title: Application of GelC-MS/MS to Proteomic Profiling of Chikungunya Virus Infection: Preparation of Peptides for Analysis.
abstract: Gel-enhanced liquid chromatography coupled with tandem mass spectrometry (GeLC-MS/MS) is a labor intensive, but relatively straightforward methodology that generates high proteome coverage which can be applied to the proteome analysis of a range of starting materials such as cells or patient specimens. Sample proteins are resolved electrophoretically in one dimension through a sodium dodecyl sulfate (SDS) polyacrylamide gel after which the lanes are sliced into sections. The sections are further diced and the gel cubes generated are subjected to in-gel tryptic digestion. The resultant peptides can then be analyzed by tandem mass spectroscopy to identify the proteins by database searching. The methodology can routinely detect several thousand proteins in one analysis. The protocol we describe here has been used with both cells in culture that have been infected with chikungunya virus and specimens from Chikungunya fever patients. This protocol details the process for generating peptides for subsequent mass spectroscopic and bioinformatic analysis.
authors.affiliation: Institute of Molecular Biosciences, Mahidol University, Salaya Campus, 25/25 Phuttamontol Sai 4, Salaya, Nakorn Pathom, Thailand.|National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathum Thani, Thailand.
authors.forename: Atchara
authors.initials: A
authors.lastname: Paemanee
authors.affiliation: Institute of Molecular Biosciences, Mahidol University, Salaya Campus, 25/25 Phuttamontol Sai 4, Salaya, Nakorn Pathom, Thailand.
authors.forename: Nitwara
authors.initials: N
authors.lastname: Wikan
authors.affiliation: National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathum Thani, Thailand.
authors.forename: Sittiruk
authors.initials: S
authors.lastname: Roytrakul
authors.affiliation: Institute of Molecular Biosciences, Mahidol University, Salaya Campus, 25/25 Phuttamontol Sai 4, Salaya, Nakorn Pathom, Thailand. duncan.smi@mahidol.ac.th.|Center for Emerging and Neglected Infectious Diseases, Mahidol University, Bangkok, Thailand. duncan.smi@mahidol.ac.th.
authors.forename: Duncan R
authors.initials: DR
authors.lastname: Smith
mesh_terms: D065632:Chikungunya Fever / Q000378:metabolism*; D002646:Chikungunya virus; D002850:Chromatography, Gel; D006801:Humans; D010455:Peptides / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D010455:Peptides
keywords: Chikungunya; GeLC-MS/MS; Mass spectrometry; Peptides; Proteome