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

Donnelly, Daniel P.; Rawlins, Catherine M.; DeHart, Caroline J.; Fornelli, Luca; Schachner, Luis F.; Lin, Ziqing; Lippens, Jennifer L.; Aluri, Krishna C.; Sarin, Richa; Chen, Bifan; Lantz, Carter; Jung, Wonhyeuk; Johnson, Kendall R.; Koller, Antonius; Wolff, Jeremy J.; Campuzano, Iain D.  G.; Auclair, Jared R.; Ivanov, Alexander R.; Whitelegge, Julian P.; Paša-Tolić, Ljiljana; Chamot-Rooke, Julia; Danis, Paul O.; Smith, Lloyd M.; Tsybin, Yury O.; Loo, Joseph A.; Ge, Ying; Kelleher, Neil L.; Agar, Jeffrey N.

doi:10.1038/s41592-019-0457-0

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

Journal Article · 27 June 2019 · Nature Methods

DOI: https://doi.org/10.1038/s41592-019-0457-0 · OSTI ID:1567191

^[1]; Rawlins, Catherine M. ^[1]; DeHart, Caroline J. ^[2]; Fornelli, Luca ^[2];

^[2]; Lin, Ziqing ^[3];

^[4];

^[5];

^[6];

^[3]; Lantz, Carter ^[7]; Jung, Wonhyeuk ^[7]; Johnson, Kendall R. ^[1]; Koller, Antonius ^[1]; Wolff, Jeremy J. ^[8];

^[4];

^[9]; Ivanov, Alexander R. ^[1];

^[7];

^[10] more »

Northeastern Univ., Boston, MA (United States)
Northeastern Univ., Evanston, IL (United States)
Univ. of Wisconsin-Madison, Madison, WI (United States)
Discovery Attribute Sciences, Thousand Oaks, CA (United States)
Northeastern Univ., Boston, MA (United States); Alnylam Pharmaceuticals, Cambridge, MA (United States)
Northeastern Univ., Boston, MA (United States); Biogen, Cambridge, MA (United States)
Univ. of California, Los Angeles, CA (United States)
Bruker Daltonics, Billerica, MA (United States)
Northeastern Univ., Burlington, MA (United States)
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Institut Pasteur, Paris (France)
Eastwoods Consulting, Boylston, MA (United States)
Spectroswiss, Lausanne (Switzerland)

One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Many laboratories, however, forgo the challenging mass analysis of intact proteins. Instead, these researchers resort to well-established methods for proteolytic digests. In this work, the Consortium for Top-Down Proteomics (CTDP) addresses the unmet need for robust protocols for obtaining intact protein mass spectra. We quantify and rationalize the signal suppression associated with common interfering substances. A decision tree, based upon sample composition and experimental goals, guides researchers to a given protocol. Cross-platform analytical benchmarks are provided to allow users to gauge their proficiency and to promote method standardization. This work should permit users at any experience level to obtain high-quality intact mass spectra of native and denatured proteins from mixtures of varying complexity, including intact antibodies and membrane proteins.

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Research Organization:: Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

Sponsoring Organization:: USDOE

Grant/Contract Number:: AC05-76RL01830

OSTI ID:: 1567191

Report Number(s):: PNNL-SA-141748

Journal Information:: Nature Methods, Vol. 16, Issue 7; ISSN 1548-7091

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

Citation Metrics:

Cited by: 181 works

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Mass Spectrometry of Intact Proteins Reveals +98 u Chemical Artifacts Following Precipitation in Acetone Güray, Melda Z.; Zheng, Shi; Doucette, Alan A. Journal of Proteome Research, Vol. 16, Issue 2 https://doi.org/10.1021/acs.jproteome.6b00841	journal	January 2017
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Post-Translational Modification by Cysteine Protects Cu/Zn-Superoxide Dismutase from Oxidative Damage Auclair, Jared R.; Johnson, Joshua L.; Liu, Qian Biochemistry, Vol. 52, Issue 36 https://doi.org/10.1021/bi4006122	journal	August 2013
In Vivo Phosphorylation Site Mapping in Mouse Cardiac Troponin I by High Resolution Top-Down Electron Capture Dissociation Mass Spectrometry: Ser22/23 Are the Only Sites Basally Phosphorylated Ayaz-Guner, Serife; Zhang, Jiang; Li, Lin Biochemistry, Vol. 48, Issue 34 https://doi.org/10.1021/bi900739f	journal	September 2009
Mass Spectrometry-Based Detection and Assignment of Protein Posttranslational Modifications Doll, Sophia; Burlingame, Alma L. ACS Chemical Biology, Vol. 10, Issue 1 https://doi.org/10.1021/cb500904b	journal	November 2014
Top Down versus Bottom Up Protein Characterization by Tandem High-Resolution Mass Spectrometry Kelleher, Neil L.; Lin, Hong Y.; Valaskovic, Gary A. Journal of the American Chemical Society, Vol. 121, Issue 4 https://doi.org/10.1021/ja973655h	journal	February 1999
Enhanced FASP (eFASP) to Increase Proteome Coverage and Sample Recovery for Quantitative Proteomic Experiments Erde, Jonathan; Loo, Rachel R. Ogorzalek; Loo, Joseph A. Journal of Proteome Research, Vol. 13, Issue 4 https://doi.org/10.1021/pr4010019	journal	March 2014
Top-down characterization of endogenous protein complexes with native proteomics Skinner, Owen S.; Haverland, Nicole A.; Fornelli, Luca Nature Chemical Biology, Vol. 14, Issue 1 https://doi.org/10.1038/nchembio.2515	journal	November 2017
Proteoform: a single term describing protein complexity Smith, Lloyd M.; Kelleher, Neil L. Nature Methods, Vol. 10, Issue 3 https://doi.org/10.1038/nmeth.2369	journal	February 2013
High-resolution mass spectrometry of small molecules bound to membrane proteins Gault, Joseph; Donlan, Joseph A. C.; Liko, Idlir Nature Methods, Vol. 13, Issue 4 https://doi.org/10.1038/nmeth.3771	journal	February 2016
Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry Hernández, Helena; Robinson, Carol V. Nature Protocols, Vol. 2, Issue 3 https://doi.org/10.1038/nprot.2007.73	journal	March 2007
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