Many of the tissue-specific proteoforms originate from genes involved in the core function of these tissues, as indicated by gene ontology enrichment (Figure , Figure ). The Subsequence proteoform search identified a series of proteoforms associated with defensins with distinct expression patterns (Figure , Figure ). Defensins are a family of small cationic host defense proteins characterized by three conserved intramolecular disulfide bonds. Six human alphadefensins have been identified to date and are subdivided into human neutrophil peptides 1 to 4 (HNP1-4) and human (enteric) defensins (HD5-6). HNPs are stored as mature peptides in granules of neutrophils and released upon activation by exocytosis. 44 HNP1 (PFR69106) was identified in both lung and spleen tissue as expected for tissues with high neutrophil content. HNP2 (PFR69109), HNP3 (PFR69079), HNP4 (PFR65983), and truncation products of HNP2 (PFR165182 and PFR165183) were observed exclusively in spleen tissue. No beta-defensin proteoforms were identified. HD5 and HD6 are produced in Paneth cells at the base of small intestinal crypts. Accordingly, HD5 and HD6 were detected exclusively in small intestinal tissue. Unlike other defensins, HD5 is stored as a propeptide, and the fully mature peptides are thought to be produced by intracellular trypsin. Consequently, the HD5 propeptide (PFR165815) and several truncated products were observed. Several of these truncated proteoforms (PFR5737351, PFR97759, and PFR97755) correspond to trypsin cleavage sites (R25, R55, and R62), while others (PFR5741069, PFR5737454, and PFR5737363) seem to correspond to other mechanisms of cleavage considering the residues at the P1 positions (D41, F46, and A61). Despite reducing samples with DTT prior to analysis, several proteoforms were observed with disulfide bridges intact (PFR4919881, PFR4919882, and PFR5026622). The disulfide linkages in these proteoforms are inconsistent with the canonical model of alpha-defensins that includes end-to-end disulfides (Figure ). Although these non-canonical disulfides might be biologically relevant, spontaneous reformation of disulfides in denatured samples is likely. Defensins are important components of the host innate immunity, so observing new proteoforms on mucosal surfaces is important in understanding their regulation and design of therapeutic mimetics. Furthermore, these findings are a good showcase for the capabilities of the presented setup to evaluate tissuespecific proteoform-related questions.