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Introduction Antibody-mediated rejection (ABMR) impacts kidney allograft outcome. The diagnosis is made based on findings from invasive kidney transplant biopsy specimens. The aim of this study was to identify a noninvasive urinary protein biomarker for ABMR after kidney transplantation. Methods We performed a multicenter case-control study to identify a urinary biomarker for ABMR (training cohort, n = 249) and an independent, prospective multicenter cohort study for validation (n = 391). We used concomitant biopsies to classify the samples according to the Banff classification. After untargeted protein identification and quantification, we used a support vector machine to train the model in the training cohort. The primary endpoint was the diagnostic accuracy of the urinary biomarker for ABMR in the validation cohort. Results We identified a set of 10 urinary proteins that accurately discriminated patients with (n = 60) and without (n = 189) ABMR in the training cohort with an area under the curve (AUC) of 0.98 (95% confidence interval [CI], 0.96–1.00). The diagnostic accuracy was maintained in the validation cohort (AUC, 0.88; 95% CI, 0.8–0.93) for discriminating the presence (n = 43) from the absence (n = 348) of ABMR. The negative predictive value of the 10-protein marker set for exclusion of ABMR was 0.99, and the positive predictive value was 0.33. The diagnostic accuracy was independent of the reason for performing the biopsy, time after transplantation, and better than the accuracy of gross proteinuria (AUC, 0.76). Conclusions We identified and validated a urinary protein biomarker set that can be used to exclude ABMR., Graphical abstract

Aneuploidy causes broad-scale disruption in the stochiometric balances of transcripts, proteins, and metabolites. These disruptions often lead to detrimental effects for the organism, but for some organisms and in certain environments, it can also cause a fitness advantage. Understanding the complex trade-off between aneuploidy fitness gains and losses requires a systems biological comprehension of its molecular impact. The protozoan parasite Leishmania provides a unique study angle to this problem, as the pathogen lacks transcriptional regulation of individual protein-coding genes and has an unusually high tolerance for aneuploidy. Here, we present the first integrated analysis of the genome, transcriptome, proteome, and metabolome of highly aneuploid Leishmania donovani strains. This 4 layer multi-omics analysis unambiguously establishes that despite this tolerance, aneuploidy in Leishmania globally and drastically impacts the transcriptome and proteome of affected chromosomes, ultimately explaining the degree of metabolic differences between strains. This impact appears to be present throughout the parasite’s life cycle as we observed it in both its proliferative and infectious life stages. We show that, through dosage compensation, protein complex subunits, secreted proteins, and non-cytoplasmic proteins responded less or even not at all to aneuploidy. In contrast to other Eukaryotes, we did not observe the widespread regulation at the transcript level that typically modulate some of the negative effects of aneuploidy. Instead, Leishmania features a surprisingly high number of regulated proteins encoded by non-aneuploid chromosomes, suggesting that aneuploidy results in extensive post-transcriptionally modulation of protein levels. Our results provide the foundation for the integrated molecular understanding of aneuploidy in Leishmania. They also highlight that the parasite is an attractive model to study processes of post-transcriptional modulation of protein levels in the context of aneuploidy and beyond.

Urine is commonly used for clinical diagnosis and biomedical research. The discovery of extracellular vesicles (EV) in urine opened a new fast-growing scientific field. In the last decade urinary extracellular vesicles (uEVs) were shown to mirror molecular processes as well as physiological and pathological conditions in kidney, urothelial and prostate tissue. Therefore, several methods to isolate and characterize uEVs have been developed. However, methodological aspects of EV separation and analysis, including normalization of results, need further optimization and standardization to foster scientific advances in uEV research and a subsequent successful translation into clinical practice. This position paper is written by the Urine Task Force of the Rigor and Standardization Subcommittee of ISEV consisting of nephrologists, urologists, cardiologists and biologists with active experience in uEV research. Our aim is to present the state of the art and identify challenges and gaps in current uEV-based analyses for clinical applications. Finally, recommendations for improved rigor, reproducibility and interoperability in uEV research are provided in order to facilitate advances in the field. ESM-U, CG, GV, GJ, IVB, MvR, and VvS, are members of the “IMMPROVE” consortium (Innovative Measurements and Markers for Prostate Cancer Diagnosis and Prognosis using Extracellular Vesicles), which is sponsored by an Alpe d'HuZes grant of the Dutch Cancer Society (grant #EMCR2015-8022). AL is supported by Norges Forskningsråd, Kreftforeningen and Helse Sør-Øst RHF (NO). UE is supported by the NIH, National Heart, Lung, and Blood Institute, Award number K23-HL-126101. CJB and EJH are supported by the Dutch Kidney Foundation (Nierstichting), Award number: CP18.05.

