Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS 66047, USA.
J Am Soc Mass Spectrom. 2012 Dec;23(12):2140-8. doi: 10.1007/s13361-012-0485-9. Epub 2012 Sep 20.
Chromatographic carry-over can severely distort measurements of amide H/D exchange in proteins analyzed by LC/MS. In this work, we explored the origin of carry-over in the online digestion of an IgG1 monoclonal antibody using an immobilized pepsin column under quenched H/D exchange conditions (pH 2.5, 0 °C). From a consensus list of 169 different peptides consistently detected during digestion of this large, ~150 kDa protein, approximately 30% of the peptic peptides exhibited carry-over. The majority of carry-over originates from the online digestion. Carry-over can be substantially decreased by washing the online digestion flow-path and pepsin column with two wash cocktails: [acetonitrile (5%)/isopropanol (5%)/acetic acid (20%) in water] and [2 M guanidine hydrochloride in 100 mM phosphate buffer pH 2.5]. Extended use of this two-step washing procedure does not adversely affect the specificity or activity of the immobilized pepsin column. The results suggest that although the mechanism of carry-over appears to be chemical in nature, and not hydrodynamic, carry-over cannot be attributed to a single factor such as mass, abundance, pI, or hydrophobicity of the peptides.
色谱峰残留会严重扭曲通过 LC/MS 分析的蛋白质酰胺 H/D 交换的测量结果。在这项工作中，我们研究了在 pH 值为 2.5、0°C 的淬灭 H/D 交换条件下，使用固定化胃蛋白酶柱在线消化 IgG1 单克隆抗体时残留的起源。在消化这种大型 (~150 kDa 蛋白)时一致检测到的 169 个不同肽的共识列表中，约 30%的肽酶肽表现出残留。大多数残留来自在线消化。通过用两种洗涤混合物（水相中的[乙腈 (5%)/异丙醇 (5%)/乙酸 (20%)]和 [2 M 盐酸胍在 100 mM 磷酸盐缓冲液 pH 2.5]）洗涤在线消化流路和胃蛋白酶柱，可以大大减少残留。这种两步洗涤程序的延长使用不会对固定化胃蛋白酶柱的特异性或活性产生不利影响。结果表明，尽管残留的机制似乎是化学性质的，而不是流体动力学的，但残留不能归因于肽的单一因素，如质量、丰度、pI 或疏水性。
