TY - JOUR
T1 - An assessment of the ultrasonic probe-based enhancement of protein cleavage with immobilized trypsin
AU - Vale, Gonçalo
AU - Santos, Hugo M.
AU - Carreira, Ricardo J.
AU - Fonseca, Luis
AU - Miró, Manuel
AU - Cerdà, Víctor
AU - Reboiro-Jato, Miguel
AU - Capelo, José L.
PY - 2011/10
Y1 - 2011/10
N2 - 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.
AB - 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.
KW - Human plasma
KW - Immobilized trypsin
KW - Protein cleavage
KW - Technology
KW - Ultrasonic Probe
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U2 - 10.1002/pmic.201100200
DO - 10.1002/pmic.201100200
M3 - Article
C2 - 21805637
AN - SCOPUS:80052973869
SN - 1615-9853
VL - 11
SP - 3866
EP - 3876
JO - Proteomics
JF - Proteomics
IS - 19
ER -