Format

Send to

Choose Destination
Anal Chem. 2008 Aug 1;80(15):6093-9. doi: 10.1021/ac702527b. Epub 2008 Jun 26.

Digestion of native proteins for proteomics using a thermocycler.

Author information

1
Department of Biochemistry, Center for Biomedical Research, University of Texas Health Science Center, Tyler, Texas 75708, USA. ot16@leicester.ac.uk

Abstract

Efficient protein digestion is a critical step for successful mass spectrometry analysis. Here we describe simultaneous tryptic digestion and gradual unfolding of native proteins by application of a temperature gradient using a single cycle of 5 min or less in a PCR thermocycler. Chemicals typically used for chromatographic techniques did not affect the digestion efficiency. Tryptic digestion was performed in a small volume (3 microL) with 1.5 microg of trypsin without denaturing agents. This rapid procedure yielded more peptides than conventional methods utilizing chemical denaturation for 18 proteins out of 20. Samples were directly spotted on the MALDI-TOF target plate, without additional purification, thus reducing losses on reversed-phase resins.

PMID:
18578500
PMCID:
PMC2613276
DOI:
10.1021/ac702527b
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

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