To specifically enrich for mature protein N-termini and neo-N-termini of proteins we developed 6 and 10-plex TMT TAILS (Terminal Amine Isotopic Labeling of Substrates) (Nature Biotech 28, 281-288 (2010); Nature Protocols 6, 1578-1611 (2011). In analyzing the N-terminome of normal human tissues we find that the N-termini of protein chains in vivo can commence at many points C terminal to the predicted start site and result from proteolytic processing to generate stable protein chains: Proteolytic processing generates new protein species with characteristic neo-N termini that are frequently accompanied by altered half-lives, function, interactions and location. We used TAILS to mechanistically dissect a severe human immunodeficiency disease. Paracaspase MALT1 proteolytic activity and molecular scaffolding are key for CARMA1–BCL10–MALT1 (CBM) complex formation. The CBM transduces signaling from lymphocyte antigen receptors. A mutation in MALT1 provided a unique opportunity to identify new MALT1 substrates through positional proteomics using TAILS. We compared B cells from the MALT1mut/mut patient with healthy MALT1+/mut family members and normal individuals using 10-plex Tandem Mass Tag TAILS with MS3 synchronous peak selection quantification, with and without antigen receptor stimulation. From the MALT1 cleaved neo-N terminal peptide (prime side) and the natural N terminus of HOIL1 identified by TAILS, and the nonprime side of the HOIL1 cleavage site identified by preTAILS shotgun proteomics, we identified HOIL1 of the linear ubiquitin chain assembly complex (LUBAC) as a novel MALT1 substrate. Upon B and T cell receptor stimulation HOIL1 cleavage resulted in disassembly of LUBAC and loss of linear ubiquitination in T and B cells and prevented reactivation of NF-kB signaling. We describe an approach to rescue the mutant MALT1 scaffolding and proteolytic activity in the patient cells. This is the unique example of positional proteomics to mechanistically decipher a human disease, possibly the first disease to be phenotyped by proteomics, and a precision medicine approach to increase patient protease function and so stabilize a patient before life saving bone marrow translatation.