title: Development and evaluation of a micro- and nanoscale proteomic sample preparation method.
abstract: 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.
authors.affiliation: Biological Sciences Division, Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
authors.forename: Haixing
authors.initials: H
authors.lastname: Wang
authors.forename: Wei-Jun
authors.initials: WJ
authors.lastname: Qian
authors.forename: Heather M
authors.initials: HM
authors.lastname: Mottaz
authors.forename: Therese R W
authors.initials: TR
authors.lastname: Clauss
authors.forename: David J
authors.initials: DJ
authors.lastname: Anderson
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Moore
authors.forename: David G
authors.initials: DG
authors.lastname: Camp
authors.forename: Arshad H
authors.initials: AH
authors.lastname: Khan
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Sforza
authors.forename: Maria
authors.initials: M
authors.lastname: Pallavicini
authors.forename: Desmond J
authors.initials: DJ
authors.lastname: Smith
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
mesh_terms: 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
chemical_list: D003902:Detergents; D010455:Peptides; D011506:Proteins; D020543:Proteome; D012997:Solvents; D014270:Trifluoroethanol