Abstract
Many proteolytic cleavage events cannot be covered with conventional trypsin-based N-terminomics workflows. These typically involve the derivatization of protein N-termini and Lys residues as an initial step, such that trypsin will cleave C-terminal of arginine but not lysine residues (ArgC-like cleavage). From 20,422 reviewed human protein sequences in Uniprot, 3597 have known N-terminal signal peptides. An in silico ArgC-like digestion of the corresponding 3597 mature protein sequences reveals that—even for these well-known and well-studied proteolytic events—trypsin-based N-terminomics workflows may miss up to 50% of signaling cleavage events as the corresponding neo-N-terminal peptides will have an unfavorable length of <7 (875 peptides) or >30 (911 peptides) amino acids. In this chapter, we provide a protocol that can be applied to all kinds of samples to improve access to this “inaccessible” N-terminome, by making use of the alternative, broad-specificity protease subtilisin for fast and reproducible digestion of proteins.
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We would like to thank the University of Manitoba and Shared Health Manitoba for financial support.
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Jiang, X., Lao, Y., Spicer, V., Zahedi, R.P. (2023). Improved Coverage of the N-Terminome by Combining ChaFRADIC with Alternative Proteases. In: Gevaert, K. (eds) Mass Spectrometry-Based Proteomics. Methods in Molecular Biology, vol 2718. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3457-8_6
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DOI: https://doi.org/10.1007/978-1-0716-3457-8_6
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