PI3Kα Translocation Mediates Nuclear PtdIns(3,4,5)P3 Effector Signaling in Colorectal Cancer.

The canonical view of PI3Kα signaling describes phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) generation and activation of downstream effectors at the plasma membrane or at microtubule-bound endosomes. Here, we show that colorectal cancer (CRC) cell lines exhibit a diverse plasma membrane-nuclear distribution of PI3Kα, controlling corresponding levels of subcellular PtdIns(3,4,5)P3 pools. PI3Kα nuclear translocation was mediated by the importin ß-dependent nuclear import pathway. By PtdIns(3,4,5)P3 affinity capture mass spectrometry done in the presence of SDS on CRC cell lines with PI3Kα nuclear localization, we identified 867 potential nuclear PtdIns(3,4,5)P3 effector proteins. Nuclear PtdIns(3,4,5)P3 interactome proteins were characterized by noncanonical PtdIns(3,4,5)P3-binding domains and showed overrepresentation for nuclear membrane, nucleolus, and nuclear speckles. The nuclear PtdIns(3,4,5)P3 interactome was enriched for proteins related to RNA metabolism, with splicing reporter assays and SC-35 foci staining suggesting a role of epidermal growth factor-stimulated nuclear PI3Kα signaling in modulating pre-mRNA splicing. In patient tumors, nuclear p110α staining was associated with lower T stage and mucinous histology. These results indicate that PI3Kα translocation mediates nuclear PtdIns(3,4,5)P3 effector signaling in human CRC, modulating signaling responses.

HDX-MS study on garadacimab binding to activated FXII reveals potential binding interfaces through differential solvent exposure.

Hageman factor (FXII) is an essential component in the intrinsic coagulation cascade and a therapeutic target for the prophylactic treatment of hereditary angioedema (HAE). CSL312 (garadacimab) is a novel high-affinity human antibody capable of blocking activated FXII activity that is currently undergoing Phase 3 clinical trials in HAE. Structural studies using hydrogen/deuterium exchange coupled to mass spectrometry revealed evidence of interaction between the antibody and regions surrounding the S1 specificity pocket of FXII, including the 99-loop, 140-loop, 180-loop, and neighboring regions. We propose complementarity-determining regions (CDRs) in heavy-chain CDR2 and CDR3 as potential paratopes on garadacimab, and the 99-loop, 140-loop, 180-loop, and 220-loop as binding sites on the beta chain of activated FXII (ß-FXIIa).

Endocytic membrane repair by ESCRT-III controls antigen export to the cytosol during antigen cross-presentation.

Despite its crucial role in initiation of cytotoxic immune responses, the molecular pathways underlying antigen cross-presentation remain incompletely understood. The mechanism of antigen exit from endocytic compartments into the cytosol is a long-standing matter of controversy, confronting two main models: transfer through specific channels/transporters or rupture of endocytic membranes and leakage of luminal content. By monitoring the occurrence of intracellular damage in conventional dendritic cells (cDCs), we show that cross-presenting cDC1s display more frequent endomembrane injuries and increased recruitment of endosomal sorting complex required for transport (ESCRT)-III, the main repair system for intracellular membranes, relative to cDC2s. Silencing of CHMP2a or CHMP4b, two effector subunits of ESCRT-III, enhances cytosolic antigen export and cross-presentation. This phenotype is partially reversed by chemical inhibition of RIPK3, suggesting that endocytic damage is related to basal activation of the necroptosis pathway. Membrane repair therefore proves crucial in containing antigen export to the cytosol and cross-presentation in cDCs.

Differential protein expression due to Se deficiency and Se toxicity in rat liver.

