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The effect of pressure cycling on proteolytic cleavage efficiency, reaction time and protein sequence coverage
Author(s) -
Biringer Roger,
Hühmer Andreas F. R.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.670.1
Subject(s) - proteolysis , proteases , chemistry , cleavage (geology) , chromatography , biochemistry , sequence (biology) , biophysics , enzyme , biology , paleontology , fracture (geology)
Over the years many methodologies have been advanced to aid in the enhancement of proteolysis cleavage efficiency, reaction time, and protein sequence coverage for LC‐MS/MS based analysis. The series of experiments presented here examine the effect pressure cycling on each of these factors. Several individual proteins and a simple protein mixture were digested with different proteases under high pressure using a Barocycler NEP2320 pressure cycler and compared to overnight at 1 atm and 37 °C proteolysis conditions. The resulting peptides were identified and quantified using an LC‐MS/MS workflow with a LTQ XL with ETD. The results indicate that digestions using pressure cycling produce more distinct peptides and equivalent or higher protein sequence coverage when compared to standard overnight digestions.