Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals.

Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope-labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study, we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [13C6]lysine or [2H2]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first-order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates.

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.

Identification and Characterization of Canine Ligament Progenitor Cells and Their Extracellular Matrix Niche.

Ligaments are prone to injury and degeneration in humans and animals, however the healing potential of ligament is poor and current treatment options ineffective. Stem cell-based therapies hold potential for treatment of ligament injuries. This study aimed to characterize a ligament progenitor cell (LPC) population and to identify specific niche components which could promote the survival and function of LPCs. LPCs were isolated from canine cranial cruciate ligament and characterized for clonogenicity, multipotency and marker expression. The extracellular matrix (ECM) composition was characterized by the novel application of a metabolic labeling and mass spectrometry technique. LPCs demonstrated clonogenicity, multipotency, and stem cell marker expression. A number of different collagens, glycoproteins, and proteoglycans were identified in the LPC niche using proteomics. Metabolic labeling of cells demonstrated unique turnover profiles for distinct ECM protein groups, indicating the importance of certain niche components for LPC survival and function. The newly synthesized niche components identified in this study could be exploited to aid identification of LPCs and to promote their survival and function for potential ligament repair strategies.

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.

Proteome Dynamics: Tissue Variation in the Kinetics of Proteostasis in Intact Animals.

Understanding the role of protein turnover in the maintenance of proteostasis requires accurate measurements of the rates of replacement of proteins in complex systems, such as intact animals. Moreover, any investigation of allometric scaling of protein turnover is likely to include species for which fully annotated proteomes are not available. We have used dietary administration of stable isotope labeled lysine to assess protein turnover rates for proteins from four tissues in the bank vole,Myodes glareolus The annotated genome for this species is not available, so protein identification was attained through cross-species matching to the mouse. For proteins for which confident identifications were derived, the pattern of lysine incorporation over 40 days was used to define the rate of synthesis of individual proteins in the four tissues. The data were heavily filtered to retain a very high quality dataset of turnover rates for 1088 proteins. Comparative analysis of the four tissues revealed different median rates of degradation (kidney: 0.099 days(-1); liver 0.136 days(-1); heart, 0.054 days(-1), and skeletal muscle, 0.035 days(-1)). These data were compared with protein degradation rates from other studies on intact animals or from cells in culture and indicate that both cell type and analytical methodology may contribute to variance in turnover data between different studies. These differences were not only due to tissue-specific proteins but were reflected in gene products common to all tissues. All data are available via ProteomeXchange with identifier PXD002054.

The mzqLibrary--An open source Java library supporting the HUPO-PSI quantitative proteomics standard.

The mzQuantML standard has been developed by the Proteomics Standards Initiative for capturing, archiving and exchanging quantitative proteomic data, derived from mass spectrometry. It is a rich XML-based format, capable of representing data about two-dimensional features from LC-MS data, and peptides, proteins or groups of proteins that have been quantified from multiple samples. In this article we report the development of an open source Java-based library of routines for mzQuantML, called the mzqLibrary, and associated software for visualising data called the mzqViewer. The mzqLibrary contains routines for mapping (peptide) identifications on quantified features, inference of protein (group)-level quantification values from peptide-level values, normalisation and basic statistics for differential expression. These routines can be accessed via the command line, via a Java programming interface access or a basic graphical user interface. The mzqLibrary also contains several file format converters, including import converters (to mzQuantML) from OpenMS, Progenesis LC-MS and MaxQuant, and exporters (from mzQuantML) to other standards or useful formats (mzTab, HTML, csv). The mzqViewer contains in-built routines for viewing the tables of data (about features, peptides or proteins), and connects to the R statistical library for more advanced plotting options. The mzqLibrary and mzqViewer packages are available from https://code.google.com/p/mzq-lib/.

Absolute quantification of the glycolytic pathway in yeast: deployment of a complete QconCAT approach.

The availability of label-free data derived from yeast cells (based on the summed intensity of the three strongest, isoform-specific peptides) permitted a preliminary assessment of protein abundances for glycolytic proteins. Following this analysis, we demonstrate successful application of the QconCAT technology, which uses recombinant DNA techniques to generate artificial concatamers of large numbers of internal standard peptides, to the quantification of enzymes of the glycolysis pathway in the yeast Saccharomyces cerevisiae. A QconCAT of 88 kDa (59 tryptic peptides) corresponding to 27 isoenzymes was designed and built to encode two or three analyte peptides per protein, and after stable isotope labeling of the standard in vivo, protein levels were determined by LC-MS, using ultra high performance liquid chromatography-coupled mass spectrometry. We were able to determine absolute protein concentrations between 14,000 and 10 million molecules/cell. Issues such as efficiency of extraction and completeness of proteolysis are addressed, as well as generic factors such as optimal quantotypic peptide selection and expression. In addition, the same proteins were quantified by intensity-based label-free analysis, and both sets of data were compared with other quantification methods.

