Glycoproteomics - Adding Carbohydrates to Proteins

Introduction:

The field of glycoproteomics is experiencing rapid growth as it helps to detect and characterize glycosylation events on a proteome-wide scale. In recent years, significant strides have been made in developing novel technologies, enrichment methods, and analytical strategies to improve the quality of glycoproteomic investigations. This review explores the latest advancements in glycoproteomics and the current state-of-the-art approaches for analyzing glycosylated substances.

The studies underscore notable enhancements in mass spectrometry equipment, combined with enrichment techniques for crucial glycosylation classes such as mucin-O-glycosylation, O-GlcNAc glycosylation, and N-linked glycosylation.

These advancements now enable the identification and quantification of hundreds to thousands of glycosylation sites within individual experiments. Ultimately, studies outline emerging trends in glycoproteomics that demonstrate a shift from the mere identification of glycosylated substances to the exploration of specific mechanisms and disease conditions.

What Is Glycoproteomics?

Protein glycosylation is the process of adding carbohydrates to proteins that constitutes a diverse array of protein modifications crucial in shaping proteome functionality. Initially believed to be exclusive to eukaryotic systems, glycosylation is now acknowledged to occur across all domains of life, including bacteria and archaea.

Various classes of glycosylation have been identified, differing in the targeted residues for modification, site locations within protein domains, and types of carbohydrates involved in glycan chains. These modifications can significantly impact a protein's biological function and are known to regulate processes in cellular development, cancer biology, and microbial infectivity.

Understanding these complex biological systems necessitates the identification, characterization, and quantification of glycosylation events, underscoring the critical role of glycoproteomics in providing comprehensive insights into glycosylation at a systems level.

In recent years, glycoproteomics has transitioned from a specialized field to a prominent subdiscipline within glycobiology and proteomics.

This evolution has brought about shifts in research focus and technical methodologies. While earlier studies in glycoproteomics primarily aimed at identifying glycosylation sites, known as de-glycoproteomics, advancements in experimental techniques have enabled investigations into the roles of glycoforms and previously challenging glycosylation classes.

This review highlights two rapidly progressing areas in glycoproteomics:

(I) Advancements in enrichment methods for analyzing key glycosylation classes.

(II) The current state-of-the-art mass spectrometry (MS)/proteomic strategies are utilized to identify glycopeptides.

What Are the Clinical Applications Of Glycoproteomics?

Glycoproteins exhibit a variety of glycoforms, where potential glycosylation sites may or may not be occupied by different glycans. These diverse forms can possess distinct biological functions or lead to clinical abnormalities.

Glycoproteins hold clinical significance for several reasons:

(i) Protein glycosylation alters protein properties and governs various biological functions through specific protein-carbohydrate interactions.

(ii) Aberrant glycosylation of glycoproteins is fundamental to disease development and progression.

(iii) Extracellular proteins are commonly glycosylated and can potentially enter the bloodstream, offering biomarkers for certain diseases.

(iv) Focusing on the glycoprotein subproteome in glycoproteomic analysis significantly reduces sample complexity, enhancing the detection sensitivity for low-abundance proteins. Numerous publications have highlighted the clinical applications of glycoproteomic analyses, showcasing the usefulness of various glycoproteomic methods.

The following are the clinical applications of glycoproteomics:

• Plasma Biomarker Discovery - Human blood has been a focal point for uncovering disease-related biomarkers due to the relatively non-invasive nature of blood sampling and its inclusion of important indicators of physiological and pathological states. An illustrative instance of a plasma biomarker utilized in disease diagnosis is the prostate-specific antigen (PSA), which is employed for screening and monitoring prostate cancer. PSA is exclusively expressed in prostate tissue, and its presence in blood correlates with disease advancement, rendering it clinically valuable. Several recent studies have demonstrated the direct identification of disease-related glycoproteins from sera or plasma.

For instance, several studies have observed distinct glycosylation profiles in human normal and lung adenocarcinoma sera. Employing wheat germ agglutinin lectin, the researchers identified 39 differentially expressed glycoproteins in lung adenocarcinoma serum samples, comprising 27 upregulated and 12 downregulated proteins. Furthermore, they validated the associations with lung cancer development of three upregulated glycoproteins (adiponectin, ceruloplasmin, and glycosylphosphatidyl-inositol-80) and two downregulated glycoproteins (cyclin H and Fyn) through western blot analysis. These specific glycoproteins hold the potential for early lung cancer detection and disease progression monitoring. Another example involves the utilization of a mixed column of Jacalin, Con A, and wheat germ agglutinin for a comparative glycoproteomic analysis of sera from breast cancer patients.

• Mapping Glycosylation Sites On Proteins Of Interest - Protein glycosylation exhibits diversity, with proteins containing multiple potential glycosylation sites and various carbohydrate modifications at each site. The analysis of protein glycosylation has pursued two primary avenues: one involves examining proteins and glycans within glycoprotein mixtures, while the other focuses on assessing the occupancy of glycosylation sites or determining the expression levels of proteins with specific glycosylation sites. Both approaches are believed to be associated with disease states. Disruptions in glycoconjugate biosynthesis lead to diseases characterized by heterogeneous biochemical and clinical features. Congenital disorders of glycosylation (CDGs), a group of defects in N-linked glycosylation, are linked with severe clinical manifestations. In a study by Hulsmeier et al., the correlation between the degree of N-glycosylation site occupancy and disease severity was investigated. They quantified the extent of under-glycosylation in CDG and healthy controls using isotopically labeled standard peptides and multiple reaction monitoring (MRM). In healthy controls, peptides such as transferrin and alpha-1-antitrypsin displayed 98 to 100 % occupancy of all N-glycosylation sites. In contrast, CDG samples exhibited decreased glycosylation site occupancy to varying degrees for each N-glycosylation site, with the extent of decrease correlating with the severity of the disease.

• The Future Direction Of Glycoproteomics In Clinical Applications - Alterations in protein glycosylation represent a prominent aspect of disease advancement. These modifications carry significant biological roles and hold promise as targets for diagnosing, treating, and managing various human conditions. Looking ahead, glycoproteomics will transition towards investigating the functional aspects of glycoproteins. It will serve as a crucial tool in elucidating the role of glycosylation in pathological mechanisms like tumor metastasis and inflammation. Overcoming the challenge posed by the dynamic range of detection in plasma proteomics is imperative. Furthermore, there is a need for analytical techniques capable of achieving enhanced precision and reproducibility in quantification.

Conclusion:

In conclusion, with the growing acknowledgment of the significance of glycosylation in biological functions, there is a corresponding demand for the creation of novel instruments to explore glycoproteomics. Encouragingly, recent advancements and tools offer the scientific community numerous reliable methods to monitor and detect glycosylation across various systems. While our capabilities have substantially progressed over the past decade, transitioning from identifying hundreds to thousands of glycosylation events, they still need to be truly exhaustive.