While many proteases are used in bottom-up mass spectrometric (MS) analysis, trypsin (4,5) is the de facto protease of choice for most applications. There are several reasons for this: Trypsin is highly efficient, active, and specific. Tryptic peptides produced after proteolysis are ideally suited, in terms of both size (350–1,600 Daltons) and charge (+2 to +4), for MS analysis. One significant drawback to trypsin digestion is the long sample preparation times, which typically range from 4 hours to overnight for most protocols. Achieving efficient digestion usually requires that protein substrates first be unfolded either with surfactants or denaturants such as urea or guanidine. These chemical additives can have negative effects, including protein modification, inhibition of trypsin or incompatibility with downstream LC-MS/MS. Accordingly, additional steps are typically required to remove these compounds prior to analysis.Continue reading “Why Wait? Sample Prep/Protein Digestion in as Little as 30 Minutes!”
Improvements in Protein Bioprocessing
As more and more protein-based therapeutics enter research pipelines, more efficient protocols are needed. In particular, we need better protocols for the characterization of protein structure and function, as well as means of quantitation. One main step in this pipeline, proteolysis of these proteins into peptides, presents a bottleneck and can require optimization of multiple steps including reduction, alkylation and digestion time.
We have developed a new trypsin reagent, Rapid Digestion–Trypsin, that streamlines the protein sample preparation process. This new development significantly reduces the time to achieve proteolysis to about 1 hour, a remarkable improvement over existing overnight sample preparation times.
How Does it Work?
With this new trypsin product, proteolysis is performed at 70°C, incorporating both denaturation and rapid digestion. The protocol can be used with multiple protein types, including pure proteins and complex mixtures, and is compatible with digestion under native, reduced or nonreduced conditions.Continue reading “Widening the Proteolysis Bottleneck: A New Protein Sample Preparation Tool”