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).
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.
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