scholarly journals Community evaluation of glycoproteomics informatics solutions reveals high-performance search strategies for serum glycopeptide analysis

2021 ◽  
Author(s):  
Rebeca Kawahara ◽  
Anastasia Chernykh ◽  
Kathirvel Alagesan ◽  
Marshall Bern ◽  
Weiqian Cao ◽  
...  
Keyword(s):  
Data Analysis ◽  
High Performance ◽  
Mass Spectra ◽  
Search Strategies ◽  
High Coverage ◽  
Tandem Mass Spectra ◽  
Software Packages ◽  
Community Evaluation ◽  
Key Variables ◽  
Glycopeptide Analysis

AbstractGlycoproteomics is a powerful yet analytically challenging research tool. Software packages aiding the interpretation of complex glycopeptide tandem mass spectra have appeared, but their relative performance remains untested. Conducted through the HUPO Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates solutions for system-wide glycopeptide analysis. The same mass spectrometrybased glycoproteomics datasets from human serum were shared with participants and the relative team performance for N- and O-glycopeptide data analysis was comprehensively established by orthogonal performance tests. Although the results were variable, several high-performance glycoproteomics informatics strategies were identified. Deep analysis of the data revealed key performance-associated search parameters and led to recommendations for improved ‘high-coverage’ and ‘high-accuracy’ glycoproteomics search solutions. This study concludes that diverse software packages for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies and specifies key variables that will guide future software developments and assist informatics decision-making in glycoproteomics.

scholarly journals Community Evaluation of Glycoproteomics Informatics Solutions Reveals High-Performance Search Strategies of Glycopeptide Data

2021 ◽  
Author(s):  
Rebeca Kawahara ◽  
Kathirvel Alagesan ◽  
Marshall Bern ◽  
Weiqian Cao ◽  
Robert J Chalkley ◽  
...  
Keyword(s):  
Data Analysis ◽  
Team Performance ◽  
High Performance ◽  
Serum Proteins ◽  
Search Strategies ◽  
Tandem Mass Spectra ◽  
Community Evaluation ◽  
Key Variables ◽  
The Many ◽  
Human Proteome Project

AbstractGlycoproteome profiling (glycoproteomics) remains a considerable analytical challenge that hinders rapid progress in glycobiology. The complex tandem mass spectra generated from glycopeptide mixtures require sophisticated analysis pipelines for structural determination. Diverse informatics solutions aiding the process have appeared, but their relative strengths and weaknesses remain untested. Conducted through the Human Proteome Project – Human Glycoproteomics Initiative, this community study comprising both developers and expert users of glycoproteomics software is the first to evaluate the relative performance of current informatics solutions for comprehensive glycopeptide analysis. High-quality LC-MS/MS-based glycoproteomics datasets of N- and O-glycopeptides from serum proteins were shared with all teams. The relative team performance for efficient glycopeptide data analysis was systematically established through multiple orthogonal performance tests. Excitingly, several high-performance glycoproteomics informatics solutions and tools displaying a considerable performance potential were identified. While the study illustrated that significant informatics challenges remain in the analysis of glycopeptide data as indicated by a high discrepancy between the reported glycopeptides, a substantial list of commonly reported high-confidence glycopeptides could be extracted from the team reports. Further, the team performance profiles were correlated to the many study variables, which revealed important performance-associated search settings and search output variables, some intuitive others unexpected. This study concludes that diverse informatics solutions for comprehensive glycopeptide data analysis exist within the community, points to several high-performance search strategies, and specifies key variables that may guide future software developments and assist the experimental decision-making of practitioners in glycoproteomics.

α-Methylene-β-Lactone Probe for Measuring Live-Cell Reactions of Small Molecules

2020 ◽  
Author(s):  
Lei Wang ◽  
Louis Riel ◽  
Bekim Bajrami ◽  
Bin Deng ◽  
Amy Howell ◽  
...  
Keyword(s):  
Mass Spectra ◽  
Live Cell ◽  
Live Cells ◽  
The Novel ◽  
Proteomics Analysis ◽  
Chemical Proteomics ◽  
Tandem Mass Spectra ◽  
Modified Peptides ◽  
Covalent Probes ◽  
Ht 29

The novel use of the α-methylene-β-lactone (MeLac) moiety as a warhead of multiple electrophilic sites is reported. In this study, we demonstrate that a MeLac-alkyne is a competent covalent probe and reacts with diverse proteins in live cells. Proteomics analysis of affinity-enriched samples identifies probe-reacted proteins, resolves their modified peptides/residues, and thus characterizes probe-protein reactions. Unique methods are developed to evaluate confidence in the identification of the reacted proteins and modified peptides. Tandem mass spectra of the peptides reveal that MeLac reacts with nucleophilic cysteine, serine, lysine, threonine, and tyrosine residues, through either Michael addition or acyl addition. A peptide-centric proteomics platform, using MeLac-alkyne as the measurement probe, successfully analyzes the Orlistat selectivity in live HT-29 cells. MeLac is a versatile warhead demonstrating enormous potential to expedite the development of covalent probes and inhibitors in interrogating protein (re)activity. MeLac-empowered platforms in chemical proteomics are widely adaptable for measuring the live-cell action of reactive molecules.

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