Glycosylation is one of the most significant and abundant posttranslational modifications in mammalian cells. It mediates a wide range of biofunctions, including cell adhesion, cell communication, immune cell trafficking, and protein stability. Also, aberrant glycosylation has been associated with various diseases such as diabetes, Alzheimer's disease, inflammation, immune deficiencies, congenital disorders, and cancers. The alterations in the distributions of glycan and glycopeptide isomers are involved in the development and progression of several human diseases. However, the microheterogeneity of glycosylation brings a great challenge to glycomic and glycoproteomic analysis, including the characterization of isomers. Over several decades, different methods and approaches have been developed to facilitate the characterization of glycan and glycopeptide isomers.Mass spectrometry (MS) has been a powerful tool utilized for glycomic and glycoproteomic isomeric analysis due to its high sensitivity and rich structural information using different fragmentation techniques. However, a comprehensive characterization of glycan and glycopeptide isomers remains a challenge when utilizing MS alone. Therefore, various separation methods, including liquid chromatography, capillary electrophoresis, and ion mobility, were developed to resolve glycan and glycopeptide isomers before MS. These separation techniques were coupled to MS for a better identification and quantitation of glycan and glycopeptide isomers. Additionally, bioinformatic tools are essential for the automated processing of glycan and glycopeptide isomeric data to facilitate isomeric studies in biological cohorts. Here in this review, we discuss commonly employed MS-based techniques, separation hyphenated MS methods, and software, facilitating the separation, identification, and quantitation of glycan and glycopeptide isomers.

Changes in the glycome of human proteins and cells are associated with the progression of multiple diseases such as Alzheimer's, diabetes mellitus, many types of cancer, and those caused by viruses. Consequently, several studies have shown essential modifications to the isomeric glycan moieties for diseases in different stages. However, the elucidation of extensive isomeric glycan profiles remains challenging because of the lack of analytical techniques with sufficient resolution power to separate all glycan and glycopeptide iso‐forms. Therefore, the development of sensitive and accurate approaches for the characterization of all the isomeric forms of glycans and glycopeptides is essential to tracking the progression of pathology in glycoprotein‐related diseases. This review describes the isomeric separation achievements reported in glycomics and glycoproteomics in the last decade. It focuses on the mass spectrometry–based analytical strategies, stationary phases, and derivatization techniques that have been developed to enhance the separation mechanisms in liquid chromatography systems and the detection capabilities of mass spectrometry systems.

Currently, surveillance strategies have inadequate performance for cirrhosis and early detection of hepatocellular carcinoma (HCC). The glycosylation of serum haptoglobin has shown to have significant differences between cirrhosis and HCC, thus can be used for diagnosis. We performed a comprehensive liquid chromatography—parallel reaction monitoring—mass spectrometry (LC-PRM-MS) approach, where a targeted parallel reaction monitoring (PRM) strategy was coupled to a powerful LC system, to study the site-specific isomerism of haptoglobin (Hp) extracted from cirrhosis and HCC patients. We found that our strategy was able to identify a large number of isomeric N-glycopeptides, mainly located in the Hp glycosylation site Asn207. Four N-glycopeptides were found to have significant changes in abundance between cirrhosis and HCC samples (p < 0.05). Strategic combinations of the significant N-glycopeptides, either with alpha-fetoprotein (AFP) or themselves, better estimate the areas under the curve (AUC) of their respective receiver operating characteristic (ROC) curves with respect to AFP. The combination of AFP with the isomeric sialylated fucosylated N-glycopeptides Asn207 + 5-6-1-2 and Asn207 + 5-6-1-3, resulted with an AUC value of 0.98, while the AUC value for AFP alone was 0.85. When comparing cirrhosis vs. early HCC, the isomeric N-glycopeptide Asn207 + 5-6-0-1 better estimated AUC with respect to AFP (AUCAFP = 0.81, and AUCAsn207 + 5-6-0-1 = 0.88, respectively).

No abstract

Protein glycosylation is one of the most common posttranslational modifications, and plays an essential role in a wide range of biological processes such as immune response, intercellular signaling, inflammation, host-pathogen interaction, and protein stability. Glycoproteomics is a proteomics subfield dedicated to identifying and characterizing the glycans and glycoproteins in a given cell or tissue. Aberrant glycosylation has been associated with various diseases such as Alzheimer's disease, viral infections, inflammation, immune deficiencies, congenital disorders, and cancers. However, glycoproteomic analysis remains challenging because of the low abundance, site-specific heterogeneity, and poor ionization efficiency of glycopeptides during LC-MS analyses. Therefore, the development of sensitive and accurate approaches to efficiently characterize protein glycosylation is crucial. Methods such as metabolic labeling, enrichment, and derivatization of glycopeptides, coupled with different mass spectrometry techniques and bioinformatics tools, have been developed to achieve sophisticated levels of quantitative and qualitative analyses of glycoproteins. This review attempts to update the recent developments in the field of glycoproteomics reported between 2017 and 2021.

Cerebrospinal fluid (CSF) contains valuable biological and neurological information. However, its glycomics analysis is hampered due to the low amount of protein in the biofluid, as has been demonstrated by other glycomics studies using a substantial amount of CSF. In this work, we investigated different N-glycan sample preparation approaches to develop a more sensitive method. These methods, one with an increased amount of buffer solution during the N-glycan release step with a lower amount of sample volume and the other with Filter-Aided N-Glycan Separation (FANGS), were compared with recent work to demonstrate their effectiveness. It was demonstrated that an increased amount of buffer solution showed higher intensity in comparison to the previously published method and FANGS. This suggested that digestion efficiency during the N-glycan release step was not in an optimal condition from the previously published method, and that there is a substantial loss of sample with FANGS when preparing N-glycans from CSF.

Currently surveillance strategies have inadequate performance for the early detection of hepatocellular carcinoma (HCC). Protein glycosylation is a potential source of biomarkers to differentiate between cirrhosis and HCC. We performed a comprehensive LC-PRM-MS approach where a targeted parallel reaction monitoring (PRM) strategy was coupled to a powerful LC system to study the microheterogeneity of haptoglobin (Hp) extracted from 15 patients with cirrhosis and 15 with HCC. We found that our strategy was able to identify a large number of isomeric N-glycopeptides mainly located in the glycosylation site Asn207. Nine out of twelve N-glycopeptides, located in the Asn207 site, had significant differences in abundance between patients with cirrhosis and HCC (p < 0.05). The area under the curve (AUC) of alpha-fetoprotein (AFP) was alone 0.85, which improved to 0.95 (95% CI: 0.88, 1) when NLF_5613 Isomer 1 was combined with AFP. When comparing the early HCC vs. cirrhosis, four sialylated-fucosylated glycopeptides better estimated AUCs with respect to AFP (AUCAFP = 0.66, and AUCN−glycopeptides = 0.86, 0.84, 0.88, and 0.80, respectively). Further large scale validation of glycopeptides for the early detection of HCC is warranted.

No abstract