Proteomics Using Protease Alternatives to Trypsin Benefits from Sequential Digestion with Trypsin

Area of Science:

• Proteomics and enzyme specificity analysis
• Biochemistry and protein identification techniques

Background:

• Trypsin is the standard enzyme for proteomics, but alternative proteases are used in specific cases.
• Understanding protease cleavage specificity is crucial for optimizing protein identification.

Purpose of the Study:

• To evaluate five alternative proteases to trypsin for protein identification and sequence coverage.
• To analyze the impact of protease specificity on protein identification in *S. pombe* whole cell lysates.
• To investigate the benefits of sequential digestion with trypsin and other proteases.

Main Methods:

• Analysis of five protease alternatives (AspN, GluC, chymotrypsin, proteinase K, elastase) to trypsin.
• Application of proteases to *S. pombe* whole cell lysates for protein identification.
• Comparison of single-digest versus sequential-digest strategies, including trypsin predigestion.

Main Results:

• Protease specificity directly influenced the number of identified proteins; higher specificity yielded more identifications.
• Sequential digestion with trypsin significantly improved protein identification for AspN (+62%), GluC (+80%), chymotrypsin (+21%), and proteinase K (+731%).
• A sequential digest of trypsin followed by AspN identified more proteins than trypsin alone.

Conclusions:

• Protease specificity is a key factor in maximizing protein identification in proteomics.
• Sequential digestion strategies, particularly involving trypsin predigestion, substantially enhance protein identification coverage.
• The combination of trypsin and AspN offers a powerful approach for comprehensive protein identification.