Format

Send to

Choose Destination
J Proteome Res. 2014 Apr 4;13(4):1885-95. doi: 10.1021/pr4010019. Epub 2014 Mar 6.

Enhanced FASP (eFASP) to increase proteome coverage and sample recovery for quantitative proteomic experiments.

Author information

1
Department of Chemistry and Biochemistry and ‡Department of Biological Chemistry, University of California-Los Angeles , Los Angeles, California 90095, United States.

Abstract

The integrity of quantitative proteomic experiments depends on the reliability and the robustness of the protein extraction, solubilization, and digestion methods utilized. Combinations of detergents, chaotropes, and mechanical disruption can yield successful protein preparations; however, the methods subsequently required to eliminate these added contaminants, in addition to the salts, nucleic acids, and lipids already in the sample, can result in significant sample losses and incomplete contaminant removal. A recently introduced method for proteomic sample preparation, filter-aided sample preparation (FASP), cleverly circumvents many of the challenges associated with traditional protein purification methods but is associated with significant sample loss. Presented here is an enhanced FASP (eFASP) approach that incorporates alternative reagents to those of traditional FASP, improving sensitivity, recovery, and proteomic coverage for processed samples. The substitution of 0.2% deoxycholic acid for urea during eFASP digestion increases tryptic digestion efficiency for both cytosolic and membrane proteins yet obviates needed cleanup steps associated with use of the deoxycholate sodium salt. For classic FASP, prepassivating Microcon filter surfaces with 5% TWEEN-20 reduces peptide loss by 300%. An express eFASP method uses tris(2-carboxyethyl)phosphine and 4-vinylpyridine to alkylate proteins prior to deposition on the Microcon filter, increasing alkylation specificity and speeding processing.

PMID:
24552128
PMCID:
PMC3993969
DOI:
10.1021/pr4010019
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for American Chemical Society Icon for PubMed Central
Loading ...
Support Center