title: SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells.
abstract: Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.
authors.affiliation: Brown University.
authors.forename: Collins
authors.initials: C
authors.lastname: Cheruiyot
authors.affiliation: Brown University.
authors.forename: Zemplen
authors.initials: Z
authors.lastname: Pataki
authors.affiliation: Brown University.
authors.forename: Robert
authors.initials: R
authors.lastname: Williams
authors.affiliation: COBRE Center for Cancer Research, Lifespan Laboratories, Rhode Island and Miriam Hospitals; Division of Infectious Diseases, Department of Medicine, Warren Alpert Medical School, Brown University.
authors.forename: Bharat
authors.initials: B
authors.lastname: Ramratnam
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Warren Alpert Medical School, Brown University; ming_li@brown.edu.
authors.forename: Ming
authors.initials: M
authors.lastname: Li
mesh_terms: D002452:Cell Count; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods*; D055354:Exosomes / Q000737:chemistry*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D020543:Proteome