title: Purification of Tryptic Digests on Polyvinylidene Difluoride Membrane.
abstract: Minor impurities in tryptic peptide digests can affect the signal obtained in matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Therefore, it becomes necessary to purify the digests, especially those that fail to yield good mass spectra. Here, we describe a simple protocol using polyvinylidene difluoride membrane for purifying tryptic peptides prior to mass spectrometric analysis. The tryptic digest is spotted on a polyvinylidene difluoride membrane, air-dried, and washed. The membrane is then extracted with trifluoroacetic acid/acetonitrile and the extract is then subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry. This method enabled us to identify a cross-reactive D1 autoantigen on the surface of neutrophils that bound antibodies targeting Ro 60 autoantigen in systemic lupus erythematosus.
authors.affiliation: Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA, biji-kurien@omrf.org.
authors.forename: Biji T
authors.initials: BT
authors.lastname: Kurien
authors.forename: R Hal
authors.initials: RH
authors.lastname: Scofield
mesh_terms: D000906:Antibodies / Q000032:analysis / Q000276:immunology; D003429:Cross Reactions; D006801:Humans; D008180:Lupus Erythematosus, Systemic / Q000276:immunology; D008567:Membranes, Artificial; D009504:Neutrophils / Q000276:immunology; D011145:Polyvinyls / Q000737:chemistry*; D012261:Ribonucleoproteins / Q000032:analysis / Q000276:immunology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D000099207:SS-A Antigen; D005465:Fluorocarbon Polymers
chemical_list: D000906:Antibodies; D008567:Membranes, Artificial; D011145:Polyvinyls; D012261:Ribonucleoproteins; D000099207:SS-A Antigen; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers