ArticleJournal of proteome research2023
Optimization of Higher-Energy Collisional Dissociation Fragmentation Energy for Intact Protein-Level Tandem Mass Tag Labeling.
Article in Journal of proteome research, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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Who cites it
12 citing papers in PubMed, 16 citations in OpenAlex.
- Methionine Oxidation Footprinting in Intact Proteins (MOFIP) Using Top-Down Proteomics.Proteomics · 2025Article
- Top-Down Thermal Proteome Profiling (TD-TPP) for Functional Characterization of the Intact Proteoforms in Complex Samples.Journal of mass spectrometry : JMS · 2025Article
- Radical-Initiated Photodissociation at Tryptophan Residues within Intact Proteins in the Gas Phase.Journal of the American Chemical Society · 2025Article
- Cysteine-Directed Isobaric Labeling Combined with GeLC-FAIMS-MS for Quantitative Top-Down Proteomics.Journal of proteome research · 2025Article
- Quantitative Top-down Proteomics Revealed Kinase Inhibitor-Induced Proteoform-Level Changes in Cancer Cells.Journal of proteome research · 2025Article
- Characterizing age-related changes in intact mitochondrial proteoforms in murine hearts using quantitative top-down proteomics.Clinical proteomics · 2024Article
- Mass spectrometry-intensive top-down proteomics: an update on technology advancements and biomedical applications.Analytical methods : advancing methods and applications · 2024Review
- Pilot Evaluation of the Long-Term Reproducibility of Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Top-Down Proteomics of a Complex Proteome Sample.Journal of proteome research · 2024Article
- Top-down proteomics.Nature reviews. Methods primers · 2024Article
- Very Low-Pressure CID Experiments: High Energy Transfer and Fragmentation Pattern at the Single Collision Regime.Molecules (Basel, Switzerland) · 2023Article
- Multidimensional Separations in Top-Down Proteomics.Analytical science advances · 2023Article
- Toward the analysis of functional proteoforms using mass spectrometry-based stability proteomics.Frontiers in analytical science · 2023Article
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Authors and funding
7 authors at 1 institution in 1 country.
Funding
Abstract
Isobaric chemical tag labeling (e.g., TMT) is a commonly used approach in quantitative proteomics, and quantification is enabled through detection of low-mass reporter ions generated after MS2 fragmentation. Recently, we have introduced and optimized an intact protein-level TMT labeling platform that demonstrated >90% labeling efficiency in complex samples with top-down proteomics. Higher-energy collisional dissociation (HCD) is commonly utilized for isobaric tag-labeled peptide fragmentation because it produces accurate reporter ion intensities and avoids loss of low mass ions. HCD energies have been optimized for isobaric tag labeled-peptides but have not been systematically evaluated for isobaric tag-labeled intact proteins. In this study, we report a systematic evaluation of normalized HCD fragmentation energies (NCEs) on TMT-labeled HeLa cell lysate using top-down proteomics. Our results suggested that reporter ions often result in higher ion intensities at higher NCEs. Optimal fragmentation of intact proteins for identification, however, required relatively lower NCE. We further demonstrated that a stepped NCE scheme with energies from 30% to 50% resulted in optimal quantification and identification of TMT-labeled HeLa proteins. These parameters resulted in an average reporter ion intensity of ∼4E4 and average proteoform spectrum matches (PrSMs) of >1000 per RPLC-MS/MS run with a 1% false discovery rate (FDR) cutoff.
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