Enabling Proteomics: The Need for an Extendable ‘Workbench’ for User-Configurable Solutions

Proteomics has the capability to generate overwhelming quantities of data in relatively short timescales, and it is not uncommon to see experimenters investing substantially more time in data analysis than in data gathering. Although several sophisticated tools for data reduction and analysis are available, they lack the flexibility to cope with increasingly innovative experimental strategies and new database resources that encode both qualitative and quantitative data. I will outline a specification of a flexible proteomics tool that could address many current bottlenecks and deficiencies

Making progress in genetic kin recognition among vertebrates

A recent study in BMC Evolutionary Biology has shown that genetically similar individual ring-tailed lemurs are also more similar in their scent composition, suggesting a possible mechanism of kin recognition. Theoretical and experimental studies reveal challenges ahead in achieving a true systems-level understanding of this process and its outcomes

A proteomics study of the response of North Ronaldsay sheep to copper challenge

BACKGROUND: The objective of this proteomics study was to identify proteins that changed expression as a result of copper challenge in the uniquely copper sensitive North Ronaldsay sheep and further, to compare those changes in expression with the more copper tolerant Cambridge breed. Such data gives us a proteome-centered perspective of the pathogenesis of copper-induced oxidative stress in this breed. RESULTS: Many proteins respond to copper challenge, but this study focuses on those exhibiting a differential response between the two breeds, related to liver copper content. As copper accumulated in the tissue, the pattern of expression of several proteins was markedly different, in North Ronaldsay sheep as compared to the Cambridge breed. CONCLUSION: The pattern of changes was consistent with the greatly enhanced susceptibility of North Ronaldsay sheep to copper-induced oxidative stress, focused on mitochondrial disturbance with consequent activation of hepatic stellate cells. The expression profiles were sufficiently complex that the response could not simply be explained as a hypersensitivity to copper in North Ronaldsay sheep

The male sex pheromone darcin stimulates hippocampal neurogenesis and cell proliferation in the subventricular zone in female mice

The integration of newly generated neurons persists throughout life in the mammalian olfactory bulb and hippocampus, regions involved in olfactory and spatial learning. Social cues can be potent stimuli for increasing adult neurogenesis; for example, odors from dominant but not subordinate male mice increase neurogenesis in both brain regions of adult females. However, little is known about the role of neurogenesis in social recognition or the assessment of potential mates. Dominant male mice scent-mark territories using urine that contains a number of pheromones including darcin (MUP20), a male-specific major urinary protein that stimulates rapid learned attraction to the spatial location and individual odor signature of the scent owner. Here we investigate whether exposure to darcin stimulates neurogenesis in the female brain. Hippocampal neurons and cellular proliferation in the lateral ventricles that supply neurons to the olfactory bulbs increased in females exposed for 7 days to male urine containing at least 0.5 μg/μl darcin. Darcin was effective whether presented alone or in the context of male urine, but other information in male urine appeared to modulate the proliferative response. When exposed to urine from wild male mice, hippocampal proliferation increased only if urine was from the same individual over 7 days, suggesting that consistency of individual scent signatures is important. While 7 days exposure to male scent initiated the first stages of increased neurogenesis, this caused no immediate increase in female attraction to the scent or in the strength or robustness of spatial learning in short-term conditioned place preference tests. The reliable and consistent stimulation of neurogenesis by a pheromone important in rapid social learning suggests that this may provide an excellent model to explore the relationship between the integration of new neurons and plasticity in spatial and olfactory learning in a socially-relevant context

Rigorous Determination of the Stoichiometry of Protein Phosphorylation Using Mass Spectrometry

Quantification of the stoichiometry of phosphorylation is usually achieved using a mixture of phosphatase treatment and differential isotopic labeling. Here, we introduce a new approach to the concomitant determination of absolute protein concentration and the stoichiometry of phosphorylation at predefined sites. The method exploits QconCAT to quantify levels of phosphorylated and nonphosphorylated peptide sequences in a phosphoprotein. The nonphosphorylated sequence is used to determine the absolute protein quantity and serves as a reference to calculate the extent of phosphorylation at the second peptide. Thus, the stoichiometry of phosphorylation and the absolute protein concentration can be determined accurately in a single experiment