There is a U-shaped dose-response between selenium (Se) status and health outcomes, but underlying metabolic processes are unclear. This study aims to identify candidate proteins in liver regulated by dietary Se, ranging from deficiency to toxic. Male rats (n=4) were fed graded Se concentrations as selenite for 28 days. Bulk Se analysis was performed by ICP-MS on both soluble and insoluble fractions. Soluble fraction samples were chromatographically separated for identification of selenocompounds by SEC-ICP-MS and protein quantification by LC-MS/MS. Bioinformatics analysis compared low-Se (0 and 0.08 µg Se g-1) and high-Se (0.8, 2 and 5 µg Se g-1) with adequate-Se (0.24 µg Se g-1) diets. Major breakpoints for Se were seen at 0.8 and 2 µg Se g-1 in the insoluble and soluble fractions, respectively. Glutathione peroxidase 1 protein abundance reached a plateau at ≥0.08 µg Se g-1diet; Se bound to selenium binding protein 2 was observed with 2 and 5 µg Se g-1 Se. The extreme diets presented the highest number of differentially expressed (P value <0.05, FC ≥1.2) proteins in comparison to the adequate-Se diet (0 µg Se g-1: 45 proteins; 5 µg Se g-1: 59 proteins); 13 proteins were commonly affected in 0 and 5 µg Se g-1 treatments. Network analysis revealed that the metabolism of glutathione, xenobiotics and amino acids were enriched in both 0 and 5 µg Se g-1 diets, indicating a U-shape effect of Se. This similarity is likely due to down-stream effects of lack of essential selenoproteins in Se deficiency and due to toxic effects of Se that exceeds the capacity to cope with excess Se.

20S proteasomes secreted by the malaria parasite promote its growth.

Mature red blood cells (RBCs) lack internal organelles and canonical defense mechanisms, making them both a fascinating host cell, in general, and an intriguing choice for the deadly malaria parasite Plasmodium falciparum (Pf), in particular. Pf, while growing inside its natural host, the human RBC, secretes multipurpose extracellular vesicles (EVs), yet their influence on this essential host cell remains unknown. Here we demonstrate that Pf parasites, cultured in fresh human donor blood, secrete within such EVs assembled and functional 20S proteasome complexes (EV-20S). The EV-20S proteasomes modulate the mechanical properties of naïve human RBCs by remodeling their cytoskeletal network. Furthermore, we identify four degradation targets of the secreted 20S proteasome, the phosphorylated cytoskeletal proteins ß-adducin, ankyrin-1, dematin and Epb4.1. Overall, our findings reveal a previously unknown 20S proteasome secretion mechanism employed by the human malaria parasite, which primes RBCs for parasite invasion by altering membrane stiffness, to facilitate malaria parasite growth.

BAK core dimers bind lipids and can be bridged by them.

BAK and BAX are essential mediators of apoptosis that oligomerize in response to death cues, thereby causing permeabilization of the mitochondrial outer membrane. Their transition from quiescent monomers to pore-forming oligomers involves a well-characterized symmetric dimer intermediate. However, no essential secondary interface that can be disrupted by mutagenesis has been identified. Here we describe crystal structures of human BAK core domain (α2-α5) dimers that reveal preferred binding sites for membrane lipids and detergents. The phospholipid headgroup and one acyl chain (sn2) associate with one core dimer while the other acyl chain (sn1) associates with a neighboring core dimer, suggesting a mechanism by which lipids contribute to the oligomerization of BAK. Our data support a model in which, unlike for other pore-forming proteins whose monomers assemble into oligomers primarily through protein-protein interfaces, the membrane itself plays a role in BAK and BAX oligomerization.

Establishing Signature Fragments for Identification and Sequencing of Dityrosine Cross-Linked Peptides Using Ultraviolet Photodissociation Mass Spectrometry.

