Tryptic peptide purification using polyvinylidene difluoride membrane for matrix-assisted laser desorption ionization time of flight mass spectrometry.
Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS) is extremely sensitive to minor impurities in tryptic peptide digests, resulting in suppression of the signal obtained. Therefore, it becomes necessary to purify the sample, especially those samples that fail to yield good mass spectra. Here, we describe a simple protocol using polyvinylidene difluoride (PVDF) membrane for purifying tryptic peptides prior to mass spectrometric analysis. The tryptic digest is spotted on a PVDF membrane, air-dried, and washed. The membrane is then extracted with trifluoroacetic acid/acetonitrile and the extract is then subjected to MALDI TOF MS. Using this procedure, we were able to identify a cross-reactive D1 autoantigen on the surface of neutrophils that bound antibodies targeting Ro 60 autoantigen in systemic lupus erythematosus.
Kurien
Biji T
BT
Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK, 73104, USA. biji-kurien@omrf.org
Scofield
R Hal
RH
D001324:Autoantigens / Q000378:metabolism; D006801:Humans; D009504:Neutrophils / Q000166:cytology; D010446:Peptide Fragments / Q000032:analysis / Q000302:isolation & purification*; D011145:Polyvinyls / Q000737:chemistry*; D020733:RNA, Small Cytoplasmic / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods; D000099207:SS-A Antigen; D005465:Fluorocarbon Polymers
D001324:Autoantigens; D010446:Peptide Fragments; D011145:Polyvinyls; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins; C116006:RO60 protein, human; D000099207:SS-A Antigen; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers