Influence of the protein staining in the fast ultrasonic sample treatment for protein identification through peptide mass fingerprint and matrix-assisted laser desorption ionization time of flight mass spectrometry

M. Galesio, Diana V. Vieira, R. Rial-Otero, C. Lodeiro, I. Moura, J. L. Capelo

Research output: Contribution to journalArticlepeer-review

21 Citations (Scopus)

Abstract

The influence of the protein staining used to visualize protein bands, after in-gel protein separation, for the correct identification of proteins by peptide mass fingerprint (PMF) after application of the ultrasonic in-gel protein protocol was studied. Coomassie brilliant blue and silver nitrate, both visible stains, and the fluorescent dyes Sypro Red and Sypro Orange were evaluated. Results obtained after comparison with the overnight in-gel protocol showed that good results, in terms of protein sequence coverage and number of peptides matched, can be obtained with anyone of the four stains studied. Two minutes of enzymatic digestion time was enough for proteins stained with coomassie blue, while 4 min was necessary when silver or Sypro stainings were employed in order to reach equivalent results to those obtained for the overnigh in-gel protein protocol. For the silver nitrate stain, the concentration of silver present in the staining solution must be 0.09% (w/v) to minimize background in the MALDI mass spectra.

Original languageEnglish
Pages (from-to)2097-2106
Number of pages10
JournalJournal Of Proteome Research
Volume7
Issue number5
DOIs
Publication statusPublished - 1 May 2008

Keywords

  • Coomassie blue
  • MALDI-TOF-MS
  • Silver nitrate
  • Sypro orange
  • Sypro red
  • Ultrasonic in-gel protein digestion

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