Identification of protein terminal peptides provides key information on protein stability and function. Our degradomics methods enrich and annotate terminomes—Terminal Amine Isotopic Labelling of Substrates (TAILS, Kleifeld etal 2010), Amino Terminal Orientated Mass Spectrometry (ATOMS), Proteome-wideIdentification of Protease Cleavage Specificity (PICS, Schilling_&_Overall 2008), LysargiNase, the new protease for proteomics we discovered (Huesgen etal 2015), and our N and C-termini database TopFIND (http://clipserve.clip.ubc.ca/topfind) our studies reveal widespread truncation and generation of termini in normal and diseased tissues.
Certain N terminal semi-tryptic peptides exhibit beneficial m/z, ionization and fragmentation properties over tryptic peptides, rendering these peptides and proteins identifiable. In the C-HPP, TAILS was used to identify proteoforms and also provide MS evidence for the expression of PE2-4 ‘missing proteins’, in rare tissues and cells.
Using PICS we discovered active protease domains in bacterial flagella of >200 species.~1,000 cleavage sites for “flagellinolysin” being identified. With ~20,000 flagellin copies/~10-μm flagella this assembles the largest proteolytic complex known with potential for numerous roles in saprophytic bacteria and in pathogens.
ATOMS was used to identify function-modifying cleavages by metalloproteinases in moonlighting extracellular tRNA synthetases. When moonlighting outside the cell, “intracellular” tyrosyl tRNA synthetase was proteolytically activated as a proinflammatory mediator signaling through Toll-like receptor-2 (TLR2), resulting in NF-κB activation and TNα/chemokine release from macrophages. In tryptophan tRNA synthetase, cleavage inactivated TLR signalling and proinflammatory functions.
By TAILS we find that the innate immune cell MMPs orchestrate leukocyte chemotaxis by cleavage of most human chemokines. For CCL chemokines, inactivation/generation of antagonists are common sequalae, whereas CXCL activation by the neutrophil specific MMP8 is followed by inactivating cleavages within the ELR motif by MMP12 regulates neutrophil chemoattraction in vivo. More recently IFNα and IFNγ, and complement proteins were discovered as new MMP substrates. MMP processing of these bioactive substrates dampens inflammation essential for terminating inflammatory responses.