Complementary Proteome and Glycoproteome Access Revealed Through Comparative Analysis of Reversed Phase and Porous Graphitic Carbon Chromatography

Aided by improvements in instrumental and analytical techniques, rigorous connections have been drawn between glycoprotein expression, modification heterogeneity, and a variety of human illnesses. These illnesses, such as various forms of cancer, are often associated with poor prognoses, prompting the need for more comprehensive characterization of the glycoproteome. Instrumental advancements, optimal and nascent dissociation techniques, and computational strategies present the largest area of glycoprotein profiling innovation, often neglecting potential benefits provided by alternative chromatography techniques. Porous graphitic carbon (PGC) represents a separation regime that has gained significant 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 glycoproteomics experiments, leaving the potential benefits unexplored. Performing comparative analysis of single and biphasic separation regimes at a range of column temperatures illustrates the 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 when in both single and biphasic PGC separation, providing a means towards facile, multiplex 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.

随着仪器与分析技术的持续进步，研究人员已严谨确立了糖蛋白（glycoprotein）表达、修饰异质性与多种人类疾病之间的紧密关联。此类疾病（如多种癌症）往往预后不佳，因此亟需对糖蛋白组（glycoproteome）进行更为全面的表征。当前，仪器技术革新、成熟与新兴的解离技术以及计算策略是糖蛋白谱分析创新的核心领域，但此类研究往往忽视了替代色谱技术所能带来的潜在价值。多孔石墨碳（Porous Graphitic Carbon, PGC）作为一种分离体系，凭借其流动相适配性强、对极性分析物的保留能力优异，且在高温下可实现更精准的结构解析，已在糖组学（Glycomics）研究中获得广泛关注。但在高通量自下而上糖蛋白组学（bottom-up glycoproteomics）实验中，目前尚未有研究将PGC与标准反相液相色谱（Reversed Phase Liquid Chromatography, RPLC）进行系统的单独对比或联用评估，因此其潜在优势尚未得到充分发掘。通过在不同柱温条件下对单相与双相分离体系开展对比分析，可明确两种方法各自的优势所在。实验结果表明，PGC分离可选择性保留更短、亲水性更强的糖肽（glycopeptide）物种，使其平均电荷更高，且对已鉴定糖基化位点的微异质性覆盖度更广。此外，本研究证实，无论是单相还是双相PGC分离体系，均可实现糖肽异构体的液相分离，为简便、高通量的糖肽表征提供了可行路径。除此之外，本研究还展示了如何通过结合多种分离体系与不同柱温条件，实现对致瘤性与侵袭性前列腺癌细胞的糖蛋白组表征。