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      Best practices and benchmarks for intact protein analysis for top-down mass spectrometry

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          Abstract

          One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.

          Abstract

          The Consortium for Top-Down Proteomics presents a decision-tree-based guide to sample preparation and analysis protocols for researchers performing top-down mass-spectrometry-based analysis of intact proteins.

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          Most cited references57

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          Electrospray ionization for mass spectrometry of large biomolecules

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            Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry.

            The growing number of applications to determine the stoichiometry, interactions and even subunit architecture of protein complexes from mass spectra suggests that some general guidelines can now be proposed. In this protocol, we describe the necessary steps required to maintain interactions between subunits in the gas phase. We begin with the preparation of suitable solutions for electrospray (ES) and then consider the transmission of complexes through the various stages of the mass spectrometer until their detection. Subsequent steps are also described, including the dissociation of these complexes into multiple subcomplexes for generation of interaction networks. Throughout we highlight the critical experimental factors that determine success. Overall, we develop a generic protocol that can be carried out using commercially available ES mass spectrometers without extensive modification.
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              Practical implications of some recent studies in electrospray ionization fundamentals.

              In accomplishing successful electrospray ionization analyses, it is imperative to have an understanding of the effects of variables such as analyte structure, instrumental parameters, and solution composition. Here, we review some fundamental studies of the ESI process that are relevant to these issues. We discuss how analyte chargeability and surface activity are related to ESI response, and how accessible parameters such as nonpolar surface area and reversed phase HPLC retention time can be used to predict relative ESI response. Also presented is a description of how derivitizing agents can be used to maximize or enable ESI response by improving the chargeability or hydrophobicity of ESI analytes. Limiting factors in the ESI calibration curve are discussed. At high concentrations, these factors include droplet surface area and excess charge concentration, whereas at low concentrations ion transmission becomes an issue, and chemical interference can also be limiting. Stable and reproducible non-pneumatic ESI operation depends on the ability to balance a number of parameters, including applied voltage and solution surface tension, flow rate, and conductivity. We discuss how changing these parameters can shift the mode of ESI operation from stable to unstable, and how current-voltage curves can be used to characterize the mode of ESI operation. Finally, the characteristics of the ideal ESI solvent, including surface tension and conductivity requirements, are discussed. Analysis in the positive ion mode can be accomplished with acidified methanol/water solutions, but negative ion mode analysis necessitates special constituents that suppress corona discharge and facilitate the production of stable negative ions. Copyright 2002 Wiley Periodicals, Inc.
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                Author and article information

                Contributors
                j.agar@northeastern.edu
                Journal
                Nat Methods
                Nat. Methods
                Nature Methods
                Nature Publishing Group US (New York )
                1548-7091
                1548-7105
                27 June 2019
                27 June 2019
                2019
                : 16
                : 7
                : 587-594
                Affiliations
                [1 ]ISNI 0000 0001 2173 3359, GRID grid.261112.7, Barnett Institute of Chemical and Biological Analysis and Departments of Chemistry & Chemical Biology and Pharmaceutical Sciences, , Northeastern University, ; Boston, MA USA
                [2 ]ISNI 0000 0001 2299 3507, GRID grid.16753.36, Departments of Chemistry and Molecular Biosciences and the Proteomics Center of Excellence, , Northwestern University, ; Evanston, IL USA
                [3 ]ISNI 0000 0001 2167 3675, GRID grid.14003.36, Department of Cell and Regenerative Biology, Department of Chemistry, Human Proteomics Program, , University of Wisconsin-Madison, ; Madison, WI USA
                [4 ]ISNI 0000 0001 0657 5612, GRID grid.417886.4, Amgen Research, Discovery Attribute Sciences, Amgen, ; Thousand Oaks, CA USA
                [5 ]ISNI 0000 0004 0506 3000, GRID grid.417897.4, Alnylam Pharmaceuticals, ; Cambridge, MA USA
                [6 ]ISNI 0000 0004 0384 8146, GRID grid.417832.b, Biogen, ; Cambridge, MA USA
                [7 ]ISNI 0000 0000 9632 6718, GRID grid.19006.3e, Department of Chemistry and Biochemistry, Department of Biological Chemistry, and UCLA/DOE Institute of Genomics and Proteomics, , University of California, Los Angeles, ; Los Angeles, CA USA
                [8 ]Bruker Daltonics, Billerica, MA USA
                [9 ]ISNI 0000 0001 2173 3359, GRID grid.261112.7, Biopharmaceutical Analysis Training Laboratory, , Northeastern University, ; Burlington, MA USA
                [10 ]ISNI 0000 0000 9632 6718, GRID grid.19006.3e, The Pasarow Mass Spectrometry Laboratory, The Jane and Terry Semel Institute for Neuroscience and Human Behavior, , David Geffen School of Medicine, University of California, Los Angeles, ; Los Angeles, CA USA
                [11 ]ISNI 0000 0001 2218 3491, GRID grid.451303.0, Environmental Molecular Sciences Laboratory, , Pacific Northwest National Laboratory, ; Richland, WA USA
                [12 ]Mass Spectrometry for Biology Unit, Institut Pasteur, USR 2000, CNRS, Paris, France
                [13 ]Eastwoods Consulting, Boylston, MA USA
                [14 ]ISNI 0000 0001 2167 3675, GRID grid.14003.36, Department of Chemistry, Genome Center of Wisconsin, , University of Wisconsin-Madison, ; Madison, WI USA
                [15 ]GRID grid.483150.b, Spectroswiss, ; Lausanne, Switzerland
                Author information
                http://orcid.org/0000-0002-7846-322X
                http://orcid.org/0000-0001-6157-0937
                http://orcid.org/0000-0002-6805-7922
                http://orcid.org/0000-0002-7460-8727
                http://orcid.org/0000-0002-2953-428X
                http://orcid.org/0000-0003-0379-0821
                http://orcid.org/0000-0003-4310-8540
                http://orcid.org/0000-0002-3094-1544
                http://orcid.org/0000-0003-2763-7733
                http://orcid.org/0000-0001-9853-5457
                http://orcid.org/0000-0002-9427-543X
                http://orcid.org/0000-0001-7533-0774
                http://orcid.org/0000-0001-9989-1437
                http://orcid.org/0000-0001-5211-6812
                http://orcid.org/0000-0002-8815-3372
                http://orcid.org/0000-0003-2645-1873
                Article
                457
                10.1038/s41592-019-0457-0
                6719561
                31249407
                04ca20bb-c95d-4b57-aee7-c154f97bf4ba
                © The Author(s), under exclusive licence to Springer Nature America, Inc. 2019

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 29 December 2017
                : 21 May 2019
                Categories
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                © The Author(s), under exclusive licence to Springer Nature America, Inc. 2019

                Life sciences
                proteomics,proteomic analysis,proteins,mass spectrometry
                Life sciences
                proteomics, proteomic analysis, proteins, mass spectrometry

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