Application of pressurized solvents for ultrafast trypsin hydrolysis in proteomics: proteomics on the fly.
A new method for rapid proteolytic digestion of proteins under high pressure that uses pressure cycling technology in the range of 5-35 kpsi was demonstrated for proteomic analysis. Successful in-solution digestions of single proteins and complex protein mixtures were achieved in 60 s and then analyzed by reversed phase liquid chromatography-electrospray ionization ion trap-mass spectrometry. Method performance in terms of the number of Shewanella oneidensis peptides and proteins identified in a shotgun approach was evaluated relative to a traditional "overnight" sample preparation method. Advantages of the new method include greatly simplified sample processing, easy implementation, no cross contamination among samples, and cost effectiveness.
López-Ferrer
Daniel
D
Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
Petritis
Konstantinos
K
Hixson
Kim K
KK
Heibeck
Tyler H
TH
Moore
Ronald J
RJ
Belov
Mikhail E
ME
Camp
David G
DG
Smith
Richard D
RD
D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis; D002021:Buffers; D002853:Chromatography, Liquid; D006454:Hemoglobins / Q000032:analysis; D006736:Horses; D006868:Hydrolysis; D011312:Pressure; D017510:Protein Folding; D040901:Proteomics / Q000379:methods*; D020592:Shewanella / Q000737:chemistry; D012997:Solvents; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry*
D001426:Bacterial Proteins; D002021:Buffers; D006454:Hemoglobins; D012997:Solvents; D014357:Trypsin