Colon tumour secretopeptidome: Insights into endogenous proteolytic cleavage events in the colon tumour microenvironment
journal contributionposted on 2021-08-12, 05:55 authored by David GreeningDavid Greening, EA Kapp, H Ji, TP Speed, Richard SimpsonRichard Simpson
The secretopeptidome comprises endogenous peptides derived from proteins secreted into the tumour microenvironment through classical and non-classical secretion. This study characterised the low-Mr (< 3 kDa) component of the human colon tumour (LIM1215, LIM1863) secretopeptidome, as a first step towards gaining insights into extracellular proteolytic cleavage events in the tumour microenvironment. Based on two biological replicates, this secretopeptidome isolation strategy utilised differential centrifugal ultrafiltration in combination with analytical RP-HPLC and nanoLC-MS/MS. Secreted peptides were identified using a combination of Mascot and post-processing analyses including MSPro re-scoring, extended feature sets and Percolator, resulting in 474 protein identifications from 1228 peptides (≤ 1% q-value, ≤ 5% PEP) - a 36% increase in peptide identifications when compared with conventional Mascot (homology ionscore thresholding). In both colon tumour models, 122 identified peptides were derived from 41 cell surface protein ectodomains, 23 peptides (12 proteins) from regulated intramembrane proteolysis (RIP), and 12 peptides (9 proteins) generated from intracellular domain proteolysis. Further analyses using the protease/substrate database MEROPS, (http://merops.sanger.ac.uk/), revealed 335 (71%) proteins classified as originating from classical/non-classical secretion, or the cell membrane. Of these, peptides were identified from 42 substrates in MEROPS with defined protease cleavage sites, while peptides generated from a further 205 substrates were fragmented by hitherto unknown proteases. A salient finding was the identification of peptides from 88 classical/non-classical secreted substrates in MEROPS, implicated in tumour progression and angiogenesis (FGFBP1, PLXDC2), cell-cell recognition and signalling (DDR1, GPA33), and tumour invasiveness and metastasis (MACC1, SMAGP); the nature of the proteases responsible for these proteolytic events is unknown. To confirm reproducibility of peptide fragment abundance in this study, we report the identification of a specific cleaved peptide fragment in the secretopeptidome from the colon-specific GPA33 antigen in 4/14 human CRC models. This improved secretopeptidome isolation and characterisation strategy has extended our understanding of endogenous peptides generated through proteolysis of classical/non-classical secreted proteins, extracellular proteolytic processing of cell surface membrane proteins, and peptides generated through RIP. The novel peptide cleavage site information in this study provides a useful first step in detailing proteolytic cleavage associated with tumourigenesis and the extracellular environment. This article is part of a Special Issue entitled: An Updated Secretome. © 2013 Elsevier B.V.
Funding was provided, in part, by the Australian National Health and Medical Research Council under Program Grant 487922 (RJS, JH and DWG). We acknowledge the NHMRC-funded Australian Proteomics Computational Facility (APCF) under Enabling Grant 381413. This work was supported by funds from the Operational Infrastructure Support Program provided by the Victorian Government, Australia.
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Rights StatementThe Author reserves all moral rights over the deposited text and must be credited if any re-use occurs. Documents deposited in OPAL are the Open Access versions of outputs published elsewhere. Changes resulting from the publishing process may therefore not be reflected in this document. The final published version may be obtained via the publisher’s DOI. Please note that additional copyright and access restrictions may apply to the published version.
Science & TechnologyLife Sciences & BiomedicineBiochemistry & Molecular BiologyBiophysicsPeptidomeSecretomeLow-molecular weightProteolytic cleavageEctodomainProteolysisCELL-ADHESION MOLECULEE-CADHERIN FRAGMENTA33 ANTIGENPEPTIDE IDENTIFICATIONEMERGING ROLESAPC MUTATIONSPROTEINECTODOMAINEXPRESSIONPREDICTIONColonCell Line, TumorHumansColonic NeoplasmsPeptidesMembrane GlycoproteinsProteomeChromatography, High Pressure LiquidProteomicsProtein Processing, Post-TranslationalAmino Acid SequenceMolecular Sequence DataTandem Mass SpectrometrySecretory PathwayGPI-Linked ProteinsTumor MicroenvironmentADAMAPPBSGCALRCDH1CEACMCRCCTFDGDSG2ECMFDRGDF15GPA33GPC1GPIHNPCCI-CLiPsICDIDEMMPMT-MMPN-terminal fragmentNMWLNTFPEPPMPSMRIPTGFBITICTMHMMTNFRSF9TP53I11XCorra disintegrin and metalloproteinaseamyloid precursor proteinbasigincalreticulincarboxy-terminal fragmentcarcinoembryonic antigencell surface A33 antigencolorectal cancercross-correlation scoreculture mediumdesmoglein-2dystroglycanepithelial cadherinextracellular matrixfalse discovery rateglycosylphosphatidylinositolglypican-1growth/differentiation factor 15hereditary nonpolyposis colorectal cancerinsulin degrading enzyme, insulysinintracellular domainintramembrane-cleaving proteasesmatrix metalloproteinasemembrane-type MMPnominal molecular weight limitpeptide spectral matchesplasma membraneposterior error probabilityregulated intramembrane proteolysissTICsummed total ion currenttotal ion currenttransforming growth factor-beta-induced protein ig-h3transmembrane hidden Markov modeltumour necrosis factor receptor superfamily member 9tumour protein p53-inducible protein 11