Online Two-Dimensional Porous Graphitic Carbon/Reversed Phase Liquid Chromatography Platform Applied to Shotgun Proteomics and Glycoproteomics

Abstract

A novel fully automatable two-dimensional liquid chromatography (2DLC) platform has been integrated into a modified commercial off-the-shelf LC instrument, incorporating porous graphitic carbon (PGC) separation and conventional low-pH reversed-phase (RP) separation for both proteomics and N-glycomics analyses; the dual-trap column configuration of this platform offers desirable high-throughput analyses with almost no idle time, in addition to a miniaturized setup and simplified operation. The total run time per analysis was only 19 h when using eight PGC fractions for unattended large-scale qualitative and quantitative proteomic analyses; the identification of 2678 nonredundant proteins and 11,984 unique peptides provided one of the most comprehensive proteome data sets for primary cerebellar granule neurons (CGNs). The effect of pH on the PGC column was investigated for the first time to improve the hydrophobic peptide coverage; the performance of the optimized system was first benchmarked using tryptic digests of Saccharomyces cerevisiae cell lysates and then evaluated through duplicate analyses of Macaca fascicularis cerebral cortex lysates using isobaric tags for relative and absolute quantitation (iTRAQ) technology. An additional plug-and-play PGC module functioned in a complementary manner to recover unretained hydrophilic solutes from the low-pH RP column; synchronization of the fractionations between the PGC-RP system and the PGC module facilitated simultaneous analyses of hydrophobic and hydrophilic compounds from a single sample injection event. This methodology was applied to perform, for the first time, detailed glycomics analyses of Macaca fascicularis plasma, resulting in the identification of a total 130 N-glycosylated plasma proteins, 705 N-glycopeptides, and 254 N-glycosylation sites.

Quantitative method for analysis of tobacco‐specific N‐nitrosamines in mainstream cigarette smoke by using heart‐cutting two‐dimensional liquid chromatography with tandem mass spectrometry

“…And heart‐cutting 2D‐LC system is a quite simple and fast method to remove those interferences. It was recently widely used to analysis of complex mixtures . TSNAs had fair chromatographic retentions in both SCX and RPLC column.…”

Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease

“…Furthermore, attempts to generate universal workflows using Pronase digestion and C18-PGC LC-MS/MS instead of the conventional RP-LC-MS/MS of tryptic glycopeptides were described (39,134). In addition, data-independent acquisition (DIA), referred to as "SWATH-MS" on Sciex MS platforms and "MS E " on Waters MS platforms, has also been trialed to monitor both micro- (135,136) and macro-(136 -138) heterogeneity of protein N-glycosylation using LC-MS/MS instrumentation. However, common for these less used, but still very exciting analytical choices, is the fact that much work is still needed to accurately document their potential in glycoproteomics relative to their already implemented counterparts.…”

Glycoprotein Enrichment Analytical Techniques

“…The abovementioned common used HPLC separation techniques are limited to its natural disadvantages in separating glycopeptides. Therefore, the combination of these techniques is applied to glycomic studies (Lam, Lau, Siu, Ng, Kong, Chiu et al, 2011; Liu, Wang, Zhu, Mao, Liu, Cheng et al, 2014; Y. Zhao, Szeto, Kong, Law, Li, Quan et al, 2014).…”

“…The C18-RPLC is majorly used for glycopeptide separation which the PGC component is used as purification materials (Liu et al, 2014) or a secondary dimension of separation. (Stavenhagen et al, 2015; Y. Zhao et al, 2014).…”