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.

Darcin: a male pheromone that stimulates female memory and sexual attraction to an individual male's odour.

BACKGROUND: Among invertebrates, specific pheromones elicit inherent (fixed) behavioural responses to coordinate social behaviours such as sexual recognition and attraction. By contrast, the much more complex social odours of mammals provide a broad range of information about the individual owner and stimulate individual-specific responses that are modulated by learning. How do mammals use such odours to coordinate important social interactions such as sexual attraction while allowing for individual-specific choice? We hypothesized that male mouse urine contains a specific pheromonal component that invokes inherent sexual attraction to the scent and which also stimulates female memory and conditions sexual attraction to the airborne odours of an individual scent owner associated with this pheromone. RESULTS: Using wild-stock house mice to ensure natural responses that generalize across individual genomes, we identify a single atypical male-specific major urinary protein (MUP) of mass 18893Da that invokes a female's inherent sexual attraction to male compared to female urinary scent. Attraction to this protein pheromone, which we named darcin, was as strong as the attraction to intact male urine. Importantly, contact with darcin also stimulated a strong learned attraction to the associated airborne urinary odour of an individual male, such that, subsequently, females were attracted to the airborne scent of that specific individual but not to that of other males. CONCLUSIONS: This involatile protein is a mammalian male sex pheromone that stimulates a flexible response to individual-specific odours through associative learning and memory, allowing female sexual attraction to be inherent but selective towards particular males. This 'darcin effect' offers a new system to investigate the neural basis of individual-specific memories in the brain and give new insights into the regulation of behaviour in complex social mammals.See associated Commentary http://www.biomedcentral.com/1741-7007/8/71.

Probing the biogenesis pathway and dynamics of thylakoid membranes.

How thylakoid membranes are generated to form a metabolically active membrane network and how thylakoid membranes orchestrate the insertion and localization of protein complexes for efficient electron flux remain elusive. Here, we develop a method to modulate thylakoid biogenesis in the rod-shaped cyanobacterium Synechococcus elongatus PCC 7942 by modulating light intensity during cell growth, and probe the spatial-temporal stepwise biogenesis process of thylakoid membranes in cells. Our results reveal that the plasma membrane and regularly arranged concentric thylakoid layers have no physical connections. The newly synthesized thylakoid membrane fragments emerge between the plasma membrane and pre-existing thylakoids. Photosystem I monomers appear in the thylakoid membranes earlier than other mature photosystem assemblies, followed by generation of Photosystem I trimers and Photosystem II complexes. Redistribution of photosynthetic complexes during thylakoid biogenesis ensures establishment of the spatial organization of the functional thylakoid network. This study provides insights into the dynamic biogenesis process and maturation of the functional photosynthetic machinery.

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.

Decoding the stoichiometric composition and organisation of bacterial metabolosomes.

Some enteric bacteria including Salmonella have evolved the propanediol-utilising microcompartment (Pdu MCP), a specialised proteinaceous organelle that is essential for 1,2-propanediol degradation and enteric pathogenesis. Pdu MCPs are a family of bacterial microcompartments that are self-assembled from hundreds of proteins within the bacterial cytosol. Here, we seek a comprehensive understanding of the stoichiometric composition and organisation of Pdu MCPs. We obtain accurate stoichiometry of shell proteins and internal enzymes of the natural Pdu MCP by QconCAT-driven quantitative mass spectrometry. Genetic deletion of the major shell protein and absolute quantification reveal the stoichiometric and structural remodelling of metabolically functional Pdu MCPs. Decoding the precise protein stoichiometry allows us to develop an organisational model of the Pdu metabolosome. The structural insights into the Pdu MCP are critical for both delineating the general principles underlying bacterial organelle formation, structural robustness and function, and repurposing natural microcompartments using synthetic biology for biotechnological applications.

Structural variability, coordination and adaptation of a native photosynthetic machinery.

