Why wait ? Sample prep/protein digestion in as little as 30 minutes!

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.

To shorten the time required to prepare samples for LC-MS/MS analysis, we have developed a specialized trypsin preparation that supports rapid and efficient digestion at temperatures as high as 70°C. There are several benefits to this approach. First, proteolytic reaction times are dramatically shortened. Second, because no chemical denaturants have been added, off -line sample cleanup is not necessary, leading to shorter preparation times and diminished sample losses.

The Rapid Digestion trypsin protocols are highly flexible. They can accommodate a variety of additives including reducing and alkylating agents. There are no restrictions on sample volume or substrate concentrations with these kits. Furthermore, the protocol is simple to follow and requires no laboratory equipment beyond a heat block. Digestion is achieved completely using an in-solution approach, and since the enzyme is not immobilized on beads, the protocol does not have strict requirements for rapid shaking and off -line filtering to remove beads.

In addition to the benefits of this flexibility, we also developed a Rapid Digestion–Trypsin/Lys-C mixture. Like the Trypsin/Lys-C Mix previously developed to prepare maximally efficiently proteolytic digests, particularly for complex mixtures, Rapid Digestion–Trypsin/Lys C is ideally suited for studies that require improved reproducibility across samples.

 

Widening the Proteolysis Bottleneck: A New Protein Sample Preparation Tool

The poster featured in this blog provides background information and data on development of Rapid Digestion-Trypsin.

The poster featured in this blog provides background information and data on development of Rapid Digestion-Trypsin.

Improvements in Protein Bioprocessing

As more and more protein-based therapeutics enter research pipelines, more efficient protocols are needed for 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, reducing 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.

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