Using Guanidine-Hydrochloride for Fast and Efficient Protein Digestion and Single-step Affinity-purification Mass Spectrometry

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• 作者：Jon W. Poulsen ; Christian T. Madsen ; Clifford Young ; Flemming M. Poulsen ; Michael L. Nielsen
• 刊名：Journal of Proteome Research
• 出版年：2013
• 出版时间：February 1, 2013
• 年：2013
• 卷：12
• 期：2
• 页码：1020-1030
• 全文大小：589K
• 年卷期：v.12,no.2(February 1, 2013)
• ISSN：1535-3907

文摘

Protein digestion is an integral part of the 鈥渟hotgun鈥?proteomics approach and commonly requires overnight incubation prior to mass spectrometry analysis. Quadruplicate 鈥渟hotgun鈥?proteomic analysis of whole yeast lysate demonstrated that Guanidine-Hydrochloride (Gnd-HCl) protein digestion can be optimally completed within 30 min with endoprotease Lys-C. No chemical artifacts were introduced when samples were incubated in Gnd-HCl at 95 掳C, making Gnd-HCl an appropriate digestion buffer for shotgun proteomics. Current methodologies for investigating protein鈥損rotein interactions (PPIs) often require several preparation steps, which prolongs any parallel operation and high-throughput interaction analysis. Gnd-HCl allow the efficient elution and subsequent fast digestion of PPIs to provide a convenient high-throughput methodology for affinity-purification mass spectrometry (AP-MS) experiments. To validate the Gnd-HCl approach, label-free PPI analysis of several GFP-tagged yeast deubiquitinating enzymes was performed. The identification of known interaction partners demonstrates the utility of the optimized Gnd-HCl protocol that is also scalable to the 96 well-plate format.

Keywords:

protein digestion; shotgun proteomics; guanidine hydrochloride; urea; endoproteinase Lys-C; protein鈭抪rotein interactions; deubiquitinating enzymes; Saccharomyces cerevisiae