Protocol for N-terminomic analysis of protease substrates. This method is an adaption of the Weng et al. 2019 Mol. Cell. Proteomics (https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC6823850\/) method for N-terminomics, modified to allow sample multiplexing and quantification using TMTpro reagents rather than the original implementation from the Lange lab which used dimethyl or DIA-based quantification. This method was used in Meyer et al. 2020 - https:\/\/www.biorxiv.org\/content\/10.1101\/2020.09.16.297945v1.If you use this protocol, please cite both the Meyer et al. and Weng et al. publications.This adaption has advantages for the analysis of protease substrates, permitting sample multiplexing early in the protocol and allowing all samples to be processed subsequently as a single sample. This protocol has disadvantages compared to its parental protocol for analysis of native N-termini where blocking of the native N-terminus by acetylation or pyroglutamine prevents TMT labelling, and thus quantification of non-lysine-containing peptides. As neo-N-termini generated by viral proteases are not expected to possess natively-blocked N-termini (aside from a possible minority of pyroglutamine N-termini), these are not a consideration here.In brief:1. Cells are lysed in SDS and heated2. Samples are reduced and alkylated.3. Protein is precipitated on SP3 beads4. Protein is resolubilised and TMTpro labelling is performed at the protein level to block N-termini and lysine residues5. Unreacted TMTpro reagent is quenched and washed away.6. Samples are pooled.7. Overnight Tryptic digestion.8. Unblocked N-termini generated by tryptic digestion are hydrophlabelled with undecannal.9. Undecannal-labelled N-termini are depleted by passing the peptides over C18 resin in 50% ethanol, resulting in flow-through containing natively-blocked and TMTpro-labelled N-termini.This protocol can be accomplished in aproximately two days.