title: An assessment of the ultrasonic probe-based enhancement of protein cleavage with immobilized trypsin.
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, 12 h) 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.
authors.affiliation: REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, Monte da Caparica, Portugal.
authors.forename: Gonçalo
authors.initials: G
authors.lastname: Vale
authors.forename: Hugo M
authors.initials: HM
authors.lastname: Santos
authors.forename: Ricardo J
authors.initials: RJ
authors.lastname: Carreira
authors.forename: Luis
authors.initials: L
authors.lastname: Fonseca
authors.forename: Manuel
authors.initials: M
authors.lastname: Miró
authors.forename: Víctor
authors.initials: V
authors.lastname: Cerdà
authors.forename: Miguel
authors.initials: M
authors.lastname: Reboiro-Jato
authors.forename: José L
authors.initials: JL
authors.lastname: Capelo
mesh_terms: D000818:Animals; D001798:Blood Proteins / Q000032:analysis / Q000378:metabolism; D002417:Cattle; D004800:Enzymes, Immobilized / Q000378:metabolism*; D004867:Equipment Design; D006801:Humans; D007768:Lactalbumin / Q000032:analysis / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014357:Trypsin / Q000378:metabolism*; D014465:Ultrasonics / Q000295:instrumentation*
chemical_list: D001798:Blood Proteins; D004800:Enzymes, Immobilized; D010446:Peptide Fragments; D011506:Proteins; D012710:Serum Albumin, Bovine; D007768:Lactalbumin; D014357:Trypsin