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

Abstract

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.

Authors:
ORCiD logo [1];  [1];  [2];  [2]; ORCiD logo [2];  [3]; ORCiD logo [4]; ORCiD logo [5]; ORCiD logo [6]; ORCiD logo [3];  [7];  [7];  [1];  [1];  [8]; ORCiD logo [4]; ORCiD logo [9];  [1]; ORCiD logo [7]; ORCiD logo [10] more »; ORCiD logo [11];  [12];  [3]; ORCiD logo [13]; ORCiD logo [12]; ORCiD logo [3]; ORCiD logo [2]; ORCiD logo [1] « less
  1. Northeastern Univ., Boston, MA (United States)
  2. Northeastern Univ., Evanston, IL (United States)
  3. Univ. of Wisconsin-Madison, Madison, WI (United States)
  4. Discovery Attribute Sciences, Thousand Oaks, CA (United States)
  5. Northeastern Univ., Boston, MA (United States); Alnylam Pharmaceuticals, Cambridge, MA (United States)
  6. Northeastern Univ., Boston, MA (United States); Biogen, Cambridge, MA (United States)
  7. Univ. of California, Los Angeles, CA (United States)
  8. Bruker Daltonics, Billerica, MA (United States)
  9. Northeastern Univ., Burlington, MA (United States)
  10. Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
  11. Institut Pasteur, Paris (France)
  12. Eastwoods Consulting, Boylston, MA (United States)
  13. Spectroswiss, Lausanne (Switzerland)
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1567191
Report Number(s):
PNNL-SA-141748
Journal ID: ISSN 1548-7091
Grant/Contract Number:  
AC05-76RL01830
Resource Type:
Accepted Manuscript
Journal Name:
Nature Methods
Additional Journal Information:
Journal Volume: 16; Journal Issue: 7; Journal ID: ISSN 1548-7091
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

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., and Agar, Jeffrey N. Best practices and benchmarks for intact protein analysis for top-down mass spectrometry. United States: N. p., 2019. Web. doi:10.1038/s41592-019-0457-0.
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. Best practices and benchmarks for intact protein analysis for top-down mass spectrometry. United States. doi:10.1038/s41592-019-0457-0.
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., and Agar, Jeffrey N. Thu . "Best practices and benchmarks for intact protein analysis for top-down mass spectrometry". United States. doi:10.1038/s41592-019-0457-0. https://www.osti.gov/servlets/purl/1567191.
@article{osti_1567191,
title = {Best practices and benchmarks for intact protein analysis for top-down mass spectrometry},
author = {Donnelly, Daniel P. and Rawlins, Catherine M. and DeHart, Caroline J. and Fornelli, Luca and Schachner, Luis F. and Lin, Ziqing and Lippens, Jennifer L. and Aluri, Krishna C. and Sarin, Richa and Chen, Bifan and Lantz, Carter and Jung, Wonhyeuk and Johnson, Kendall R. and Koller, Antonius and Wolff, Jeremy J. and Campuzano, Iain D. G. and Auclair, Jared R. and Ivanov, Alexander R. and Whitelegge, Julian P. and Paša-Tolić, Ljiljana and Chamot-Rooke, Julia and Danis, Paul O. and Smith, Lloyd M. and Tsybin, Yury O. and Loo, Joseph A. and Ge, Ying and Kelleher, Neil L. and Agar, Jeffrey N.},
abstractNote = {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.},
doi = {10.1038/s41592-019-0457-0},
journal = {Nature Methods},
number = 7,
volume = 16,
place = {United States},
year = {2019},
month = {6}
}

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Figures / Tables:

Fig. 1 Fig. 1: Common buffer components suppress MS signal. a, MgCl2 reduces signal (and S/N) in a concentration-dependent manner. b, Fit of experimental data to determine the concentration of MgCl2 required for 50% signal suppression (SC50; black arrow), c, Table of common buffer components and the concentration threshold for 50% SC50more » (experimental data curves and their fits are shown in Supplementary Fig. 1) and calculations in Supplementary Note 2. Detergents compatible with mass spectrometry are discussed in Protocol 2b. *Signal suppression by detergents is less pronounced above their critical micellar concentration (CMC) (described in Protocol 2b).« less

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