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Glycans are complex molecules composed of various monosaccharides and exhibit diverse, branched polymer structures. Extensive research has been conducted on mass spectrometry (MS)-based qualitative and quantitative glycan analysis due to their critical biological functions. However, traditional data-dependent acquisition (DDA) in MS analysis primarily selects a limited subset of abundant ions during MS1 scans for fragmentation in subsequent MS2 stages. In this study, we introduce an advanced isobaric labeling strategy that incorporates a large amount of content-relevant sample labeled with one isobaric tag channel as an additional boosting channel. This innovation enhances the efficiency of isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tagging in quantitative glycomics. Notably, this approach significantly improves the characterization of low-abundance N-glycans and enables the detection of subtle quantitative differences in N-glycan profiling.
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http://dx.doi.org/10.1021/jasms.5c00153 | DOI Listing |
bioRxiv
July 2025
Department of Molecular Biology, Princeton University, Princeton, NJ, USA.
Shotgun proteomics hinges on complete enzymatic digestion of proteins into peptides. Incomplete digestion narrows proteome coverage and inflates variability in quantitative workflows, whether label-free DIA or multiplexing with isobaric tags. Sequential Lys-C/Trypsin digestions mitigate missed cleavages at lysine residues, but arginine sites remain a persistent challenge.
View Article and Find Full Text PDFJ Am Soc Mass Spectrom
September 2025
Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States.
Glycans are complex molecules composed of various monosaccharides and exhibit diverse, branched polymer structures. Extensive research has been conducted on mass spectrometry (MS)-based qualitative and quantitative glycan analysis due to their critical biological functions. However, traditional data-dependent acquisition (DDA) in MS analysis primarily selects a limited subset of abundant ions during MS1 scans for fragmentation in subsequent MS2 stages.
View Article and Find Full Text PDFJ Proteome Res
August 2025
Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, United States.
Isobaric labeling of biospecimens followed by mass spectrometry (MS) has become the method of choice for large-scale, untargeted, quantitative proteomic profiling. However, subtle variation in experimental conditions can amplify sample variability and introduce systematic biases. Motivated by the challenges and opportunities arose in a recent proteogenomic study, we developed ProMix, a flexible analytical framework designed to improve protein normalization by leveraging two key experimental design features: (1) the inclusion of an additional reference sample to serve as an internal standard, and (2) the incorporation of replicates of each specimen.
View Article and Find Full Text PDFbioRxiv
May 2025
Department of Pathology, University of Michigan, Ann Arbor, MI, USA.
Isobaric mass tags, such as iTRAQ and TMT, are widely utilized for peptide and protein quantification in multiplex quantitative proteomics. We present TMT-Integrator, a bioinformatics tool for processing quantitation results from TMT and iTRAQ experiments, offering integrative reports at the gene, protein, peptide, and post-translational modification site levels. We demonstrate the versatility of TMT-Integrator using five publicly available TMT datasets: an dataset with 13 spike-in proteins, the clear cell renal cell carcinoma (ccRCC) whole proteome and phosphopeptide-enriched datasets from the Clinical Proteomic Tumor Analysis Consortium, and two human cell lysate datasets showcasing the latest advances with the Astral instrument and TMT 35-plex reagents.
View Article and Find Full Text PDFCell Chem Biol
May 2025
Department of Chemistry, Molecular Sciences Research Hub, Imperial College London, 82 Wood Lane, London W12 0BZ, UK. Electronic address:
Cellular signaling by membrane G protein-coupled receptors (GPCRs) is governed by a complex and diverse array of mechanisms. The dynamics of a GPCR interactome, as it evolves over time and space in response to an agonist, provide a unique perspective on pleiotropic signaling decoding and functional selectivity at the cellular level. In this study, we utilized proximity-based APEX2 proteomics to investigate the interaction network of the luteinizing hormone receptor (LHR) on a minute-to-minute timescale.
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