A clean, more efficient method for in-solution digestion of protein mixtures without detergent or urea.

A clean, more efficient method for in-solution digestion of protein mixtures without detergent or urea. Research Abstract Details 

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A clean, more efficient method for in-solution digestion of protein mixtures without detergent or urea. Abstract Text:

Sung Chan Kim,Yue Chen,Shama Mirza,Yingda Xu,Jaeick Lee,Pingsheng Liu,Yingming Zhao,

Proteolytic digestion of a complicated protein mixture from an organelle or whole-cell lysate is usually carried out in a dilute solution of a denaturing buffer, such as 1-2 M urea. Urea must be subsequently removed by C18 beads before downstream analysis such as HPLC/MS/MS or complete methylation followed by IMAC isolation of phosphopeptides. Here we describe a procedure for digesting a complicated protein mixture in the absence of denaturants. Proteins in the mixture are precipitated with trichloroacetic acid/acetone for denaturation and salt removal and resuspended in NH4HCO3 buffer. After trypsinolysis, the resulting peptides are not contaminated by urea or other nonvolatile salts and can be dried in a SpeedVac to remove NH4HCO3. When this protocol was applied to an extract of A431 cells, 96.8% of the tryptic peptides were completely digested (i.e., had no missed cleavage sites), in contrast to 87.3% of those produced by digestion in urea buffer. We successfully applied this digestion method to analysis of the phosphoproteome of adiposomes from HeLa cells, identifying 33 phosphorylation sites in 28 different proteins. Our digestion method avoids the need to remove urea before HPLC/MS/MS analysis or methylation and IMAC, increasing throughput while reducing sample loss and contamination from sample handling. We believe that this method should be valuable for proteomics studies.

A clean, more efficient method for in-solution digestion of protein mixtures without detergent or urea. Publishing Authors By Initials

SC Kim,Y Chen,S Mirza,Y Xu,J Lee,P Liu,Y Zhao,

For similar enzymes and coenzymes: enzymes: hydrolases: peptide hydrolases: endopeptidases: serine endopeptidases: trypsin research abstracts see: enzymes and coenzymes: enzymes: hydrolases: peptide hydrolases: endopeptidases: serine endopeptidases: trypsin research

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A clean, more efficient method for in-solution digestion of protein mixtures without detergent or urea. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Journal of proteome research

VOLUME: 5

Page Numbers: 3446-52

Journal Abbreviation: J. Proteome Res.

ISSN: 1535-3893

DAY: 3

MONTH: Dec

YEAR: 2006

A clean, more efficient method for in-solution digestion of protein mixtures without detergent or urea. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 101128775

A clean, more efficient method for in-solution digestion of protein mixtures without detergent or urea. Keywords Mesh Terms:

KEYWORDS: Trypsin

MESH TERMS: methods

Chemical & Substance for Abstract: A clean, more efficient method for in-solution digestion of protein mixtures without detergent or urea. Information

Substance Name: Trypsin

Registry Number: EC 3.4.21.4

Grant and Affiliation Information for A clean, more efficient method for in-solution digestion of protein mixtures without detergent or urea.

AFFILIATION: Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9038, USA.

Country: United States

AGENCY: United States NCI

GRANT: CA 107943

ACRONYM: CA

MEDLINETA: J Proteome Res

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