Rapid sample processing for LC-MS-based quantitative proteomics using high intensity focused ultrasound.
A new sample processing workflow that uses high intensity focused ultrasound to rapidly reduce and alkylate cysteines, digest proteins and then label peptides with (18)O was developed for quantitative proteomics applications. Each step was individually refined to minimize reaction times, peptide loses and undesired byproducts or modifications. When this novel workflow was used, mouse plasma proteins were successfully denatured, alkylated, in-solution digested, and (18)O-labeled in <10 min for subsequent analysis by liquid chromatography-electrospray ionization high resolution mass spectrometry. Performance was evaluated in terms of the number of mouse plasma peptides and proteins identified in a shotgun approach and the quantitative dynamic range. The results were compared with previously published results obtained using conventional sample preparation methods and were found to be similar. Advantages of the new method include greatly simplified and accelerated sample processing, as well as being readily amenable to automation.
López-Ferrer
Daniel
D
Biological Science Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
Heibeck
Tyler H
TH
Petritis
Konstantinos
K
Hixson
Kim K
KK
Qian
Weijun
W
Monroe
Matthew E
ME
Mayampurath
Anoop
A
Moore
Ronald J
RJ
Belov
Mikhail E
ME
Camp
David G
DG
Smith
Richard D
RD
D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D051379:Mice; D008969:Molecular Sequence Data; D040901:Proteomics; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*