Dityrosine cross-linking of Aß peptides and α-synuclein is increasingly becoming recognized as a biomarker of neuropathological diseases. However, there remains a need for the development of analytical methods that enable the specific and selective identification of dityrosine cross-linked proteins and peptides in complex biological samples. Here, we report that the gas-phase fragmentation of protonated dityrosine cross-linked peptides under ultraviolet photodissociation (UVPD) tandem mass spectrometry (MS/MS) conditions results in the cleavage across Cα and Cß atoms of the dityrosine residue. This Cα-Cß cleavage in UVPD-MS/MS results in the formation of diagnostic pairs of product ions, providing information on the two individual peptides involved in the cross-linking, resolving the intrinsic "n2 problem" plaguing the identification of this post-translational modification (PTM) by tandem mass spectrometry. Sequencing of a heterodimeric dityrosine cross-linked peptide was demonstrated using hybrid UVPD-MS/MS and CID-MS3 on a diagnostic pair of product ions. In combination with dedicated MS-cleavable MSn software, UVPD-MSn therefore provides an avenue to selectively discover and describe dityrosine cross-linked peptides. Additionally, observation of dityrosine-specific "reporter ions" at m/z 240.1019 and m/z 223.0752 in UVPD-MS/MS will be useful for the validation of the dityrosine cross-linked peptides.

Proteomics Standards Initiative Extended FASTA Format.

Mass-spectrometry-based proteomics enables the high-throughput identification and quantification of proteins, including sequence variants and post-translational modifications (PTMs) in biological samples. However, most workflows require that such variations be included in the search space used to analyze the data, and doing so remains challenging with most analysis tools. In order to facilitate the search for known sequence variants and PTMs, the Proteomics Standards Initiative (PSI) has designed and implemented the PSI extended FASTA format (PEFF). PEFF is based on the very popular FASTA format but adds a uniform mechanism for encoding substantially more metadata about the sequence collection as well as individual entries, including support for encoding known sequence variants, PTMs, and proteoforms. The format is very nearly backward compatible, and as such, existing FASTA parsers will require little or no changes to be able to read PEFF files as FASTA files, although without supporting any of the extra capabilities of PEFF. PEFF is defined by a full specification document, controlled vocabulary terms, a set of example files, software libraries, and a file validator. Popular software and resources are starting to support PEFF, including the sequence search engine Comet and the knowledge bases neXtProt and UniProtKB. Widespread implementation of PEFF is expected to further enable proteogenomics and top-down proteomics applications by providing a standardized mechanism for encoding protein sequences and their known variations. All the related documentation, including the detailed file format specification and example files, are available at http://www.psidev.info/peff .

The inner ear proteome of fish.

The mechanisms that underpin the formation, growth and composition of otoliths, the biomineralized stones in the inner ear of fish, are largely unknown, as only a few fish inner ear proteins have been reported. Using a partial transcriptome for the inner ear of black bream (Acanthopagrus butcheri), in conjunction with proteomic data, we discovered hundreds of previously unknown proteins in the otolith. This allowed us to develop hypotheses to explain the mechanisms of inorganic material supply and daily formation of growth bands. We further identified a likely protein mediator of crystal nucleation and an explanation for the apparent metabolic inertness of the otolith. Due to the formation of both daily and annual increments, otoliths are routinely employed as natural chronometers, being used for age and growth estimation, fisheries stock assessments, and the reconstruction of habitat use, movement, diet and the impacts of climate change. Our findings provide an unprecedented view of otolith molecular machinery, aiding in the interpretation of these essential archived data.

Aspartyl Protease 5 Matures Dense Granule Proteins That Reside at the Host-Parasite Interface in Toxoplasma gondii.

