Fixed-Charge Trimethyl Pyrilium Modification for Enabling Enhanced Top-Down Mass Spectrometry Sequencing of Intact Protein Complexes

Anal Chem. 2018 Feb 20;90(4):2756-2764. doi: 10.1021/acs.analchem.7b04806. Epub 2018 Feb 2.

Abstract

Mass spectrometry of intact proteins and protein complexes has the potential to provide a transformative level of information on biological systems, ranging from sequence and post-translational modification analysis to the structures of whole protein assemblies. This ambitious goal requires the efficient fragmentation of both intact proteins and the macromolecular, multicomponent machines they collaborate to create through noncovalent interactions. Improving technologies in an effort to achieve such fragmentation remains perhaps the greatest challenge facing current efforts to comprehensively analyze cellular protein composition and is essential to realizing the full potential of proteomics. In this work, we describe the use of a trimethyl pyrylium (TMP) fixed-charge covalent labeling strategy aimed at enhancing fragmentation for challenging intact proteins and intact protein complexes. Combining analysis of TMP-modified and unmodified protein complexes results in a greater diversity of regions within the protein that give rise to fragments, and results in an up to 2.5-fold increase in sequence coverage when compared to unmodified protein alone, for protein complexes up to 148 kDa. TMP modification offers a simple and powerful platform to expand the capabilities of existing mass spectrometric instrumentation for the complete characterization of intact protein assemblies.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Alcohol Dehydrogenase / chemistry*
  • Animals
  • Avidin / chemistry*
  • Chickens
  • Mass Spectrometry
  • Molecular Structure
  • Ovalbumin / chemistry*
  • Pyrans / chemistry*
  • Saccharomyces cerevisiae / enzymology

Substances

  • Pyrans
  • trimethyl pyrylium
  • Avidin
  • Ovalbumin
  • Alcohol Dehydrogenase