Chapter title |
Purification of Tryptic Digests on Polyvinylidene Difluoride Membrane
|
---|---|
Chapter number | 28 |
Book title |
Detection of Blotted Proteins
|
Published in |
Methods in molecular biology, January 2015
|
DOI | 10.1007/978-1-4939-2718-0_28 |
Pubmed ID | |
Book ISBNs |
978-1-4939-2717-3, 978-1-4939-2718-0
|
Authors |
Biji T. Kurien, R. Hal Scofield, Kurien, Biji T., Scofield, R. Hal |
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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