Higher Temperature Porous Graphitic Carbon Separations Differentially Impact Distinct Glycopeptide Classes

Abstract

Mass spectrometry-based discovery glycoproteomics is highly dependent on the use of chromatography paradigms amenable to analyte retention and separation. When compared against established stationary phases such as reversed-phase and hydrophilic interaction liquid chromatography, reports utilizing porous graphitic carbon have detailed its numerous advantages. Recent efforts have highlighted the utility in porous graphitic carbon in high-throughput glycoproteomics, principally through enhanced profiling depth and liquid-phase resolution at higher column temperatures. However, increasing column temperature has been shown to impart disparaging effects in glycopeptide identification. Herein we further elucidate this trend, describing qualitative and semiquantitative effects of increased column temperature on glycopeptide identification rates, signal intensity, resolution, and spectral count linear response. Through analysis of enriched bovine and human glycopeptides, species with high mannose and sialylated glycans were shown to most significantly benefit and suffer from high column temperatures, respectively. These results provide insight as to how porous graphitic carbon separations may be appropriately leveraged for glycopeptide identification while raising concerns over quantitative and semiquantitative label-free comparisons as the temperature changes. RAW MS glycoproteomic data are available via ProteomeXchange with identifier PXD034354.

Analysis of Sialic Acid Linkage in N-Linked Glycopeptides Using Liquid Chromatography–Electron-Activated Dissociation Time-of-Flight Mass Spectrometry

“…We assumed that each peak in the separated LC peaks of isomeric N -glycopeptides indicated the difference in Sa linkages because this was also frequently reported in HPLC employing a HILIC column − or GC column − or electrophoresis . We analyzed a digested fetuin sample with α(2–3)-linked Sa removal as a proof of concept.…”

“…Our current data is not sufficient for the assignment of the Sa to one of the two antennas. Improvements in LC , combined with cross-ring cleavage of the mannose at the branch, which can be observed in ECD experiments, may lead to such identification in the future.…”

“…For characterizing glycans further on the sugar linkages, especially for Sa linkage to Gal in the α(2–3) linkage or α(2–6) linkage, a high-pressure LC (HPLC) system with a hydrophilic interaction liquid chromatography (HILIC) column − or a porous graphitized carbon column (PGC) is used. ,− Electrophoresis is also used. We tested a HILIC column; however, electrospray ionization (ESI) produced lower precursor ion charge states than a reversed-phase HPLC.…”

Inclusion of Porous Graphitic Carbon Chromatography Yields Greater Protein Identification and Compartment and Process Coverage and Enables More Reflective Protein-Level Label-Free Quantitation

“…Porous graphitic carbon (PGC) chromatography is an emerging chromatographic regime that has gained popularity for its ability to retain polar, hydrophilic analytes 32−34 particularly favorable for the analysis of released glycans. 35−45 This separation strategy was shown to be suitable for the analysis of tryptic glycopeptides, 46,47 suggesting the utility of PGC may extend beyond metabolomic and glycomic analyses. With a growing understanding of the retention mechanism, it was recently hypothesized that chromatography of this nature may be a suitable complement to traditional RPLC in untargeted, high-throughput analyses.…”

“…Electrostatic repulsion–hydrophilic interaction chromatography (ERLIC) − is a recent addition to the chromatographic toolbox, reporting greater retention of hydrophilic peptides and polar analytes. − A limitation of both HILIC and ERLIC is the requirement of salt-containing buffers to mitigate charge effects , or to maintain and improve separation capacity, which can hinder mass spectrometry detection. Porous graphitic carbon (PGC) chromatography is an emerging chromatographic regime that has gained popularity for its ability to retain polar, hydrophilic analytes − particularly favorable for the analysis of released glycans. − This separation strategy was shown to be suitable for the analysis of tryptic glycopeptides, , suggesting the utility of PGC may extend beyond metabolomic and glycomic analyses. With a growing understanding of the retention mechanism, it was recently hypothesized that chromatography of this nature may be a suitable complement to traditional RPLC in untargeted, high-throughput analyses.…”