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.

Figure 1

Base peak chromatograms of LCQ LC-MS/MS results of mouse brain tissues prepared using, (A) 7 M urea, 2 M thiourea, 40 mM Tris, 4% CHAPS in 50 mM NH₄HCO₃ buffer, and (B) 5 mM PBS with 50% (v/v) TFE buffer.

Figure 2

(A) Base peak chromatogram of LTQ LC-MS/MS analysis of 5000 MCF-7 human breast cancer cells prepared using the TFE protocol. (B) 2-D plot of identified peptides from 5000 MCF-7 cancer cells using the TFE protocol. Peptides observed in the LC-FTICR analysis were displayed based on their monoisotopic masses and normalized elution times (NET).