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Development and Evaluation of a Micro- and Nanoscale Proteomic Sample Preparation Method

2005; American Chemical Society; Volume: 4; Issue: 6 Linguagem: Inglês

10.1021/pr050160f

1535-3907

Hai Wang, Weijun Qian, Heather M. Mottaz, Therese RW Clauss, David J. Anderson, Ronald J. Moore, David Camp, Arshad H. Khan, Daniel M. Sforza, Maria G. Pallavicini, Desmond Smith, Richard Smith,

Biosensors and Analytical Detection

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 μg 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 (∼30 μg total protein content) and also for samples of ∼5000 MCF-7 human breast cancer cells (∼500 ng total protein content), where the detergent-based methods were ineffective due to losses during cleanup and transfer steps. Keywords: micro- and nanoscale protein extraction • sample preparation • organic cosolvent • LC−MS/MS • proteomics