期刊
JOURNAL OF PROTEOME RESEARCH
卷 12, 期 2, 页码 1020-1030出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr300883y
关键词
protein digestion; shotgun proteomics; guanidine hydrochloride; urea; endoproteinase Lys-C; protein-protein interactions; deubiquitinating enzymes; Saccharomyces cerevisiae
资金
- Novo Nordisk Foundation Center for Protein Research
- Lundbeck Foundation
- European Union [262067, HEALTH-F4-2010-241504]
Protein digestion is an integral part of the shotgun proteomics approach and commonly requires overnight incubation prior to mass spectrometry analysis. Quadruplicate shotgun 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 degrees C, making Gnd-HCl an appropriate digestion buffer for shotgun proteomics. Current methodologies for investigating protein protein 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.
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