ArticleNature protocols2020
Direct characterization of overproduced proteins by native mass spectrometry.
Article in Nature protocols, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 21 papers.
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Who cites it
21 citing papers in PubMed, 46 citations in OpenAlex.
- Native Multidimensional Protein Complex Identification and Topology Characterization (Native MudPIT).Analytical chemistry · 2026Article
- Advancing Collision-Induced Affinity Selection Mass Spectrometry for Quantitative Ligand Analysis in Complex Mixtures.Analytical chemistry · 2026Article
- Direct analysis of biotransformations with mass spectrometry-DiBT-MS.Nature protocols · 2025Review
- Deciphering the safeguarding role of cysteine residues in p53 against HCommunications chemistry · 2025Article
- Controlling the incorporation of fluorinated amino acids in human cells and its structural impact.Protein science : a publication of the Protein Society · 2024Article
- Native mass spectrometry interrogation of complexes formed during targeted protein degradation.Rapid communications in mass spectrometry : RCM · 2023Article
- Dissecting the structural heterogeneity of proteins by native mass spectrometry.Protein science : a publication of the Protein Society · 2023Review
- Ion Mobility Mass Spectrometry (IM-MS) for Structural Biology: Insights Gained by Measuring Mass, Charge, and Collision Cross Section.Chemical reviews · 2023Review
- AlacatDesigner─Computational Design of Peptide Concatamers for Protein Quantitation.Journal of proteome research · 2023Article
- Native Mass Spectrometry: Recent Progress and Remaining Challenges.Annual review of biophysics · 2022Review
- Surface-induced Dissociation Mass Spectrometry as a Structural Biology Tool.Chemical reviews · 2022Review
- Approaches to Heterogeneity in Native Mass Spectrometry.Chemical reviews · 2022Review
- High-Resolution Native Mass Spectrometry.Chemical reviews · 2022Review
- Characterizing Endogenous Protein Complexes with Biological Mass Spectrometry.Chemical reviews · 2022Review
- Surface-Induced Dissociation for Protein Complex Characterization.Methods in molecular biology (Clifton, N.J.) · 2022Article
- Direct-MS analysis of antibody-antigen complexes.Proteomics · 2021Article
- Intracellular Protein-Drug Interactions Probed by Direct Mass Spectrometry of Cell Lysates.Angewandte Chemie (International ed. in English) · 2021Article
- Analysis of Tagged Proteins Using Tandem Affinity-Buffer Exchange Chromatography Online with Native Mass Spectrometry.Biochemistry · 2021Article
- Structural Proteomics Methods to Interrogate the Conformations and Dynamics of Intrinsically Disordered Proteins.Frontiers in chemistry · 2021Review
- Use of tandem affinity-buffer exchange chromatography online with native mass spectrometry for optimizing overexpression and purification of recombinant proteins.Methods in enzymology · 2021Article
Corrections and comments
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
Abstract
Proteins derived by recombinant technologies must be characterized to ensure quality, consistency and optimal production. These properties are usually assayed following purification procedures that are time consuming and labor intensive. Here, we describe a native mass spectrometry (MS) approach, direct-MS, for rapid characterization of intact overexpressed proteins immediately from crude samples. In this protocol, we discuss the multiple applications of the method and outline the necessary steps required for sample preparation, data collection and interpretation of results. We begin with the sample preparation workflows, which are relevant for recombinant proteins produced within bacteria, those analyzed straight from crude cell lysate, and secreted proteins generated in eukaryotic expression systems that are assessed directly from the growth culture medium. We continue with the mass acquisition steps that enable immediate definition of properties such as expressibility, solubility, assembly state, folding, overall structure, stability, post-translational modifications and associations with biomolecules. We demonstrate the applicability of the method by presenting the characterization of a computationally designed toxin-antitoxin heterodimer, activity and protein-interaction determination of a regulatory protein and detailed glycosylation analysis of a designed intact antibody. Overall, we describe a simple and rapid protocol that is relevant to both prokaryotic and eukaryotic expression systems and can be carried out on multiple mass spectrometers, such as Orbitrap and quadrupole time-of-flight (QTOF)-based mass spectroscopy platforms, that enable intact protein detection. The procedure takes from 30 min to several hours, from sample collection to data acquisition, depending on the depth of MS analysis.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.