Characterisation of infection associated microRNA and protein cargo in extracellular vesicles of Theileria annulata infected leukocytes.

The protozoan parasites Theileria annulata and Theileria parva are unique amongst intracellular eukaryotic pathogens as they induce a transformation-like phenotype in their bovine host cell. T. annulata causes tropical theileriosis, which is frequently fatal, with infected leukocytes becoming metastatic and forming foci in multiple organs resulting in destruction of the lymphoid system. Exosomes, a subset of extracellular vesicles (EV), are critical in metastatic progression in many cancers. Here, we characterised the cargo of EV from a control bovine lymphosarcoma cell line (BL20) and BL20 infected with T. annulata (TBL20) by comparative mass spectrometry and microRNA (miRNA) profiling (data available via ProteomeXchange, identifier PXD010713 and NCBI GEO, accession number GSE118456, respectively). Ingenuity pathway analysis that many infection-associated proteins essential to migration and extracellular matrix digestion were upregulated in EV from TBL20 cells compared with BL20 controls. An altered repertoire of host miRNA, many with known roles in tumour and/or infection biology, was also observed. Focusing on the tumour suppressor miRNA, bta-miR-181a and bta-miR-181b, we identified putative messenger RNA targets and confirmed the interaction of bta-miR181a with ICAM-1. We propose that EV and their miRNA cargo play an important role in the manipulation of the host cell phenotype and the pathobiology of Theileria infection.

Stable Isotope Dynamic Labeling of Secretomes (SIDLS) Identifies Authentic Secretory Proteins Released by Cancer and Stromal Cells.

Analysis of secretomes critically underpins the capacity to understand the mechanisms determining interactions between cells and between cells and their environment. In the context of cancer cell micro-environments, the relevant interactions are recognized to be an important determinant of tumor progression. Global proteomic analyses of secretomes are often performed at a single time point and frequently identify both classical secreted proteins (possessing an N-terminal signal sequence), as well as many intracellular proteins, the release of which is of uncertain biological significance. Here, we describe a mass spectrometry-based method for stable isotope dynamic labeling of secretomes (SIDLS) that, by dynamic SILAC, discriminates the secretion kinetics of classical secretory proteins and intracellular proteins released from cancer and stromal cells in culture. SIDLS is a robust classifier of the different cellular origins of proteins within the secretome and should be broadly applicable to nonproliferating cells and cells grown in short term culture.

Tumour compartment transcriptomics demonstrates the activation of inflammatory and odontogenic programmes in human adamantinomatous craniopharyngioma and identifies the MAPK/ERK pathway as a novel therapeutic target.

Adamantinomatous craniopharyngiomas (ACPs) are clinically challenging tumours, the majority of which have activating mutations in CTNNB1. They are histologically complex, showing cystic and solid components, the latter comprised of different morphological cell types (e.g. ß-catenin-accumulating cluster cells and palisading epithelium), surrounded by a florid glial reaction with immune cells. Here, we have carried out RNA sequencing on 18 ACP samples and integrated these data with an existing ACP transcriptomic dataset. No studies so far have examined the patterns of gene expression within the different cellular compartments of the tumour. To achieve this goal, we have combined laser capture microdissection with computational analyses to reveal groups of genes that are associated with either epithelial tumour cells (clusters and palisading epithelium), glial tissue or immune infiltrate. We use these human ACP molecular signatures and RNA-Seq data from two ACP mouse models to reveal that cell clusters are molecularly analogous to the enamel knot, a critical signalling centre controlling normal tooth morphogenesis. Supporting this finding, we show that human cluster cells express high levels of several members of the FGF, TGFB and BMP families of secreted factors, which signal to neighbouring cells as evidenced by immunostaining against the phosphorylated proteins pERK1/2, pSMAD3 and pSMAD1/5/9 in both human and mouse ACP. We reveal that inhibiting the MAPK/ERK pathway with trametinib, a clinically approved MEK inhibitor, results in reduced proliferation and increased apoptosis in explant cultures of human and mouse ACP. Finally, we analyse a prominent molecular signature in the glial reactive tissue to characterise the inflammatory microenvironment and uncover the activation of inflammasomes in human ACP. We validate these results by immunostaining against immune cell markers, cytokine ELISA and proteome analysis in both solid tumour and cystic fluid from ACP patients. Our data support a new molecular paradigm for understanding ACP tumorigenesis as an aberrant mimic of natural tooth development and opens new therapeutic opportunities by revealing the activation of the MAPK/ERK and inflammasome pathways in human ACP.

Autologous chondrocyte implantation-derived synovial fluids display distinct responder and non-responder proteomic profiles.

