Efficient digestion and mass spectral analysis of vesicular glutamate transporter 1: a recombinant membrane protein expressed in yeast

Journal of Proteome Research
Holly D CoxCharles M Thompson

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 the...Continue Reading

References

May 5, 1982·Journal of Molecular Biology·J Kyte, R F Doolittle
Nov 1, 1994·Protein Science : a Publication of the Protein Society·R R LooP C Andrews
Apr 21, 1999·Rapid Communications in Mass Spectrometry : RCM·M PuchadesP Davidsson
Oct 9, 2001·Bioinformatics·G E Tusnády, I Simon
Jan 31, 2002·Analytical Chemistry·A LiR J Fisher
Dec 4, 2002·Protein Expression and Purification·Wenke FengZhao-Hui Song
Sep 14, 2004·Journal of Proteome Research·Jun Han, Kevin L Schey
Sep 21, 2004·Proteomics·Holly D CoughenourCharles M Thompson
Dec 23, 2004·The Journal of Peptide Research : Official Journal of the American Peptide Society·S FilppulaA Makriyannis
Feb 22, 2005·Protein Expression and Purification·Tae-Kang KimZhao-Hui Song
May 17, 2005·Brain Research. Molecular Brain Research·Keith H ChristoffersRichard D Howells
Aug 17, 2005·Current Medicinal Chemistry·Charles M ThompsonJohn M Gerdes
Mar 2, 2006·Biological & Pharmaceutical Bulletin·Sun-Kyung JungHiroshi Omote

❮ Previous
Next ❯

Citations

Jan 3, 2019·Antonie van Leeuwenhoek·Mariana Caroline Tocantins AlvimWendel Batista da Silveira
Jan 20, 2018·Journal of Proteome Research·Mamiyo KawakamiTakaaki Miyaji

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.