Protein identification by in-gel digestion, high-performance liquid chromatography, and mass spectrometry: peptide analysis by complementary ionization techniques

Research output: Contribution to journalArticle

Abstract

A biologically active protein fraction was isolated from rabbit intestine, purified by one-dimensional SDS-PAGE and stained with Coomassie Brilliant Blue. A predominant band of approximately 110-130 kDa was excised and digested in-gel with trypsin. The resulting peptides were extracted then separated by microbore reversed-phase high-performance liquid chromatography (HPLC). Mass spectrometric data from one HPLC fraction obtained by two different ionization techniques proved to be complementary. Matrix-assisted laser desorption/ionization (MALDI) showed nine peptide masses, which by post source decay analysis and database searching were attributed to two proteins. Nanoflow electrospray analysis performed on a hybrid tandem mass spectrometer of quadrupole-quadrupole-orthogonal acceleration time-of-flight (QqTOF) geometry detected six additional peptide components. On the basis of the additional peptides and superior quality collision-induced dissociation spectra typical of this instrument type, two further proteins were identified. The resolution afforded by the QqTOF instrument permitted charge state determination for the fragment ions while preserving the high detection sensitivity that was essential in obtaining the composition of this mixture of proteins.

Details

Authors
  • Katalin F. Medzihradszky
  • Hakon Leffler
  • Michael A. Baldwin
  • A L Burlingame
External organisations
  • External Organization - Unknown
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Microbiology in the medical area
  • Immunology in the medical area
Original languageEnglish
Pages (from-to)215-221
JournalJournal of the American Society for Mass Spectrometry
Volume12
Issue number2
Publication statusPublished - 2001
Publication categoryResearch
Peer-reviewedYes
Externally publishedYes