Chen Rui, Stupak Jacek, Williamson Sam, Twine Susan M, Li Jianjun
Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, Ontario, Canada.
Rapid Commun Mass Spectrom. 2019 Aug 15;33(15):1240-1247. doi: 10.1002/rcm.8459.
Porous graphic carbon chromatography (PGC) has a different mechanism in the retention of tryptic peptides compared with reversed-phase chromatography and in this study we show that coupling PGC with tandem mass spectrometry offer advantages for the quantitation of phosphorylation stoichiometry and characterization of site-specific glycosylation.
Digests of protein standards (horse myoglobin, bovine fetuin and β-casein) were analyzed with a capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) system by coupling an Agilent 1100 HPLC system to a Synapt G2-Si HDMS (Waters). Peptides were separated using a HyperCarb PGC column (300 μm i.d. × 100 mm) packed with 3 μm particles. MS/MS data were collected in data-dependent mode and three MS/MS scans were acquired after the full MS scan. RAW data were transformed to .mgf by PLGS (Waters) and searched against the Swissprot database by Mascot. Chromatograms and MS/MS spectra of identified compounds were extracted with Masslynx (Waters) and imported to Origin for analysis. Glycan composition and peptide sequence were manually annotated.
PGC/MS/MS enabled accurate quantitation of the stoichiometry of specific phosphorylation sites from β-casein by efficient separation of the phosphopeptide and its non-phosphorylated counterpart, which cannot be achieved by reversed-phase chromatography. PGC/MS/MS also enabled comprehensive characterization of protein sialoglycosylation as isomeric glycopeptides with different combinations of α2-3- and α2-6-linked sialic acids can be separated and the ratios of each combination were verified by exoglycosidase digestion.
PGC has demonstrated superior separation of peptides with phosphorylation and glycosylation and can be used as an alternative in the proteomic characterization of post-translational modifications (PTMs) by polar groups.
与反相色谱法相比，多孔石墨化碳色谱法（PGC）在保留胰蛋白酶肽段方面具有不同的机制，在本研究中我们表明，将PGC与串联质谱联用在磷酸化化学计量的定量分析和位点特异性糖基化的表征方面具有优势。
通过将安捷伦1100高效液相色谱系统与Synapt G2-Si HDMS（沃特世公司）联用的毛细管液相色谱/串联质谱（LC/MS/MS）系统分析蛋白质标准品（马肌红蛋白、牛胎球蛋白和β-酪蛋白）的酶解产物。使用填充3μm颗粒的HyperCarb PGC柱（内径300μm×100mm）分离肽段。以数据依赖模式采集MS/MS数据，在全扫描质谱后采集三次MS/MS扫描。通过PLGS（沃特世公司）将原始数据转换为.mgf格式，并通过Mascot在Swissprot数据库中进行搜索。用Masslynx（沃特世公司）提取已鉴定化合物的色谱图和MS/MS谱图，并导入Origin进行分析。手动注释聚糖组成和肽序列。
PGC/MS/MS能够通过有效分离磷酸化肽段及其非磷酸化对应物，准确地对β-酪蛋白特定磷酸化位点的化学计量进行定量分析，这是反相色谱法无法实现的。PGC/MS/MS还能够对蛋白质唾液酸化糖基化进行全面表征，因为具有不同α2-3-和α2-6-连接唾液酸组合的异构体糖肽可以被分离，并且每种组合的比例通过外切糖苷酶消化得到验证。
PGC已证明在分离具有磷酸化和糖基化修饰的肽段方面具有优势，可作为通过极性基团进行蛋白质翻译后修饰（PTM）蛋白质组学表征的一种替代方法。
