Chemistry Department, University of Manitoba, Winnipeg, Manitoba, Canada.
Rapid Commun Mass Spectrom. 2021 May 15;35(9):e9063. doi: 10.1002/rcm.9063.
While high-throughput proteomic methods have been widely applied to monoclonal antibodies and human immunoglobulin gamma (IgG) samples, less information is available on porcine IgG. As pigs are considered one of the most suitable species for xenotransplantation, it is important to characterize IgG amino acid sequences and glycosylation profiles, which is the focus of this study.
Three different purified porcine IgG samples, including wild-type and knockout species, were digested with trypsin and enriched for glycopeptides. Digestion mixtures were spiked with a mixture of six standard peptides. Analysis was performed using electrospray ionization liquid chromatography-tandem mass spectrometry (MS/MS) in standard MS/MS data-dependent acquisition mode on a hybrid triple quadrupole time-of-flight mass spectrometer.
To facilitate the classification of subtypes detected experimentally, UniprotKB database entries were organized using comparative alignment scores. Sequences were grouped based on 11 different subtypes as translated from GenBank entries. Proteomic searches were accomplished automatically using specialized software, whereas glycoprotein searches were performed manually by monitoring the extracted chromatograms of diagnostic MS/MS glycan fragments and studying their corresponding mass spectra; 40-50 non-glycosylated peptides and 4-5 glycosylated peptides were detected in each sample, with several glycoforms per sequence.
Proteomic analysis of porcine IgG is complicated by factors such as the presence of several subtypes, redundant heavy chain (HC) sequences in protein databases, and the lack of consistent cross-referencing between databases. Aligning and comparing HC sequences were necessary to eliminate redundancy. This study highlights the complexity of pig IgG and shows the importance of MS in proteomics and glycoproteomics.
虽然高通量蛋白质组学方法已广泛应用于单克隆抗体和人免疫球蛋白 γ（IgG）样品，但关于猪 IgG 的信息较少。由于猪被认为是最适合异种移植的物种之一，因此表征 IgG 氨基酸序列和糖基化谱非常重要，这是本研究的重点。
使用胰蛋白酶消化三种不同的纯化猪 IgG 样品，包括野生型和敲除物种，并对糖肽进行富集。消化混合物中加入了六种标准肽的混合物。在混合三重四极杆飞行时间质谱仪上，以标准 MS/MS 数据依赖型采集模式进行电喷雾电离液相色谱-串联质谱（MS/MS）分析。
为了便于对实验中检测到的亚型进行分类，使用比较比对分数对 UniprotKB 数据库条目进行了组织。根据从 GenBank 条目翻译的 11 种不同亚型对序列进行分组。使用专门的软件自动完成蛋白质组搜索，而糖蛋白搜索则通过监测诊断 MS/MS 糖基化片段的提取色谱图并研究其相应的质谱图手动进行；每个样品中检测到 40-50 个非糖基化肽和 4-5 个糖基化肽，每个序列有几个糖型。
猪 IgG 的蛋白质组学分析受到多种因素的影响，例如存在几种亚型、蛋白质数据库中冗余的重链（HC）序列以及数据库之间缺乏一致的交叉引用。对齐和比较 HC 序列对于消除冗余是必要的。本研究强调了猪 IgG 的复杂性，并展示了 MS 在蛋白质组学和糖蛋白质组学中的重要性。