Studying the proteome–the entire set of proteins in cells, tissues, organs and body fluids—is of great relevance in cancer research, as differential forms of proteins are expressed in response to specific intrinsic and extrinsic signals. Discovering protein signatures/pathways responsible for cancer transformation may lead to a better understanding of tumor biology and to a more effective diagnosis, prognosis, recurrence and response to therapy. Moreover, proteins can act as a biomarker or potential drug targets. Hence, it is of major importance to implement proteomic, particularly mass spectrometric, approaches in cancer research, to provide new crucial insights into tumor biology. Recently, mass spectrometry imaging (MSI) approaches were implemented in cancer research, to provide individual molecular characteristics of each individual tumor while retaining molecular spatial distribution, essential in the context of personalized disease management and medicine.

Urinary extracellular vesicles (EVs) are an attractive source of biomarkers for urological diseases. A crucial step in biomarker discovery studies is the determination of the variation parameters to perform a sample size calculation. In this way, a biomarker discovery study with sufficient statistical power can be performed to obtain biologically significant biomarkers. Here, a variation study was performed on both the protein and lipid content of urinary EVs of healthy individuals, aged between 52 and 69 years. Ultrafiltration (UF) in combination with size exclusion chromatography (SEC) was used to isolate the EVs from urine. Different experimental variation set-ups were used in this variation study. The calculated standard deviations (SDs) of the 90% least variable peptides and lipids did not exceed 2 and 1.2, respectively. These parameters can be used in a sample size calculation for a well-designed biomarker discovery study at the cargo of EVs.

Naturally occurring peptides, including growth factors, hormones, and neurotransmitters, represent an important class of biomolecules and have crucial roles in human physiology. The study of these peptides in clinical samples is therefore as relevant as ever. Compared to more routine proteomics applications in clinical research, peptidomics research questions are more challenging and have special requirements with regard to sample handling, experimental design, and bioinformatics. In this review, we describe the issues that confront peptidomics in a clinical context. After these hurdles are (partially) overcome, peptidomics will be ready for a successful translation into medical practice.

Extracellular vesicles (EVs) have a great potential in clinical applications. However, their isolation from different bodily fluids and their characterisation are currently not optimal or standardised. Here, we report the results of examining the performance of ultrafiltration combined with size exclusion chromatography (UF-SEC) to isolate EVs from urine. The results reveal that UF-SEC is an efficient method and provides high purity. Furthermore, we introduce asymmetrical-flow field-flow fractionation coupled with a UV detector and multi-angle light-scattering detector (AF4/UV-MALS) as a characterisation method and compare it with current methods. We demonstrate that AF4/UV-MALS is a straightforward and reproducible method for determining size, amount and purity of isolated urinary EVs.

