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" preparatio...

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Bibliographic Details
Published in:Journal of proteome research Vol. 4; no. 6; p. 2397
Main Authors: Wang, Haixing, Qian, Wei-Jun, Mottaz, Heather M, Clauss, Therese R W, Anderson, David J, Moore, Ronald J, Camp, 2nd, David G, Khan, Arshad H, Sforza, Daniel M, Pallavicini, Maria, Smith, Desmond J, Smith, Richard D
Format: Journal Article
Language:English
Published: United States 01.11.2005
Subjects:
Animals
Brain - metabolism
Breast Neoplasms - metabolism
Cell Line, Tumor
Detergents - pharmacology
Humans
Male
Mass Spectrometry
Mice
Nanotechnology - methods
Peptides - chemistry
Proteins - chemistry
Proteome
Proteomics - instrumentation
Proteomics - methods
Solvents
Time Factors
Trifluoroethanol - chemistry
ISSN:1535-3893
Online Access:Get more information
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Summary: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.
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ISSN:1535-3893
DOI:10.1021/pr050160f