Toxoplasma gondii infects approximately 30% of the world's population, causing disease primarily during pregnancy and in individuals with weakened immune systems. Toxoplasma secretes and exports effector proteins that modulate the host during infection, and several of these proteins are processed by the Golgi-associated aspartyl protease 5 (ASP5). Here, we identify ASP5 substrates by selectively enriching N-terminally derived peptides from wild-type and Δasp5 parasites. We reveal more than 2,000 unique Toxoplasma N-terminal peptides, mapping to both natural N termini and protease cleavage sites. Several of these peptides mapped directly downstream of the characterized ASP5 cleavage site, arginine-arginine-leucine (RRL). We validate candidates as true ASP5 substrates, revealing they are not processed in parasites lacking ASP5 or in wild-type parasites following mutation of the motif from RRL to ARL. All identified ASP5 substrates are dense granule proteins, and interestingly, none appear to be exported, thus differing from the analogous system in related Plasmodium spp. Instead we show that the majority of substrates reside within the parasitophorous vacuole (PV), and its membrane (the PVM), including two kinases and one phosphatase. We show that genetic deletion of WNG2 leads to attenuation in a mouse model, suggesting that this putative kinase is a new virulence factor in Toxoplasma Collectively, these data constitute the first in-depth analyses of ASP5 substrates and shed new light on the role of ASP5 as a maturase of dense granule proteins during the Toxoplasma lytic cycle.IMPORTANCEToxoplasma gondii is one of the most successful human parasites. Central to its success is the arsenal of virulence proteins introduced into the infected host cell. Several of these virulence proteins require direct maturation by the aspartyl protease ASP5, and all require ASP5 for translocation into the host cell, yet the true number of ASP5 substrates and complete repertoire of effectors is currently unknown. Here we selectively enrich N-terminally derived peptides using Terminal Amine Isotopic Labeling of Substrates (TAILS) and use quantitative proteomics to reveal novel ASP5 substrates. We identify, using two different enrichment techniques, new ASP5 substrates and their specific cleavage sites. ASP5 substrates include two kinases and one phosphatase that reside at the host-parasite interface, which are important for infection.

The Peptidome Comes of Age: Mass Spectrometry-Based Characterization of the Circulating Cancer Peptidome.

Peptides play a seminal role in most physiological processes acting as neurotransmitters, hormones, antibiotics, and immune regulation. In the context of tumor biology, it is hypothesized that endogenous peptides, hormones, cytokines, growth factors, and aberrant degradation of select protein networks (e.g., enzymatic activities, protein shedding, and extracellular matrix remodeling) are fundamental in mediating cancer progression. Analysis of peptides in biological fluids by mass spectrometry holds promise of providing sensitive and specific diagnostic and prognostic information for cancer and other diseases. The identification of circulating peptides in the context of disease constitutes a hitherto source of new clinical biomarkers. The field of peptidomics can be defined as the identification and comprehensive analysis of physiological and pathological peptides. Like proteomics, peptidomics has been advanced by the development of new separation strategies, analytical detection methods such as mass spectrometry, and bioinformatic technologies. Unlike proteomics, peptidomics is targeted toward identifying endogenous protein and peptide fragments, defining proteolytic enzyme substrate specificity, as well as protease cleavage recognition (degradome). Peptidomics employs "top-down proteomics" strategies where mass spectrometry is applied at the proteoform level to analyze intact proteins and large endogenous peptide fragments. With recent advances in prefractionation workflows for separating peptides, mass spectrometry instrumentation, and informatics, peptidomics is an important field that promises to impact on translational medicine. This review covers the current advances in peptidomics, including top-down and imaging mass spectrometry, comprehensive quantitative peptidome analyses (developments in reproducibility and coverage), peptide prefractionation and enrichment workflows, peptidomic data analyses, and informatic tools. The application of peptidomics in cancer biomarker discovery will be discussed.

Characterization and Identification of Dityrosine Cross-Linked Peptides Using Tandem Mass Spectrometry.

