Optimized Glycopeptide Enrichment Method–It Is All About the Sauce

Abstract

Protein glycosylation is a family of posttranslational modifications that play a crucial role in many biological pathways and diseases. The enrichment and analysis of such a diverse family of modifications are very challenging because of the number of possible glycan–peptide combinations. Among the methods used for the enrichment of glycopeptides, boronic acid never lived up to its promise. While most studies focused on improving the affinity of the boronic acids to the sugars, we discovered that the buffer choice is just as important for successful enrichment if not more so. We show that an amine-less buffer allows for the best glycoproteomic coverage, in human plasma and brain specimens, improving total quantified glycopeptides by over 10-fold, and reaching 1598 N-linked glycopeptides in the brain and 737 in nondepleted plasma. We speculate that amines compete with the glycans for boronic acid binding, and therefore the elimination of them improved the method significantly.

Secretome Analysis: Reading Cellular Sign Language to Understand Intercellular Communication

“…Indeed, protein capture with single lectin species may also introduce a bias and preclude a true reflection of the stoichiometry of a glycan-modified secreted protein. In this respect, the combined use of multiple lectins for glyco-peptide level enrichment ( 114 ) and diagnostic mass triggering MS acquisition ( 115 , 116 ) may overall achieve better sensitivity and specificity of detection. In an ideal scenario, glycosylation events may evolve as biomarkers in the development of novel disease stratification tools ( 117 ).…”

An Introduction to Mass Spectrometry-Based Proteomics

“…Glycosylation can occur on a variety of amino acid residues, and glycans can assume a diverse array of varyingly branched structures . Some fundamentals of glycoproteomics have now been established: enrichment of glycopeptides or glycoproteins is often performed − and relatively gentle electron transfer dissociation (ETD) is frequently employed, often in conjunction with HCD. ,,, Recently, digestion enzymes have also been developed to enable the detection of challenging glycans such as O -linked glycans on mucins, which are resistant to tryptic digestion. − Whereas other PTMs with constant atomic composition, such as phosphorylation, require the consideration of few additional possible fragments in database search, peptide-spectrum matching in glycoproteomics is greatly complicated by the various structures of glycans; search algorithms continue to be developed and improved. ,, All steps in the glycoproteomic workflow, from digestion and enrichment to raw data acquisition and processing, continue to undergo improvements to further illuminate glycobiology …”

Recent developments in capillary and microchip electroseparations of peptides (2021–mid‐2023)

“…The achieved limit of detections (LODs) were in the range 0.1-0.5 ng/mL. Glycopeptide enrichment in the digest of human plasma and brain (glyco)proteins using polyethylenimine (PEI)-benzoboroxole beads was improved 10 times when the amine-free buffers were employed [80].…”