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Efficient Mass Spectral Analysis of Active Transporters Overexpressed in Escherichia coli
journal contribution
posted on 2018-01-19, 00:00 authored by Mamiyo Kawakami, Narinobu Juge, Yuri Kato, Hiroshi Omote, Yoshinori Moriyama, Takaaki MiyajiStructural 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.
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Keywords
glutamate transport activityα- helical proteinEfficient Mass Spectral Analysismethodacidmembrane proteinsdetergentEscherichia coli Structural analysisMALDI-TOFActive Transporters OverexpressedMSmatrix-assisted laser desorption ionization time-of-flightNi-NTA affinity chromatographyVGLUT 2Vesicular glutamate transporter 2coverageeukaryotic membrane proteins