Rapid Evaporative Ionization Mass Spectrometry (REIMS): a Potential and Rapid Tool for the Identification of Insecticide Resistance in Mosquito Larvae

Insecticide resistance is a significant challenge facing the successful control of mosquito vectors globally. Bioassays are currently the only method for phenotyping resistance. They require large numbers of mosquitoes for testing, the availability of a susceptible comparator strain, and often insectary facilities. This study aimed to trial the novel use of rapid evaporative ionization mass spectrometry (REIMS) for the identification of insecticide resistance in mosquitoes. No sample preparation is required for REIMS and analysis can be rapidly conducted within hours. Temephos resistant Aedes aegypti (Linnaeus) larvae from Cúcuta, Colombia and temephos susceptible larvae from two origins (Bello, Colombia, and the lab reference strain New Orleans) were analyzed using REIMS. We tested the ability of REIMS to differentiate three relevant variants: population source, lab versus field origin, and response to insecticide. The classification of these data was undertaken using linear discriminant analysis (LDA) and random forest. Classification models built using REIMS data were able to differentiate between Ae. aegypti larvae from different populations with 82% (±0.01) accuracy, between mosquitoes of field and lab origin with 89% (±0.01) accuracy and between susceptible and resistant larvae with 85% (±0.01) accuracy. LDA classifiers had higher efficiency than random forest with this data set. The high accuracy observed here identifies REIMS as a potential new tool for rapid identification of resistance in mosquitoes. We argue that REIMS and similar modern phenotyping alternatives should complement existing insecticide resistance management tools

An in vivo control map for the eukaryotic mRNA translation machinery

Rate control analysis defines the in vivo control map governing yeast protein synthesis and generates an extensively parameterized digital model of the translation pathway. Among other non-intuitive outcomes, translation demonstrates a high degree of functional modularity and comprises a non-stoichiometric combination of proteins manifesting functional convergence on a shared maximal translation rate. In exponentially growing cells, polypeptide elongation (eEF1A, eEF2, and eEF3) exerts the strongest control. The two other strong control points are recruitment of mRNA and tRNAi to the 40S ribosomal subunit (eIF4F and eIF2) and termination (eRF1; Dbp5). In contrast, factors that are found to promote mRNA scanning efficiency on a longer than-average 5′untranslated region (eIF1, eIF1A, Ded1, eIF2B, eIF3, and eIF5) exceed the levels required for maximal control. This is expected to allow the cell to minimize scanning transition times, particularly for longer 5′UTRs. The analysis reveals these and other collective adaptations of control shared across the factors, as well as features that reflect functional modularity and system robustness. Remarkably, gene duplication is implicated in the fine control of cellular protein synthesis

Caught in a Trap? Proteomic Analysis of Neutrophil Extracellular Traps in Rheumatoid Arthritis and Systemic Lupus Erythematosus

