Improved detection of tryptic immunoglobulin variable region peptides by chromatographic and gas-phase fractionation techniques.

Circulating antibodies provide valuable information about an individual’s immune status and constitute a significant portion of the human plasma proteome. Proteomic analysis of plasma is challenged by the large dynamic concentration range of plasma proteins, including antibodies. Peptide fractionation prior to data-independent (shotgun) proteomics has the potential to overcome this obstacle and enable increased coverage of plasma proteins. In this work, we evaluated the detection of tryptic immunoglobulin variable region peptides using multidimensional chromatography (high-pH preparative LC) or gas-phase ion mobility spectrometry (FAIMS). Typical digests of eight serum samples were obtained by (a) direct LC-MS (without prior fractionation, 1D), (b) LC-FAIMS-MS, and (c) high-pH reversed-phase fractionation into 24 fractions and subsequent measurement of each fraction. As de novo search is an essential step in antibody variable region peptide identification, all MS/MS spectra were acquired in high resolution Orbitrap mode.

循环抗体可为个体免疫状态提供关键参考信息，同时也是人类血浆蛋白质组（human plasma proteome）的重要组成部分。血浆蛋白质组学分析面临的核心挑战之一，是血浆蛋白（包括抗体）的浓度动态范围极宽。在数据非依赖型（鸟枪法）蛋白质组学（data-independent (shotgun) proteomics）分析前开展肽段分级，有望突破这一限制，实现血浆蛋白覆盖度的显著提升。本研究中，我们评估了采用多维色谱法（高pH制备液相色谱（high-pH preparative LC））或气相离子迁移谱（gas-phase ion mobility spectrometry, FAIMS）对胰蛋白酶消化的免疫球蛋白可变区肽段的检测效果。我们对8份血清样品的典型酶解产物分别采用三种方式处理：(a) 直接进行LC-MS分析（未预先分级，即一维模式，1D）；(b) 采用LC-FAIMS-MS联用分析；(c) 经高pH反相分级为24个组分，随后对每个组分分别进行质谱检测。由于从头测序（de novo）搜索是免疫球蛋白可变区肽段鉴定的必要步骤，因此所有MS/MS谱图均采用高分辨率轨道阱（Orbitrap）模式采集。