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In-House Packed Porous Graphitic Carbon Columns for Liquid Chromatography-Mass Spectrometry Analysis of N-Glycans.

ABSTRACT: Protein glycosylation is a common post-translational modification that modulates biological processes such as the immune response and protein trafficking. Altered glycosylation profiles are associated with cancer and inflammatory diseases, as well as impacting the efficacy of therapeutic monoclonal antibodies. Consisting of oligosaccharides attached to asparagine residues, enzymatically released N-linked glycans are analytically challenging due to the diversity of isomeric structures that exist. A commonly used technique for quantitative N-glycan analysis is liquid chromatography-mass spectrometry (LC-MS), which performs glycan separation and characterization. Although many reversed and normal stationary phases have been utilized for the separation of N-glycans, porous graphitic carbon (PGC) chromatography has become desirable because of its higher resolving capability, but is difficult to implement in a robust and reproducible manner. Herein, we demonstrate the analytical properties of a 15 cm fused silica capillary (75 µm i.d., 360 µm o.d.) packed in-house with Hypercarb PGC (3 µm) coupled to an Agilent 6550 Q-TOF mass spectrometer for N-glycan analysis in positive ion mode. In repeatability and intermediate precision measurements conducted on released N-glycans from a glycoprotein standard mixture, the majority of N-glycans reported low coefficients of variation with respect to retention times (≤4.2%) and peak areas (≤14.4%). N-glycans released from complex samples were also examined by PGC LC-MS. A total of 120 N-glycan structural and compositional isomers were obtained from formalin-fixed paraffin-embedded ovarian cancer tissue sections. Finally, a comparison between early- and late-stage formalin-fixed paraffin-embedded ovarian cancer tissues revealed qualitative changes in the α2,3- and α2,6-sialic acid linkage of a fucosylated bi-antennary complex N-glycan. Although the α2,3-linkage was predominant in late-stage ovarian cancer, the alternate α2,6-linkage was more prevalent in early-stage ovarian cancer. This study establishes the utility of in-house packed PGC columns for the robust and reproducible LC-MS analysis of N-glycans.

SUBMITTER: Young C

PROVIDER: S-EPMC8226321 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Continual developments in instrumental and analytical techniques have aided in establishing rigorous connections between protein glycosylation and human illness. These illnesses, such as various forms of cancer, are often associated with poor prognoses, prompting the need for more comprehensive characterization of the glycoproteome. While innovative instrumental and computational strategies have largely benefited glycoproteomic analyses, less attention is given to benefits gained through alternative, optimized chromatographic techniques. Porous graphitic carbon (PGC) chromatography has gained considerable interest in glycomics research due to its mobile phase flexibility, increased retention of polar analytes, and improved structural elucidation at higher temperatures. PGC has yet to be systematically compared against or in tandem with standard reversed phase liquid chromatography (RPLC) in high-throughput bottom-up glycoproteomic experiments, leaving the potential benefits unexplored. Performing comparative analysis of single and biphasic separation regimes at a range of column temperatures illustrates complementary advantages for each method. PGC separation is shown to selectively retain shorter, more hydrophilic glycopeptide species, imparting higher average charge, and exhibiting greater microheterogeneity coverage for identified glycosites. Additionally, we demonstrate that liquid-phase separation of glycopeptide isomers may be achieved through both single and biphasic PGC separations, providing a means towards facile, multidimensional glycopeptide characterization. Beyond this, we demonstrate how utilization of multiple separation regimes and column temperatures can aid in profiling the glycoproteome in tumorigenic and aggressive prostate cancer cells. RAW MS proteomic and glycoproteomic datasets have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD024196 (10.6019/PXD024196) and PXD024195, respectively.

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In-House Packed Porous Graphitic Carbon Columns for Liquid Chromatography-Mass Spectrometry Analysis of N-Glycans.

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