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.

蛋白质消化是鸟枪法蛋白质组学（shotgun proteomics）方法不可或缺的组成部分，通常需在质谱分析（mass spectrometry）前进行过夜孵育。对全酵母裂解液开展四次重复鸟枪法蛋白质组学分析后证实，采用内切蛋白酶Lys-C时，盐酸胍（Guanidine-Hydrochloride，Gnd-HCl）介导的蛋白质消化可在30分钟内最优完成。当样品于95℃的盐酸胍体系中孵育时，未引入任何化学伪迹，这使得盐酸胍成为适配鸟枪法蛋白质组学的理想消化缓冲液。当前用于研究蛋白质-蛋白质相互作用（protein–protein interactions, PPIs）的实验方法通常需要多步样品制备步骤，这会延长并行操作与高通量相互作用分析的整体耗时。盐酸胍可实现蛋白质-蛋白质相互作用复合物的高效洗脱与后续快速消化，为亲和纯化质谱（affinity-purification mass spectrometry, AP-MS）实验提供了一种便捷的高通量研究方案。为验证盐酸胍方法的有效性，研究人员对若干GFP标记的酵母去泛素化酶开展了无标记（label-free）蛋白质-蛋白质相互作用分析。对已知相互作用伙伴的成功鉴定，证实了优化后的盐酸胍实验方案具备良好实用性，且该方案可适配96孔板实验体系。