The use of mass spectrometry coupled with chemical cross-linking of proteins has become a powerful tool for proteins structure and interactions studies. Unlike structural analysis of proteins using chemical reagents specific for lysine or cysteine residues, identification of gas-phase fragmentation patterns of endogenous dityrosine cross-linked peptides have not been investigated. Dityrosine cross-linking in proteins and peptides are clinical markers of oxidative stress, aging, and neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. In this study, we investigated and characterized the fragmentation pattern of a synthetically prepared dityrosine cross-linked dimer of Aß(1-16) using ESI tandem mass spectrometry. We then detailed the fragmentation pattern of dityrosine cross-linked Aß(1-16), using collision induced dissociation (CID), higher-energy collision induced dissociation (HCD), electron transfer dissociation (ETD), and electron capture dissociation (ECD). Application of these generic fragmentation rules of dityrosine cross-linked peptides allowed for the identification of dityrosine cross-links in peptides of Aß and α-synuclein generated in vitro by enzymatic peroxidation. We report, for the first time, the dityrosine cross-linked residues in human hemoglobin and α-synuclein under oxidative conditions. Together these tools open up the potential for automated analysis of this naturally occurring post-translation modification in neurodegenerative diseases as well as other pathological conditions.

Selenium Levels in Serum, Red Blood Cells, and Cerebrospinal Fluid of Alzheimer's Disease Patients: A Report from the Australian Imaging, Biomarker & Lifestyle Flagship Study of Ageing (AIBL).

Selenium (Se) protects cells against oxidative stress damage through a range of bioactive selenoproteins. Increased oxidative stress is a prominent feature of Alzheimer's disease (AD), and previous studies have shown that Se deficiency is associated with age-related cognitive decline. In this study, we assessed Se status in different biofluids from a subgroup of participants in the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing. As Se in humans can either be an active component of selenoproteins or inactive via non-specific incorporation into other proteins, we used both size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) and tandem mass spectrometry to characterize selenoproteins in serum. We observed no differences in total Se concentration in serum or cerebrospinal fluid of AD subjects compared to mildly cognitively impairment patients and healthy controls. However, Se levels in erythrocytes were decreased in AD compared to controls. SEC-ICP-MS analysis revealed a dominant Se-containing fraction. This fraction was subjected to standard protein purification and a bottom-up proteomics approach to confirm that the abundant Se in the fraction was due, in part, to selenoprotein P. The lack of change in the Se level is at odds with our previous observations in a Brazilian population deficient in Se, and we attribute this to the Australian cohort being Se-replete.

ABRF Proteome Informatics Research Group (iPRG) 2015 Study: Detection of Differentially Abundant Proteins in Label-Free Quantitative LC-MS/MS Experiments.

Detection of differentially abundant proteins in label-free quantitative shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) experiments requires a series of computational steps that identify and quantify LC-MS features. It also requires statistical analyses that distinguish systematic changes in abundance between conditions from artifacts of biological and technical variation. The 2015 study of the Proteome Informatics Research Group (iPRG) of the Association of Biomolecular Resource Facilities (ABRF) aimed to evaluate the effects of the statistical analysis on the accuracy of the results. The study used LC-tandem mass spectra acquired from a controlled mixture, and made the data available to anonymous volunteer participants. The participants used methods of their choice to detect differentially abundant proteins, estimate the associated fold changes, and characterize the uncertainty of the results. The study found that multiple strategies (including the use of spectral counts versus peak intensities, and various software tools) could lead to accurate results, and that the performance was primarily determined by the analysts' expertise. This manuscript summarizes the outcome of the study, and provides representative examples of good computational and statistical practice. The data set generated as part of this study is publicly available.

Synthesis of an inositol hexakisphosphate (IP6) affinity probe to study the interactome from a colon cancer cell line.

Inositol hexakisphosphate (InsP6 or IP6) is an important signalling molecule in vesicular trafficking, neurotransmission, immune responses, regulation of protein kinases and phosphatases, activation of ion channels, antioxidant functions and anticancer activities. An IP6 probe was synthesised from myo-inositol via a derivatised analogue, which was immobilised through a terminal amino group onto Dynabeads. Systematic analysis of the IP6 interactome has been performed using the IP6 affinity probe using cytosolic extracts from the LIM1215 colonic carcinoma cell line. LC/MS/MS analysis identified 77 proteins or protein complexes that bind to IP6 specifically, including AP-2 complex proteins and ß-arrestins as well as a number of novel potential IP6 interacting proteins. Bioinformatic enrichment analysis of the IP6 interactome reinforced the concept that IP6 regulates a number of biological processes including cell cycle and division, signal transduction, intracellular protein transport, vesicle-mediated transport and RNA splicing.

