glycopeptides. Acetic acid is a frequently employed sample matrix for both capillary electrophoresis and electrospray ionization-mass spectrometry (ESI-MS). Acetic acid appears to enhance the spontaneous hydrolysis of sialic acids from the nonreducing termini of glycopeptides in a time- and concentration-dependent manner, even at 5 degrees C, as evidenced by changes in the electrophoretic mobility and ESI-MS spectra of the resulting glycopeptides. The observed parallel electrophoretic mobility changes for specific glycoforms are consistent with the induction of peptide structure with time. Asialoglycopeptide mobilities were stable in acetic acid. Electrophoretic mobilities can be stabilized with propionic acid sample matrix with no apparent structural changes observed by ESI-MS within 31 h. Migration time reproducibility was in the range of 0.1% relative standard deviation (N = 7) with excellent peak shapes and enhanced glycopeptide resolution.
糖蛋白消化产物的高效毛细管电泳（CE）常常显示出与特定蛋白质糖基化位点相关的广泛微观不均一性。样品基质的选择会影响电泳迁移时间、峰形和分辨率，以及产物糖肽的物理稳定性。乙酸是毛细管电泳和电喷雾电离质谱（ESI-MS）常用的样品基质。乙酸似乎会以时间和浓度依赖的方式促进糖肽非还原末端唾液酸的自发水解，即使在5℃时也是如此，这可由所得糖肽的电泳迁移率和ESI-MS谱图的变化得到证明。观察到的特定糖型的平行电泳迁移率变化与肽结构随时间的诱导一致。去唾液酸糖肽在乙酸中的迁移率是稳定的。使用丙酸样品基质可使电泳迁移率稳定，在31小时内ESI-MS未观察到明显的结构变化。迁移时间重现性在相对标准偏差0.1%的范围内（N = 7），峰形极佳，糖肽分辨率提高。
