Methods to improve quantitative glycoprotein coverage from bottom‐up LC‐MS data

Abstract

Advances in mass spectrometry instrumentation, methods development, and bioinformatics have greatly improved the ease and accuracy of site-specific, quantitative glycoproteomics analysis. Data-dependent acquisition is the most popular method for identification and quantification of glycopeptides; however, complete coverage of glycosylation site glycoforms remains elusive with this method. Targeted acquisition methods improve the precision and accuracy of quantification, but at the cost of throughput and discoverability. Dataindependent acquisition (DIA) holds great promise for more complete and highly quantitative site-specific glycoproteomics analysis, while maintaining the ability to discover novel glycopeptides without prior knowledge. We review additional features that can be used to increase selectivity and coverage to the DIA workflow: retention time modeling, which would simplify the interpretation of complex tandem mass spectra, and ion mobility separation, which would maximize the sampling of all precursors at a giving chromatographic retention time.The instrumentation and bioinformatics to incorporate these features into glycoproteomics analysis exist. These improvements in quantitative, site-specific analysis will enable researchers to assess glycosylation similarity in related biological systems, answering new questions about the interplay between glycosylation state and biological function.

Mucinomics as the Next Frontier of Mass Spectrometry

“…The general workflow of an MSn measurement consists of the isolation of the species and fragmentation of the precursor ions, followed by detection and data acquisition. Detailed reviews regarding MS approaches to glycomic and glycoproteomic analysis can be found elsewhere. ,,, Also, we note that data independent acquisition (DIA), despite being in its infancy, is an emerging technique to analyze glycopeptides; though we do not cover DIA here, we recommend recent reviews − on the topic.…”

Dual roles of drug or its metabolite−protein conjugate: Cutting‐edge strategy of drug discovery using shotgun proteomics

“…Such strategies can potentially be employed in the identification of DMPCs on membranes. Besides, the extraction methods of other proteins, such as total proteins, hydrophobic proteins, and glycoproteins, are also established and applied for proteomics studies 93 . Here, we summarized some solvents and commercial kits that are commonly used for different protein extraction in Table 2.…”

Advances in mass spectrometry‐based glycoproteomics: An update covering the period 2017–2021

“…In TIMS tunnel, an electric field is applied in an opposite direction to the drag of gas flow. The precursor ions are trapped at different positions based on the different CCS, then released consequentially to fragmentation or detection with the decreasing of electric field strength [34]. TIMS-MS has been successfully applied to analyze targeted peptides from biological samples [142].…”

“…The two main strategies for glycoproteomic analysis using MS are known as bottom-up and top-down approaches [29][30][31]. The bottom-up methodology uses proteolytic enzymes to digest glycoproteins in peptides and glycopeptides that are analyzed by MS; due the complexity of the data, the quantification needs to be addressed using computational tools [32][33][34]. In the top-down method, intact glycoproteins are directly analyzed in the MS system, thus enabling the identification of glycoproteins with minimal sample preparation [35][36][37].…”