No evidence of enemy release in pathogen and microbial communities of common wasps (Vespula vulgaris) in their native and introduced range.

When invasive species move to new environments they typically experience population bottlenecks that limit the probability that pathogens and parasites are also moved. The invasive species may thus be released from biotic interactions that can be a major source of density-dependent mortality, referred to as enemy release. We examined for evidence of enemy release in populations of the common wasp (Vespula vulgaris), which attains high densities and represents a major threat to biodiversity in its invaded range. Mass spectrometry proteomic methods were used to compare the microbial communities in wasp populations in the native (Belgium and England) and invaded range (Argentina and New Zealand). We found no evidence of enemy release, as the number of microbial taxa was similar in both the introduced and native range. However, some evidence of distinctiveness in the microbial communities was observed between countries. The pathogens observed were similar to a variety of taxa observed in honey bees. These taxa included Nosema, Paenibacillus, and Yersina spp. Genomic methods confirmed a diversity of Nosema spp., Actinobacteria, and the Deformed wing and Kashmir bee viruses. We also analysed published records of bacteria, viruses, nematodes and fungi from both V. vulgaris and the related invader V. germanica. Thirty-three different microorganism taxa have been associated with wasps including Kashmir bee virus and entomophagous fungi such as Aspergillus flavus. There was no evidence that the presence or absence of these microorganisms was dependent on region of wasp samples (i.e. their native or invaded range). Given the similarity of the wasp pathogen fauna to that from honey bees, the lack of enemy release in wasp populations is probably related to spill-over or spill-back from bees and other social insects. Social insects appear to form a reservoir of generalist parasites and pathogens, which makes the management of wasp and bee disease difficult.

Proteome informatics research group (iPRG)_2012: a study on detecting modified peptides in a complex mixture.

The proteome informatics research group of the Association of Biomolecular Resource Facilities conducted a study to assess the community's ability to detect and characterize peptides bearing a range of biologically occurring post-translational modifications when present in a complex peptide background. A data set derived from a mixture of synthetic peptides with biologically occurring modifications combined with a yeast whole cell lysate as background was distributed to a large group of researchers and their results were collectively analyzed. The results from the twenty-four participants, who represented a broad spectrum of experience levels with this type of data analysis, produced several important observations. First, there is significantly more variability in the ability to assess whether a results is significant than there is to determine the correct answer. Second, labile post-translational modifications, particularly tyrosine sulfation, present a challenge for most researchers. Finally, for modification site localization there are many tools being employed, but researchers are currently unsure of the reliability of the results these programs are producing.

Sulindac modulates secreted protein expression from LIM1215 colon carcinoma cells prior to apoptosis.