BACKGROUND: Autologous chondrocyte implantation (ACI) can be used in the treatment of focal cartilage injuries to prevent the onset of osteoarthritis (OA). However, we are yet to understand fully why some individuals do not respond well to this intervention. Identification of a reliable and accurate biomarker panel that can predict which patients are likely to respond well to ACI is needed in order to assign the patient to the most appropriate therapy. This study aimed to compare the baseline and mid-treatment proteomic profiles of synovial fluids (SFs) obtained from responders and non-responders to ACI. METHODS: SFs were derived from 14 ACI responders (mean Lysholm improvement of 33 (17-54)) and 13 non-responders (mean Lysholm decrease of 14 (4-46)) at the two stages of surgery (cartilage harvest and chondrocyte implantation). Label-free proteome profiling of dynamically compressed SFs was used to identify predictive markers of ACI success or failure and to investigate the biological pathways involved in the clinical response to ACI. RESULTS: Only 1 protein displayed a ≥2.0-fold differential abundance in the preclinical SF of ACI responders versus non-responders. However, there is a marked difference between these two groups with regard to their proteome shift in response to cartilage harvest, with 24 and 92 proteins showing ≥2.0-fold differential abundance between Stages I and II in responders and non-responders, respectively. Proteomic data has been uploaded to ProteomeXchange (identifier: PXD005220). We have validated two biologically relevant protein changes associated with this response, demonstrating that matrix metalloproteinase 1 was prominently elevated and S100 calcium binding protein A13 was reduced in response to cartilage harvest in non-responders. CONCLUSIONS: The differential proteomic response to cartilage harvest noted in responders versus non-responders is completely novel. Our analyses suggest several pathways which appear to be altered in non-responders that are worthy of further investigation to elucidate the mechanisms of ACI failure. These protein changes highlight many putative biomarkers that may have potential for prediction of ACI treatment success.

QconCATs: design and expression of concatenated protein standards for multiplexed protein quantification.

Systems biology requires knowledge of the absolute amounts of proteins in order to model biological processes and simulate the effects of changes in specific model parameters. Quantification concatamers (QconCATs) are established as a method to provide multiplexed absolute peptide standards for a set of target proteins in isotope dilution standard experiments. Two or more quantotypic peptides representing each of the target proteins are concatenated into a designer gene that is metabolically labelled with stable isotopes in Escherichia coli or other cellular or cell-free systems. Co-digestion of a known amount of QconCAT with the target proteins generates a set of labelled reference peptide standards for the unlabelled analyte counterparts, and by using an appropriate mass spectrometry platform, comparison of the intensities of the peptide ratios delivers absolute quantification of the encoded peptides and in turn the target proteins for which they are surrogates. In this review, we discuss the criteria and difficulties associated with surrogate peptide selection and provide examples in the design of QconCATs for quantification of the proteins of the nuclear factor κB pathway.

Acetone precipitation of proteins and the modification of peptides.

Acetone precipitation is a common method for precipitation and concentration of proteins. We show here that a trace amount of residual acetone in the precipitated protein, can, after proteolysis, lead to selective modification of peptides predominantly those in which a glycine residue is the second amino acid, probably generating a relatively stable derivative that, under gas phase conditions, generates a y(1) ion of the same mass as proline. This modification is detectable by either MALDI-ToF or ESI-ion trap mass spectrometry and under normal sample preparation conditions is incomplete. The derivatization occurs in the condensed phase and is sufficiently stable that the modified peptide can elute on reversed phase chromatography at a different time to the unmodified peptide. Acetone precipitation is such a commonly used procedure in protein sample preparation for proteomics that some caution may be warranted. A significant number of peptides (about 5% of a typical proteome) meet the requirements for this reaction and could, therefore, change the outcome of studies.

QCAL--a novel standard for assessing instrument conditions for proteome analysis.

If proteome datasets are to be collated, shared, and merged for higher level proteome analyses, there is a need for generally accepted strategies and reagents for optimization and standardization of instrument performance. At present, there is no single protein or peptide standard set that is capable of assessing instrument performance for peptide separation and analysis in this manner. To create such a standard, we have used the recently described QconCAT methodology to generate an artificial protein, QCAL. This protein, a concatenation of tryptic peptides that is expressed in E. coli, provides a stoichiometrically controlled mixture of peptides that are amenable to analysis by all commonly used instrumentation platforms for proteomics.

Protein quantification by selective isolation and fragmentation of isotopic pairs using FT-ICR MS.

Isolation of tryptic peptide ions, along with their differentially labeled analogs derived from an artificial QconCAT protein, is performed using multiple correlated harmonic excitation fields in an FT-ICR cell. Simultaneous fragmentation of the isolated unlabeled and labeled peptide pairs using IRMPD yields specific y-series fragment ions useful for quantification. The mass increment attributed to stable isotope labeling at the C-terminus is maintained in the C-terminal fragment ions, providing multiple measurements of labeled/unlabeled intensity ratios during highly selective detection. The utility of this approach has been demonstrated in the absolute quantification of components of an unfractionated chicken muscle protein mixture.

Multiplexed absolute quantification for proteomics using concatenated signature peptides encoded by QconCAT genes.

An important area of proteomics involves the need for quantification, whether relative or absolute. Many methods now exist for relative quantification, but to support biomarker proteomics and systems biology, absolute quantification rather than relative quantification is required. Absolute quantification usually involves the concomitant mass spectrometric determination of signature proteotypic peptides and stable isotope-labeled analogs. However, the availability of standard labeled signature peptides in accurately known amounts is a limitation to the widespread adoption of this approach. We describe the design and synthesis of artificial QconCAT proteins that are concatamers of tryptic peptides for several proteins. This protocol details the methods for the design, expression, labeling, purification, characterization and use of the QconCATs in the absolute quantification of complex protein mixtures. The total time required to complete this protocol (from the receipt of the QconCAT expression plasmid to the absolute quantification of the set of proteins encoded by the QconCAT protein in an analyte sample) is approximately 29 d.