Protein Digestion for Mass Spectrometry

Sequence specific proteases are utilized to cleave proteins into smaller fragments, or peptides. Trypsin is the protease of choice for protein digestion. However, separate or sequential digestion with alternative proteases, such as Glu-C, LysN, Lys-C, Asp-N or Chymotrypsin, can improve individual protein sequence coverage or generate unique peptide sequences for different MS applications. The Thermo Scientific Pierce Proteases are highly purified and modified for optimal protein digestion and validated for use in MS. The Thermo Scientific EasyPep Mini MS Sample Prep Kit provides pre-formulated reagents and a robust method for the preparation of high quality samples that are ready for MS analysis in less than 3 hours.

• Comprehensive—broad range of proteases to accommodate a variety of digestion strategies
• Convenient —complete in-gel and in-solution kits are available as well as standalone proteases in multiple pack sizes
• Economical—bulk and custom packaging of some proteases provide more cost effective or optimized options
• Validated—all proteases have been fully tested and processed samples have been analyzed using Thermo Scientific Mass Spectrometers

The decision to perform in-solution or in-gel digestion is generally determined by the sample amount and/or its complexity.

In-solution digestion is recommended for small sample volumes with low to moderate complexity and is more amenable to high-throughput sample.

For highly complex samples, in-gel digestion has an inherent advantage because the SDS-PAGE workflow combines protein denaturation with separation, and provides a visual indication of the relative protein abundance in the sample,  although peptide recovery is reduced.

We offer complete kits for both in-gel and in-solution sample processing. 

Percent sequence coverage of selected proteins in an A549 cell lysate upon in-solution digestion with LysN protease or trypsin. The total (merged) sequence coverage represents the combined coverage of separate LysN and trypsin digestions followed by LC-MS/MS analysis with CID.