Cyanobacterial thylakoid membranes represent the active sites for both photosynthetic and respiratory electron transport. We used high-resolution atomic force microscopy to visualize the native organization and interactions of photosynthetic complexes within the thylakoid membranes from the model cyanobacterium Synechococcus elongatus PCC 7942. The thylakoid membranes are heterogeneous and assemble photosynthetic complexes into functional domains to enhance their coordination and regulation. Under high light, the chlorophyll-binding proteins IsiA are strongly expressed and associate with Photosystem I (PSI), forming highly variable IsiA-PSI supercomplexes to increase the absorption cross-section of PSI. There are also tight interactions of PSI with Photosystem II (PSII), cytochrome b6f, ATP synthase and NAD(P)H dehydrogenase complexes. The organizational variability of these photosynthetic supercomplexes permits efficient linear and cyclic electron transport as well as bioenergetic regulation. Understanding the organizational landscape and environmental adaptation of cyanobacterial thylakoid membranes may help inform strategies for engineering efficient photosynthetic systems and photo-biofactories.

The heparin-binding proteome in normal pancreas and murine experimental acute pancreatitis.

Acute pancreatitis (AP) is acute inflammation of the pancreas, mainly caused by gallstones and alcohol, driven by changes in communication between cells. Heparin-binding proteins (HBPs) play a central role in health and diseases. Therefore, we used heparin affinity proteomics to identify extracellular HBPs in pancreas and plasma of normal mice and in a caerulein mouse model of AP. Many new extracellular HBPs (360) were discovered in the pancreas, taking the total number of HBPs known to 786. Extracellular pancreas HBPs form highly interconnected protein-protein interaction networks in both normal pancreas (NP) and AP. Thus, HBPs represent an important set of extracellular proteins with significant regulatory potential in the pancreas. HBPs in NP are associated with biological functions such as molecular transport and cellular movement that underlie pancreatic homeostasis. However, in AP HBPs are associated with additional inflammatory processes such as acute phase response signalling, complement activation and mitochondrial dysfunction, which has a central role in the development of AP. Plasma HBPs in AP included known AP biomarkers such as serum amyloid A, as well as emerging targets such as histone H2A. Other HBPs such as alpha 2-HS glycoprotein (AHSG) and histidine-rich glycoprotein (HRG) need further investigation for potential applications in the management of AP. Pancreas HBPs are extracellular and so easily accessible and are potential drug targets in AP, whereas plasma HBPs represent potential biomarkers for AP. Thus, their identification paves the way to determine which HBPs may have potential applications in the management of AP.

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.

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.

DOSCATs: Double standards for protein quantification.

The two most common techniques for absolute protein quantification are based on either mass spectrometry (MS) or on immunochemical techniques, such as western blotting (WB). Western blotting is most often used for protein identification or relative quantification, but can also be deployed for absolute quantification if appropriate calibration standards are used. MS based techniques offer superior data quality and reproducibility, but WB offers greater sensitivity and accessibility to most researchers. It would be advantageous to apply both techniques for orthogonal quantification, but workflows rarely overlap. We describe DOSCATs (DOuble Standard conCATamers), novel calibration standards based on QconCAT technology, to unite these platforms. DOSCATs combine a series of epitope sequences concatenated with tryptic peptides in a single artificial protein to create internal tryptic peptide standards for MS as well as an intact protein bearing multiple linear epitopes. A DOSCAT protein was designed and constructed to quantify five proteins of the NF-κB pathway. For three target proteins, protein fold change and absolute copy per cell values measured by MS and WB were in excellent agreement. This demonstrates that DOSCATs can be used as multiplexed, dual purpose standards, readily deployed in a single workflow, supporting seamless quantitative transition from MS to WB.

Glareosin: a novel sexually dimorphic urinary lipocalin in the bank vole, Myodes glareolus.

The urine of bank voles (Myodes glareolus) contains substantial quantities of a small protein that is expressed at much higher levels in males than females, and at higher levels in males in the breeding season. This protein was purified and completely sequenced at the protein level by mass spectrometry. Leucine/isoleucine ambiguity was completely resolved by metabolic labelling, monitoring the incorporation of dietary deuterated leucine into specific sites in the protein. The predicted mass of the sequenced protein was exactly consonant with the mass of the protein measured in bank vole urine samples, correcting for the formation of two disulfide bonds. The sequence of the protein revealed that it was a lipocalin related to aphrodisin and other odorant-binding proteins (OBPs), but differed from all OBPs previously described. The pattern of secretion in urine used for scent marking by male bank voles, and the similarity to other lipocalins used as chemical signals in rodents, suggest that this protein plays a role in male sexual and/or competitive communication. We propose the name glareosin for this novel protein to reflect the origin of the protein and to emphasize the distinction from known OBPs.