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Simple, scalable, and ultrasensitive tip-based identification of protease substrates
Gerta Shema, Minh T. N. Nguyen, Fiorella A. Solari, Stefan Loroch, A. Saskia Venne, Laxmikanth Kollipara, Albert Sickmann, Steven H. L. Verhelst and Rene P. Zahedi
Molecular & Cellular Proteomics, (2018) 17 826-834
The authors have developed a new method for the identification of novel N-termini formed by the action of proteases upon various protein substrates, revealing information about the regulatory proteolytic networks in health and disease. The method relies on the enrichment of the N-terminal peptides followed by MS identification and quantification.
The method, charge-based fractional diagonal chromatography in a pipette tip or ChaFRAtip, utilizes well-established protein chemistry and minimal equipment: A pipette tip with a cellulose frit, strong cation exchange chromatography beads and common buffers. In conjunction with iTRAQ-8-plex labeling, the method can quantify over 2,000 N-terminal peptides across eight samples using around 4 µg of protein per condition/channel.
The authors compared the original LC/MS methodology to ChaFRAtip using SH-SY5Y cells treated with staurosporine to induce apoptosis. They achieved a similar level of technical reproducibility for both methods and quantified 975 ±24 (HPLC) and 783 ±71 (tips) unique N-terminal peptides at 1% false discovery rate.
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