Using guanidine-hydrochloride for fast and efficient protein digestion and single-step affinity-purification mass spectrometry

Jon Wriedt Poulsen, Christian Toft Madsen, Clifford Young, Flemming Martin Poulsen, Michael L Nielsen

Research output: Contribution to journalJournal articleResearchpeer-review

37 Citations (Scopus)

Abstract

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 °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.
Original languageEnglish
JournalJournal of Proteome Research
Volume12
Issue number2
Pages (from-to)1020-30
Number of pages11
ISSN1535-3893
DOIs
Publication statusPublished - 2013

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