A Clean, More Efficient Method for In-Solution Digestion of Protein Mixtures without Detergent or Urea

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• 作者：Sung Chan Kim ; Yue Chen ; Shama Mirza ; Yingda Xu ; Jaeick Lee ; Pingsheng Liu ; Yingming Zhao
• 刊名：Journal of Proteome Research
• 出版年：2006
• 出版时间：December 2006
• 年：2006
• 卷：5
• 期：12
• 页码：3446 - 3452
• 全文大小：256K
• 年卷期：v.5,no.12(December 2006)
• ISSN：1535-3907

文摘

Proteolytic digestion of a complicated proteinmixture from an organelle or whole-cell lysate is usuallycarried out in a dilute solution of a denaturing buffer, suchas 1-2 M urea. Urea must be subsequently removed byC18 beads before downstream analysis such as HPLC/MS/MS or complete methylation followed by IMAC isolationof phosphopeptides. Here we describe a procedure fordigesting a complicated protein mixture in the absenceof denaturants. Proteins in the mixture are precipitatedwith trichloroacetic acid/acetone for denaturation and saltremoval and resuspended in NH₄HCO₃ buffer. After trypsinolysis, the resulting peptides are not contaminated byurea or other nonvolatile salts and can be dried in aSpeedVac to remove NH₄HCO₃. When this protocol wasapplied to an extract of A431 cells, 96.8% of the trypticpeptides were completely digested (i.e., had no missedcleavage sites), in contrast to 87.3% of those producedby digestion in urea buffer. We successfully applied thisdigestion method to analysis of the phosphoproteome ofadiposomes from HeLa cells, identifying 33 phosphoryl-ation sites in 28 different proteins. Our digestion methodavoids the need to remove urea before HPLC/MS/MSanalysis or methylation and IMAC, increasing throughputwhile reducing sample loss and contamination fromsample handling. We believe that this method should bevaluable for proteomics studies.