Efficient Mass Spectral Analysis of Active Transporters Overexpressed in Escherichia coli

J Proteome Res. 2018 Mar 2;17(3):1108-1119. doi: 10.1021/acs.jproteome.7b00777. Epub 2018 Feb 5.

Abstract

Structural analysis of purified active membrane proteins can be performed by mass spectrometry (MS). However, no large-scale expression systems for active eukaryotic membrane proteins are available. Moreover, because membrane proteins cannot easily be digested by trypsin and ionized, they are difficult to analyze by MS. We developed a method for mass spectral analysis of eukaryotic membrane proteins combined with an overexpression system in Escherichia coli. Vesicular glutamate transporter 2 (VGLUT2/SLC17A6) with a soluble α-helical protein and histidine tag on the N- and C-terminus, respectively, was overexpressed in E. coli, solubilized with detergent, and purified by Ni-NTA affinity chromatography. Proteoliposomes containing VGLUT2 retained glutamate transport activity. For MS analysis, the detergent was removed from purified VGLUT2 by trichloroacetic acid precipitation, and VGLUT2 was then subjected to reductive alkylation and tryptic digestion. The resulting peptides were detected with 88% coverage by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS with or without liquid chromatography. Vesicular excitatory amino acid transporter and vesicular acetylcholine transporter were also detected with similar coverage by the same method. Thus this methodology could be used to analyze purified eukaryotic active transporters. Structural analysis with chemical modifiers by MS could have applications in functional binding analysis for drug discovery.

Keywords: matrix-assisted laser desorption ionization time-of-flight mass spectrometry; membrane protein; sequence coverage; trypsin digestion; vesicular glutamate transporter; vesicular neurotransmitter transporter.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chemical Precipitation
  • Cloning, Molecular
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Excitatory Amino Acid Transporter 1 / analysis*
  • Excitatory Amino Acid Transporter 1 / genetics
  • Excitatory Amino Acid Transporter 1 / metabolism
  • Gene Expression
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • Humans
  • Mice
  • Peptide Fragments / analysis*
  • Peptide Mapping
  • Proteolysis
  • Rats
  • Recombinant Proteins / analysis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Trichloroacetic Acid / chemistry
  • Trypsin / chemistry
  • Vesicular Acetylcholine Transport Proteins / analysis*
  • Vesicular Acetylcholine Transport Proteins / genetics
  • Vesicular Acetylcholine Transport Proteins / metabolism
  • Vesicular Glutamate Transport Protein 2 / analysis*
  • Vesicular Glutamate Transport Protein 2 / genetics
  • Vesicular Glutamate Transport Protein 2 / metabolism

Substances

  • Excitatory Amino Acid Transporter 1
  • Peptide Fragments
  • Recombinant Proteins
  • SLC18A3 protein, human
  • Slc17a6 protein, rat
  • Slc1a3 protein, mouse
  • Vesicular Acetylcholine Transport Proteins
  • Vesicular Glutamate Transport Protein 2
  • Trichloroacetic Acid
  • Trypsin