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.
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Acknowledgement
This work was supported by NIH grant ARO1844 and Oklahoma Center for the Advancement of Science and Technology to RHS. We also express our gratitude to Drs. Hiroyuki Matsumoto and Sadamu Kurono (Department of Biochemistry and Molecular Biology, Oklahoma City, OK, USA) for their help with mass spectrometry and analysis.
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Kurien, B.T., Scofield, R.H. (2015). Purification of Tryptic Digests on Polyvinylidene Difluoride Membrane. In: Kurien, B., Scofield, R. (eds) Detection of Blotted Proteins. Methods in Molecular Biology, vol 1314. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2718-0_28
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DOI: https://doi.org/10.1007/978-1-4939-2718-0_28
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