An assessment of the indirect high intensity ultrasonic assisted cleavage of complex proteomes with immobilized trypsin

The indirect high intensity ultrasonic assisted cleavage of complex proteomes using immobilized trypsin has been assessed. It has been found that the formation of aggregates between the beds supporting the immobilized trypsin is promoted. This affects the efficiency of the digestion process, which c...

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Bibliographic Details
Published in:Talanta (Oxford) 2013-03, Vol.106, p.163-168
Main Authors: Fernández-Costa, C., Ruiz-Romero, C., Blanco, F.J., Santos, H.M., Capelo, J.L.
Format: Article
Language:English
Subjects:
Biomarker discovery
Case-Control Studies
Cup-horn
Enzymes, Immobilized - chemistry
Humans
Immobilized trypsin
Isotope Labeling
Osteoarthritis - blood
Oxygen Isotopes
Peptide Fragments - blood
Probe
Protein cleavage
Proteolysis - radiation effects
Proteome - analysis
Sonication - methods
Sound
Trypsin - chemistry
Ultrasonic
Ultrasound
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Summary:The indirect high intensity ultrasonic assisted cleavage of complex proteomes using immobilized trypsin has been assessed. It has been found that the formation of aggregates between the beds supporting the immobilized trypsin is promoted. This affects the efficiency of the digestion process, which can be 100 times lower than the digestion efficiency obtained with in-solution trypsin. Through the use of isotopic peptide labelling with 18-O, it has been quantified that the digestion efficiency over serum samples can be 1.2–100 times higher for the 70% of the peptides when indirect ultrasonication is replaced by direct ultrasonication. Therefore, direct high intensity ultrasonic assisted cleavage of proteins is proposed as an alternative to be combined with immobilized trypsin. ► Sera samples were digested with indirect or direct high intensity ultrasonic energy. ► Sera cleavage was done with immobilized trypsin and with in-solution trypsin. ► 18-O water was used to compare the performance of the methods of cleavage assessed.
ISSN:0039-9140
1873-3573
DOI:10.1016/j.talanta.2012.12.022