Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA.
J Sep Sci. 2010 Jun;33(10):1438-47. doi: 10.1002/jssc.200900860.
Selectivity of both peptide- and glycan-targeting antibodies was examined by 2-D LC-MS/MS. Proteins selected from biological extracts immunospecifically in a first chromatography dimension using antibodies immobilized by either covalent coupling or adsorption to protein G were desorbed with a denaturing mobile phase and transferred to a 1.5 microm nonporous particle RP chromatography (NP-RPC) column in a second dimension. Protein peak capacity of the NP-RPC column was approximately 50. Peaks collected from the RPC column were tryptic digested and the peptide fragments were identified by MALDI-MS/MS. The objective of this analytical strategy was to discriminate between protein antigens and nonantigens through identification of their peptides, leading to an evaluation of the selectivity of antibodies and immunosorbents. Quantification of the relative amount of antigen and nonantigen species captured by immunosorbents was achieved by absorbance, along with the likely capture mechanism. A limitation of the approach was in discriminating between isoforms of an antigen in which neither the antibody nor the LC-MS system targeted the differentiating feature in the isoforms.
通过二维 LC-MS/MS 研究了肽和聚糖靶向抗体的选择性。使用通过共价偶联或吸附到蛋白 G 固定的抗体在第一色谱维度中对生物提取物进行免疫特异性选择的蛋白质，用变性流动相洗脱，并转移到第二维中的 1.5 微米无孔颗粒反相色谱 (NP-RPC) 柱。NP-RPC 柱的蛋白质峰容量约为 50。从 RPC 柱收集的峰进行胰蛋白酶消化，并通过 MALDI-MS/MS 鉴定肽片段。该分析策略的目的是通过鉴定其肽来区分蛋白质抗原和非抗原，从而评估抗体和免疫吸附剂的选择性。通过吸光度以及可能的捕获机制实现对免疫吸附剂捕获的抗原和非抗原种类的相对量的定量。该方法的一个限制是在区分既不是抗体也不是 LC-MS 系统针对同种型中区分特征的同种型的抗原之间。
