Paraffin-wax-coated plates as matrix-assisted laser desorption/ionization sample support for high-throughput identification of proteins by peptide mass fingerprinting☆
Section snippets
Sample preparation of purified proteins
The working solution for both bovine serum albumin and ovalbumin contained 10 pmol/μl of protein prepared from 10-fold dilution of stocks (100 pmol/μl). Bovine serum albumin or ovalbumin (60 pmol in 60 μl) was incubated with 180 μl of trypsin working solution (71 pmol/μl) in 50 mM ammonium bicarbonate buffer. After incubation, each protein solution was equally split and transferred into six siliconized microcentrifuge tubes and vacuum dried for 3–4 h.
Preparation of whole-cell proteins
Mouse C2C12 myoblast cells were bought from American
Results and discussion
We have been using the 2D gels and MALDI-TOF-MS to analyze the proteome of C2C12 cells undergoing myogenic differentiation in vitro [22], [23]. We have also been studying the proteomes of murine and human leukocytes [24], [25]. For these experiments, we routinely excise protein spots from stained 2D gels and digest the protein(s) contained in the gel fragments with trypsin. The trypsin-digested peptides are then extracted in a high-salt buffer prior to MALDI. Since high salt suppresses the MS
Acknowledgements
We gratefully acknowledge the support of Dr. Dominic M. Desiderio and the Charles B. Stout, Neuroscience Mass Spectrometry Laboratory of UTHSC. We also thank Jacquelyn Fountain for her competent and cheerful assistance in the preparation of the manuscript.
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These studies were supported by grants from the National Institute of Health, the Department of Veteran Affairs and the Center of Excellence in the Diseases of Connective Tissue of University of Tennessee Health Science Center. R.R. is a Senior Research Career Scientist of DVA.
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These authors contributed equally.