Elsevier

Analytical Biochemistry

Volume 327, Issue 2, 15 April 2004, Pages 222-232
Analytical Biochemistry

Paraffin-wax-coated plates as matrix-assisted laser desorption/ionization sample support for high-throughput identification of proteins by peptide mass fingerprinting

https://doi.org/10.1016/j.ab.2004.01.033Get rights and content

Abstract

We compared trysin-digested protein samples desalted by ZipTipC18 reverse-phase microcolumns with on-plate washing of peptides deposited either on paraffin-coated plates (PCP), Teflon-based AnchorChip plates, or stainless steel plates, before analysis by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Trypsinized bovine serum albumin and ovalbumin and 16 protein spots extracted from silver-stained two-dimensional gels of murine C2C12 myoblasts or human leukocytes, prepared by the above two methods, were subjected to MALDI on PCP, AnchorChip plates, or uncoated stainless steel plates. Although most peptide mass peaks were identical regardless of the method of desalting and concentrating of protein samples, samples washed and concentrated by the PCP-based method had peptide peaks that were not seen in the samples prepared using the ZipTipC18 columns. The mass spectra of peptides desalted and washed on uncoated stainless steel MALDI plates were consistently inferior due to loss of peptides. Some peptides of large molecular masses were apparently lost from samples desalted by ZipTipC18 microcolumns, thus diminishing the quality of the fingerprint needed for protein identification. We demonstrate that the method of washing of protein samples on paraffin-coated plates provides an easy, reproducible, inexpensive, and high-throughput alternative to ZipTipC18-based purification of protein prior to MALDI-TOF-MS analysis.

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.

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