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

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.