Colorectal cancer (CRC) is a major cause of mortality in Western populations. Growing evidence from human and rodent studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) cause regression of existing colon tumors and act as effective chemopreventive agents in sporadic colon tumor formation. Although much is known about the action of the NSAID sulindac, especially its role in inducing apoptosis, mechanisms underlying these effects is poorly understood. In previous secretome-based proteomic studies using 2D-DIGE/MS and cytokine arrays we identified over 150 proteins released from the CRC cell line LIM1215 whose expression levels were dysregulated by treatment with 1mM sulindac over 16h; many of these proteins are implicated in molecular and cellular functions such as cell proliferation, differentiation, adhesion, angiogenesis and apoptosis (Ji et al., Proteomics Clin. Appl. 2009, 3, 433-451). We have extended these studies and describe here an improved protein/peptide separation strategy that facilitated the identification of 987 proteins and peptides released from LIM1215 cells following 1mM sulindac treatment for 8h preceding the onset of apoptosis. This peptidome separation strategy involved fractional centrifugal ultrafiltration of concentrated cell culture media (CM) using nominal molecular weight membrane filters (NMWL 30K, 3K and 1K). Proteins isolated in the >30K and 3-30K fractions were electrophoretically separated by SDS-PAGE and endogenous peptides in the 1-3K membrane filter were fractioned by RP-HPLC; isolated proteins and peptides were identified by nanoLC-MS-MS. Collectively, our data show that LIM1215 cells treated with 1mM sulindac for 8h secrete decreased levels of proteins associated with extracellular matrix remodeling (e.g., collagens, perlecan, syndecans, filamins, dyneins, metalloproteinases and endopeptidases), cell adhesion (e.g., cadherins, integrins, laminins) and mucosal maintenance (e.g., glycoprotein 340 and mucins 5AC, 6, and 13). A salient finding of this study was the increased proteolysis of cell surface proteins following treatment with sulindac for 8h (40% higher than from untreated LIM1215 cells); several of these endogenous peptides contained C-terminal amino acids from transmembrane domains indicative of regulated intramembrane proteolysis (RIP). Taken together these results indicate that during the early-stage onset of sulindac-induced apoptosis (evidenced by increased annexin V binding, dephosphorylation of focal adhesion kinase (FAK), and cleavage of caspase-3), 1mM sulindac treatment of LIM1215 cells results in decreased expression of secreted proteins implicated in ECM remodeling, mucosal maintenance and cell-cell-adhesion. This article is part of a Special Issue entitled: An Updated Secretome.

Colon tumour secretopeptidome: insights into endogenous proteolytic cleavage events in the colon tumour microenvironment.

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 (<3kDa) 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.

Oncogenic H-ras reprograms Madin-Darby canine kidney (MDCK) cell-derived exosomal proteins following epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a highly conserved morphogenic process defined by the loss of epithelial characteristics and the acquisition of a mesenchymal phenotype. EMT is associated with increased aggressiveness, invasiveness, and metastatic potential in carcinoma cells. To assess the contribution of extracellular vesicles following EMT, we conducted a proteomic analysis of exosomes released from Madin-Darby canine kidney (MDCK) cells, and MDCK cells transformed with oncogenic H-Ras (21D1 cells). Exosomes are 40-100 nm membranous vesicles originating from the inward budding of late endosomes and multivesicular bodies and are released from cells on fusion of multivesicular bodies with the plasma membrane. Exosomes from MDCK cells (MDCK-Exos) and 21D1 cells (21D1-Exos) were purified from cell culture media using density gradient centrifugation (OptiPrep™), and protein content identified by GeLC-MS/MS proteomic profiling. Both MDCK- and 21D1-Exos populations were morphologically similar by cryo-electron microscopy and contained stereotypical exosome marker proteins such as TSG101, Alix, and CD63. In this study we show that the expression levels of typical EMT hallmark proteins seen in whole cells correlate with those observed in MDCK- and 21D1-Exos, i.e. reduction of characteristic inhibitor of angiogenesis, thrombospondin-1, and epithelial markers E-cadherin, and EpCAM, with a concomitant up-regulation of mesenchymal makers such as vimentin. Further, we reveal that 21D1-Exos are enriched with several proteases (e.g. MMP-1, -14, -19, ADAM-10, and ADAMTS1), and integrins (e.g. ITGB1, ITGA3, and ITGA6) that have been recently implicated in regulating the tumor microenvironment to promote metastatic progression. A salient finding of this study was the unique presence of key transcriptional regulators (e.g. the master transcriptional regulator YBX1) and core splicing complex components (e.g. SF3B1, SF3B3, and SFRS1) in mesenchymal 21D1-Exos. Taken together, our findings reveal that exosomes from Ras-transformed MDCK cells are reprogrammed with factors which may be capable of inducing EMT in recipient cells.