Development and evaluation of a micro- and nanoscale proteomic sample preparation method.
Challenges associated with the efficient and effective preparation of micro- and nanoscale (micro- and nanogram) clinical specimens for proteomic applications include the unmitigated sample losses that occur during the processing steps. Herein, we describe a simple "single-tube" preparation protocol appropriate for small proteomic samples using the organic cosolvent, trifluoroethanol (TFE) that circumvents the loss of sample by facilitating both protein extraction and protein denaturation without requiring a separate cleanup step. The performance of the TFE-based method was initially evaluated by comparisons to traditional detergent-based methods on relatively large scale sample processing using human breast cancer cells and mouse brain tissue. The results demonstrated that the TFE-based protocol provided comparable results to the traditional detergent-based protocols for larger, conventionally sized proteomic samples (>100 microg protein content), based on both sample recovery and numbers of peptide/protein identifications. The effectiveness of this protocol for micro- and nanoscale sample processing was then evaluated for the extraction of proteins/peptides and shown effective for small mouse brain tissue samples (approximately 30 microg total protein content) and also for samples of approximately 5000 MCF-7 human breast cancer cells (approximately 500 ng total protein content), where the detergent-based methods were ineffective due to losses during cleanup and transfer steps.
Wang
Haixing
H
Biological Sciences Division, Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
Qian
Wei-Jun
WJ
Mottaz
Heather M
HM
Clauss
Therese R W
TR
Anderson
David J
DJ
Moore
Ronald J
RJ
Camp
David G
DG
Khan
Arshad H
AH
Sforza
Daniel M
DM
Pallavicini
Maria
M
Smith
Desmond J
DJ
Smith
Richard D
RD
D000818:Animals; D001921:Brain / Q000378:metabolism; D001943:Breast Neoplasms / Q000378:metabolism; D045744:Cell Line, Tumor; D003902:Detergents / Q000494:pharmacology; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D036103:Nanotechnology / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D020543:Proteome; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods*; D012997:Solvents; D013997:Time Factors; D014270:Trifluoroethanol / Q000737:chemistry
D003902:Detergents; D010455:Peptides; D011506:Proteins; D020543:Proteome; D012997:Solvents; D014270:Trifluoroethanol