An assessment of the ultrasonic probe-based enhancement of protein cleavage with immobilized trypsin

Gonçalo Vale, H. M. Santos, Ricardo J. Carreira, Manuel Miró, Victor Cerdà, Miguel Reboiro-Jato, J. L. Capelo

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

The use of ultrasonic probe, in conjunction with immobilized trypsin, has been explored in this work for potential enhancement of protein digestion. Several solid supports commonly used to immobilize trypsin were subjected to different ultrasonication amplitudes and time in order to investigate their mechanical resistance to ultrasonic energy when provided by the ultrasonic probe. Glass beads and magnetic particles were found to remain intact in most conditions studied. It was found that immobilized trypsin cannot be reused after ultrasonication since the enzymatic activity was greatly diminished. For comparative purposes, vortex shaking was also explored for protein cleavage. Four standard proteins - bovine serum albumin, α-lactalbumin, carbonic anhydrase and ovalbumin - were successfully identified using peptide mass fingerprint, or peptide fragment fingerprint. In addition, the performance of the classical protein cleavage (overnight, 12h) and the ultrasonic methods was found to be similar when the digestion of a complex proteome, human plasma, was assessed through 18-O quantification. The digestion yields found were 90-117% for the ultrasonic and 5-21% for the vortex when those methods were compared with the classical overnight digestion. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Original languageEnglish
Pages (from-to)3866-3876
Number of pages11
JournalProteomics
Volume11
Issue number19
DOIs
Publication statusPublished - 2011

Keywords

  • Human plasma
  • Immobilized trypsin
  • Protein cleavage
  • Technology
  • Ultrasonic Probe
  • alpha lactalbumin
  • carbonate dehydratase
  • ovalbumin
  • peptide fragment
  • proteome
  • serum albumin
  • trypsin
  • alkylation
  • article
  • comparative study
  • enzyme activity
  • finger dermatoglyphics
  • human
  • plasma
  • priority journal
  • protein cleavage
  • protein degradation
  • reduction
  • temperature
  • ultrasound
  • vortex motion
  • Animals
  • Blood Proteins
  • Cattle
  • Enzymes, Immobilized
  • Equipment Design
  • Humans
  • Lactalbumin
  • Peptide Fragments
  • Proteins
  • Serum Albumin, Bovine
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Trypsin
  • Ultrasonics
  • Bovinae

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