title: Efficient digestion and mass spectral analysis of vesicular glutamate transporter 1: a recombinant membrane protein expressed in yeast.
abstract: Attempts to characterize recombinant integral membrane proteins (IMPs) by mass spectrometry are frequently hindered by several factors including the detergents required for extraction and purification that interferes with analysis, poor solubility, incomplete digestion, and limited identification of the transmembrane domain-spanning peptides. The goal of this study was to examine and develop methods for purification of an IMP that are amenable to downstream digestion of the protein and peptide analysis by mass spectrometry. In this study, we have overexpressed a candidate IMP, the vesicular glutamate transporter 1 (VGLUT1) in Pichia pastoris and examined conditions for the efficient affinity purification, in-solution digestion, and analysis of the protein. Analysis of the intact purified protein without detergent was performed by MALDI-TOF mass spectrometry. The purified IMP was digested with trypsin, and the resulting peptides were identified. A method that utilizes differential solubility and ionization properties of hydrophobic and hydrophilic peptides was developed. Large hydrophobic peptides were only detected in solutions containing 50% formic acid. Ionization of hydrophilic peptides was suppressed in formic acid, but they produced a strong signal in 50% acetonitrile. Eighty-seven percent sequence coverage of the protein was obtained with only one large hydrophobic peptide that remained unidentified. The results demonstrate a simple method to purify and digest a recombinant IMP for analysis by mass spectrometry.
authors.affiliation: Center for Structural and Functional Neuroscience, Department of Biomedical and Pharmaceutical Sciences, The University of Montana, Missoula, Montana 59812, USA.
authors.forename: Holly D
authors.initials: HD
authors.lastname: Cox
authors.forename: Chih-Kai
authors.initials: CK
authors.lastname: Chao
authors.forename: Sarjubhai A
authors.initials: SA
authors.lastname: Patel
authors.forename: Charles M
authors.initials: CM
authors.lastname: Thompson
mesh_terms: D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D006639:Histidine; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010766:Phosphorylation; D010843:Pichia / Q000235:genetics*; D040901:Proteomics; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D050598:Vesicular Glutamate Transport Protein 1 / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
chemical_list: D000345:Affinity Labels; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; C493466:Slc17a7 protein, rat; D050598:Vesicular Glutamate Transport Protein 1; D006639:Histidine