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

Nilesh S. Tannu, Jian Wu, Vamshi K. Rao, Himanshu S. Gadgil, Michael J. Pabst, Ivan Gerling, Rajendra Raghow

Research output: Contribution to journalArticle

56 Citations (Scopus)

Abstract

We compared trysin-digested protein samples desalted by ZipTip C18 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.

Original languageEnglish (US)
Pages (from-to)222-232
Number of pages11
JournalAnalytical Biochemistry
Volume327
Issue number2
DOIs
StatePublished - Apr 15 2004

Fingerprint

Paraffin waxes
Peptide Mapping
Waxes
Paraffin
Ionization
Desorption
Lasers
Throughput
Peptides
Stainless Steel
Proteins
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Washing
Mass spectrometry
Mass Spectrometry
Salt removal
Myoblasts
Ovalbumin
Polytetrafluoroethylene
Molecular mass

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Paraffin-wax-coated plates as matrix-assisted laser desorption/ionization sample support for high-throughput identification of proteins by peptide mass fingerprinting. / Tannu, Nilesh S.; Wu, Jian; Rao, Vamshi K.; Gadgil, Himanshu S.; Pabst, Michael J.; Gerling, Ivan; Raghow, Rajendra.

In: Analytical Biochemistry, Vol. 327, No. 2, 15.04.2004, p. 222-232.

Research output: Contribution to journalArticle

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