Neutrophil Extracellular Traps (NETs) are implicated in the development of auto-immunity in diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) through the externalization of intracellular neoepitopes e.g., dsDNA and nuclear proteins in SLE and citrullinated peptides in RA. The aim of this work was to use quantitative proteomics to identify and measure NET proteins produced by neutrophils from healthy controls, and from patients with RA and SLE to determine if NETs can be differentially-generated to expose different sets of neoepitopes. Ultra-pure neutrophils (>99%) from healthy individuals (n = 3) and patients with RA or SLE (n = 6 each) were incubated ± PMA (50 nM, PKC super-activator) or A23187 (3.8 μM, calcium ionophore) for 4 h. NETs were liberated by nuclease digestion and concentrated onto Strataclean beads prior to on-bead digestion with trypsin. Data-dependent LC-MS/MS analyses were conducted on a QExactive HF quadrupole-Orbitrap mass spectrometer, and label-free protein quantification was carried out using Progenesis QI. PMA-induced NETs were decorated with annexins, azurocidin and histone H3, whereas A23187-induced NETs were decorated with granule proteins including CAMP/LL37, CRISP3, lipocalin and MMP8, histones H1.0, H1.4, and H1.5, interleukin-8, protein-arginine deiminase-4 (PADI4), and α-enolase. Four proteins were significantly different between PMA-NETs from RA and SLE neutrophils (p < 0.05): RNASE2 was higher in RA, whereas MPO, leukocyte elastase inhibitor and thymidine phosphorylase were higher in SLE. For A23187-NETs, six NET proteins were higher in RA (p < 0.05), including CAMP/LL37, CRISP3, interleukin-8, MMP8; Thirteen proteins were higher in SLE, including histones H1.0, H2B, and H4. This work provides the first, direct comparison of NOX2-dependent (PMA) and NOX2-independent (A23187) NETs using quantitative proteomics, and the first direct comparison of RA and SLE NETs using quantitative proteomics. We show that it is the nature of the stimulant rather than neutrophil physiology that determines NET protein profiles in disease, since stimulation of NETosis in either a NOX2-dependent or a NOX2-independent manner generates broadly similar NET proteins irrespective of the disease background. We also use our proteomics pipeline to identify an extensive range of post-translationally modified proteins in RA and SLE, including histones and granule proteins, many of which are known targets of auto-antibodies in each disease

The Genetic Basis of Individual-Recognition Signals in the Mouse

SummaryThe major histocompatibility complex (MHC) is widely assumed to be a primary determinant of individual-recognition scents in many vertebrates [1–6], but there has been no functional test of this in animals with normal levels of genetic variation. Mice have evolved another polygenic and highly polymorphic set of proteins for scent communication, the major urinary proteins (MUPs) [7–12], which may provide a more reliable identity signature ([13, 14] and A.L. Sherborne, M.D.T., S. Paterson, F.J., W.E.R.O., P. Stockley, R.J.B., and J.L.H., unpublished data). We used female preference for males that countermark competitor male scents [15–17] to test the ability of wild-derived mice to recognize individual males differing in MHC or MUP type on a variable genetic background. Differences in MHC type were not used for individual recognition. Instead, recognition depended on a difference in MUP type, regardless of other genetic differences between individuals. Recognition also required scent contact, consistent with detection of involatile components through the vomeronasal system [6, 18]. Other differences in individual scent stimulated investigation but did not result in individual recognition. Contrary to untested assumptions of a vertebrate-wide mechanism based largely on MHC variation, mice use a species-specific [12] individual identity signature that can be recognized reliably despite the complex internal and external factors that influence scents [2]. Specific signals for genetic identity recognition in other species now need to be investigated

Selection on Coding and Regulatory Variation Maintains Individuality in Major Urinary Protein Scent Marks in Wild Mice

Recognition of individuals by scent is widespread across animal taxa. Though animals can often discriminate chemical blends based on many compounds, recent work shows that specific protein pheromones are necessary and sufficient for individual recognition via scent marks in mice. The genetic nature of individuality in scent marks (e.g. coding versus regulatory variation) and the evolutionary processes that maintain diversity are poorly understood. The individual signatures in scent marks of house mice are the protein products of a group of highly similar paralogs in the major urinary protein (Mup) gene family. Using the offspring of wild-caught mice, we examine individuality in the major urinary protein (MUP) scent marks at the DNA, RNA and protein levels. We show that individuality arises through a combination of variation at amino acid coding sites and differential transcription of central Mup genes across individuals, and we identify eSNPs in promoters. There is no evidence of post-transcriptional processes influencing phenotypic diversity as transcripts accurately predict the relative abundance of proteins in urine samples. The match between transcripts and urine samples taken six months earlier also emphasizes that the proportional relationships across central MUP isoforms in urine is stable. Balancing selection maintains coding variants at moderate frequencies, though pheromone diversity appears limited by interactions with vomeronasal receptors. We find that differential transcription of the central Mup paralogs within and between individuals significantly increases the individuality of pheromone blends. Balancing selection on gene regulation allows for increased individuality via combinatorial diversity in a limited number of pheromones