ArticleAnalytica chimica acta2022
Optimization of protein-level tandem mass tag (TMT) labeling conditions in complex samples with top-down proteomics.
Article in Analytica chimica acta, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 18 papers, 1 of them a synthesis that pooled it.
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Who cites it
18 citing papers in PubMed, 1 synthesis or guideline pooled it.
- Properties, Origin, and Consistency of Truncated Proteoforms Across Top-Down Proteomic Studies.Molecular & cellular proteomics : MCP · 2025Pooled it
- 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
- 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
- Influence of different sample preparation approaches on proteoform identification by top-down proteomics.Nature methods · 2024Article
- 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
- Novel insights into whey protein among Yak, Yellow Cattle, and Cattle-Yak milk.Food chemistry: X · 2024Article
- Top-down proteomics.Nature reviews. Methods primers · 2024Article
- Integrative Proteomics and Phosphoproteomics Profiling of Symptomatic Accessory Navicular Bone Based on Tandem Mass Tag Technology.International journal of general medicine · 2024Article
- An automated spray-capillary platform for the microsampling and CE-MS analysis of picoliter- and nanoliter-volume samples.Analytical and bioanalytical chemistry · 2023Article
- Cysteine Counting via Isotopic Chemical Labeling for Intact Mass Proteoform Identifications in Tissue.Analytical chemistry · 2023Article
- Multidimensional Separations in Top-Down Proteomics.Analytical science advances · 2023Article
- Optimization of Higher-Energy Collisional Dissociation Fragmentation Energy for Intact Protein-Level Tandem Mass Tag Labeling.Journal of proteome research · 2023Article
- Proteoforms expand the world of microproteins and short open reading frame-encoded peptides.iScience · 2023Review
- Toward the analysis of functional proteoforms using mass spectrometry-based stability proteomics.Frontiers in analytical science · 2023Article
- A benchmarking protocol for intact protein-level Tandem Mass Tag (TMT) labeling for quantitative top-down proteomics.MethodsX · 2022Article
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5 authors.
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Abstract
Isobaric chemical tag labels (e.g., iTRAQ and TMT) have been extensively utilized as a standard quantification approach in bottom-up proteomics, which provides high multiplexing capacity and enables MS2-level quantification while not complicating the MS1 scans. We recently demonstrated the feasibility of intact protein TMT labeling for the identification and quantification with top-down proteomics of smaller intact proteoforms (<35 kDa) in complex biological samples through the removal of large proteins prior to labeling. Still, the production of side products during TMT labeling (i.e., incomplete labeling or labeling of unintended residues) complicated the analysis of complex protein samples. In this study, we systematically evaluated the protein-level TMT labeling reaction parameters, including TMT-to-protein mass ratio, pH/concentration of quenching buffer, protein concentration, reaction time, and reaction buffer. Our results indicated that: (1) high TMT-to-protein mass ratio (e.g., 8:1, 4:1), (2) high pH/concentration of quenching buffer (pH > 9.1, final hydroxylamine concentration >0.3%), and (3) high protein concentration (e.g., > 1.0 μg/μL) resulted in optimal labeling efficiency and minimized production of over/underlabeled side products. >90% labeling efficiency was achieved for E. coli cell lysate after optimization of protein-level TMT labeling conditions. In addition, a double labeling approach was developed for efficiently labeling limited biological samples with low concentrations. This research provides practical guidance for efficient TMT labeling of complex intact protein samples, which can be readily adopted in the high-throughput quantification top-down proteomics.
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