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.

蛋白质酶解是鸟枪法蛋白质组学（shotgun proteomics）不可或缺的组成环节，通常需在质谱分析前进行过夜孵育。对全酵母裂解物进行四次重复鸟枪法蛋白质组学分析后发现，使用内切蛋白酶Lys-C时，盐酸胍（Guanidine-Hydrochloride，Gnd-HCl）介导的蛋白质酶解可在30分钟内最优完成。当样品在95℃下于Gnd-HCl体系中孵育时，未引入任何化学副产物，因此Gnd-HCl是适用于鸟枪法蛋白质组学的理想酶解缓冲液。当前用于研究蛋白质相互作用（protein–protein interactions，PPIs）的方法通常需多步样品制备，这会延长并行操作与高通量相互作用分析的周期。而Gnd-HCl可实现蛋白质相互作用复合物的高效洗脱与后续快速酶解，为亲和纯化质谱（affinity-purification mass spectrometry，AP-MS）实验提供了便捷的高通量研究方案。为验证该Gnd-HCl方法，研究人员对数个GFP标签酵母去泛素化酶开展了无标记蛋白质相互作用分析。对已知相互作用伴侣的成功鉴定，证明了该优化后的Gnd-HCl实验方案的实用性，且该方案可适配96孔板体系。