Ultrasonic-based protein quantitation by18O-labeling: Optimization and comparison between different procedures

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Abstract

Herein we report results regarding the optimization and comparison between different ultrasonic-based procedures for protein quantitation by the direct 18O-labeling approach. The labeling procedure was evaluated using different proteins, different ultrasonic devices and different reaction times: from 30 s to 10 min with the ultrasonic probe and from 30 s to 30 min with the sonoreactor. Variables such as the enzyme-to-protein ratio and protein concentration were also assessed. The results show that it is possible to accelerate the labeling reaction from 12 h to only 15 min with the sonoreactor without compromising the labeling efficiency. A larger variation in the double labeling yield was obtained among the different peptides, but the values for the smaller peptides are similar to the ones achieved with the classic methodology. These findings were further confirmed by labeling a complex protein mixture from human plasma. It was also found that the labeling reaction is affected by the sample concentration, even when performed with the classic overnight procedure.

Original languageEnglish
Pages (from-to)75-87
Number of pages13
JournalRapid Communications in Mass Spectrometry
Volume25
Issue number1
DOIs
Publication statusPublished - 15 Jan 2011

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Labeling
Ultrasonics
Proteins
Ultrasonic devices
Plasma (human)
Peptides
Enzymes

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title = "Ultrasonic-based protein quantitation by18O-labeling: Optimization and comparison between different procedures",
abstract = "Herein we report results regarding the optimization and comparison between different ultrasonic-based procedures for protein quantitation by the direct 18O-labeling approach. The labeling procedure was evaluated using different proteins, different ultrasonic devices and different reaction times: from 30 s to 10 min with the ultrasonic probe and from 30 s to 30 min with the sonoreactor. Variables such as the enzyme-to-protein ratio and protein concentration were also assessed. The results show that it is possible to accelerate the labeling reaction from 12 h to only 15 min with the sonoreactor without compromising the labeling efficiency. A larger variation in the double labeling yield was obtained among the different peptides, but the values for the smaller peptides are similar to the ones achieved with the classic methodology. These findings were further confirmed by labeling a complex protein mixture from human plasma. It was also found that the labeling reaction is affected by the sample concentration, even when performed with the classic overnight procedure.",
author = "Carreira, {R. J.} and Diniz, {M. S.} and Capelo, {J. L.}",
note = "Ricardo J. Carreira acknowledges the doctoral grant SRFH/BD/28563/2006 from FCT (Science and Technological Foundation) of Portugal. The University of Vigo is acknowledged for financial support under projects INOU/UVIGO/K919/2009 and INOU/UVIGO/K914/2009. Xunta de Galicia, Spain, is acknowledged by the Isidro Parga Pondal Program (J. L. Capelo) and for financial support given under project 09CSA043383PR. Mario S. Diniz acknowledges program Ciencia 2008 from FCT/MCTES.",
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AU - Capelo, J. L.

N1 - Ricardo J. Carreira acknowledges the doctoral grant SRFH/BD/28563/2006 from FCT (Science and Technological Foundation) of Portugal. The University of Vigo is acknowledged for financial support under projects INOU/UVIGO/K919/2009 and INOU/UVIGO/K914/2009. Xunta de Galicia, Spain, is acknowledged by the Isidro Parga Pondal Program (J. L. Capelo) and for financial support given under project 09CSA043383PR. Mario S. Diniz acknowledges program Ciencia 2008 from FCT/MCTES.

PY - 2011/1/15

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N2 - Herein we report results regarding the optimization and comparison between different ultrasonic-based procedures for protein quantitation by the direct 18O-labeling approach. The labeling procedure was evaluated using different proteins, different ultrasonic devices and different reaction times: from 30 s to 10 min with the ultrasonic probe and from 30 s to 30 min with the sonoreactor. Variables such as the enzyme-to-protein ratio and protein concentration were also assessed. The results show that it is possible to accelerate the labeling reaction from 12 h to only 15 min with the sonoreactor without compromising the labeling efficiency. A larger variation in the double labeling yield was obtained among the different peptides, but the values for the smaller peptides are similar to the ones achieved with the classic methodology. These findings were further confirmed by labeling a complex protein mixture from human plasma. It was also found that the labeling reaction is affected by the sample concentration, even when performed with the classic overnight procedure.

AB - Herein we report results regarding the optimization and comparison between different ultrasonic-based procedures for protein quantitation by the direct 18O-labeling approach. The labeling procedure was evaluated using different proteins, different ultrasonic devices and different reaction times: from 30 s to 10 min with the ultrasonic probe and from 30 s to 30 min with the sonoreactor. Variables such as the enzyme-to-protein ratio and protein concentration were also assessed. The results show that it is possible to accelerate the labeling reaction from 12 h to only 15 min with the sonoreactor without compromising the labeling efficiency. A larger variation in the double labeling yield was obtained among the different peptides, but the values for the smaller peptides are similar to the ones achieved with the classic methodology. These findings were further confirmed by labeling a complex protein mixture from human plasma. It was also found that the labeling reaction is affected by the sample concentration, even when performed with the classic overnight procedure.

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