PNNL

Abstract

Efficient and effective sample preparation of micro- and nano-scale (micro- and nano-gram) clinical specimens for proteomic applications is often difficult due to losses during the processing steps. Herein we describe a simple “single-tube” preparation protocol appropriate for small proteomic samples using the organic co-solvent, trifluoroethanol (TFE). TFE facilitates both protein extraction and protein denaturation without requiring a separate cleanup step, thus minimizing sample loss. The performance of the TFE method was initially evaluated by comparing 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 protocol provided comparable results to the traditional detergent-based protocols for larger samples (milligrams), based on both sample recovery and peptide/protein identification. The effectiveness of this protocol for micro- and nano-scale sample processing was then evaluated for the extraction of proteins/peptides and shown effective for small mouse brain tissue samples (~ 20 ?g total protein content) and also for samples of ~ 5 000 human breast cancer MCF-7 cells (~ 500 ng total protein content), where the detergent-based methods were ineffective due to losses during cleanup and transfer steps.