RATIONALE The phosphorylation of proteins is one of the most important post-translational modifications in nature. Knowledge of the quantity or degree of protein phosphorylation in biological samples is extremely important. A combination of liquid chromatography (LC) and inductively coupled plasma mass spectrometry (ICP-MS) allows the absolute and relative quantification of the phosphorus signal. METHODS A comparison between dynamic reaction cell quadrupole ICP-MS (DRC-Q-ICP-MS) and high-resolution sector field ICP-MS (SF-ICP-MS) in detecting signals of phosphorus-containing species using identical capillary LC (reversed-phase technology) and nebulizer settings was performed. RESULTS A method to diminish the reversed-phase gradient-related signal instability in phosphorus detection with LC/ICP-MS applications was developed. Bis(4-nitrophenyl)phosphate (BNPP) was used as a standard to compare signal-to-noise ratios and limits of detection (LODs) between the two instrumental setups. The LOD reaches a value of 0.8 µg L−1 when applying the DRC technology in Q-ICP-MS and an LOD of 0.09 µg L−1 was found with the SF-ICP-MS setup. This BNPP standard was further used to compare the absolute quantification possibilities of phosphopeptides in these two setups. CONCLUSIONS This one-to-one comparison of two interference-reducing ICP-MS instruments demonstrates that absolute quantification of individual LC-separated phosphopeptides is possible. However, based on the LOD values, SF-ICP-MS has a higher sensitivity in detecting phosphorus signals and thus is preferred in phosphopeptide analysis. Copyright © 2014 John Wiley & Sons, Ltd.

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective tyrosine kinase inhibitors (TKIs). To optimize the use of these TKIs, the discovery of new biomarkers for early detection and disease progression is mandatory. These plasma-isolated exosomes can be used as a non-invasive and repeatable way for the detection and followup of these biomarkers. One ml of plasma from 12 NSCLC patients, with different mutations and treatments (and 6 healthy donors as controls), were used as exosome sources. After RNAse treatment, in order to degrade circulating miRNAs, the exosomes were isolated with a commercial kit and resuspended in specific buffers for further analysis. The exosomes were characterized by western blotting for ALIX and TSG101 and by transmission electron microscopy (TEM) analysis, the standard techniques to obtain biochemical and dimensional data of these nanovesicles. Total RNA extraction was performed with a high yield commercial kit. Due to the limited miRNA-content in exosomes, we decided to perform retro-transcription PCR using an individual assay for each selected miRNA. A panel of miRNAs (30b, 30c, 103, 122, 195, 203, 221, 222), all correlated with NSCLC disease, were analyzed taking advantage of the remarkable sensitivity and specificity of Real-Time PCR analysis; mir-1228-3p was used as endogenous control and data were processed according to the formula 2(-Delta Delta ct 13). Control values were used as baseline and results are shown in logarithmic scale.

With the current expanded technical capabilities to perform mass spectrometry-based biomedical proteomics experiments, an improved focus on the design of experiments is crucial. As it is clear that ignoring the importance of a good design leads to an unprecedented rate of false discoveries which would poison our results, more and more tools are developed to help researchers designing proteomic experiments. In this review, we apply statistical thinking to go through the entire proteomics workflow for biomarker discovery and validation and relate the considerations that should be made at the level of hypothesis building, technology selection, experimental design and the optimization of the experimental parameters. peerreview_statement: The publishing and review policy for this title is described in its Aims & Scope. aims_and_scope_url: http://www.tandfonline.com/action/journalInformation?show=aimsScope&journalCode=ieru20 ispartof: Expert Review of Proteomics vol:13 issue:5 pages:495-511 ispartof: location:England status: published

et al., The possibility that nanomaterials could perturb the normal course of an inflammatory response is a key issue when assessing nanoimmunosafety. The alteration of the normal progress of an inflammatory response may have pathological consequences, since inflammation is a major defensive mechanism and its efficiency maintains the body’s health. The immunosafety of engineered nanoparticles at nontoxic concentrations was investigated with the use of a human primary monocyte-based in vitro system, which reproduces in a simplified fashion the full course of the physiological inflammatory response, from initiation and development to resolution. The kinetics of expression and production of inflammatory and anti-inflammatory cytokines and the proteomic profiles were used for describing the inflammatory defensive response. We assessed the ability of gold and silver nanoparticles to trigger inflammation and to interfere with the course of an ongoing defensive reaction. While neither nanoparticle type was able to directly activate monocytes, silver nanoparticles could exacerbate the inflammatory response of monocytes but did not interfere with the resolution of the inflammatory reaction. These findings support the use of human primary monocyte-based in vitro assays for realistically investigating the effects of engineered nanoparticles on human innate immune responses, in order to predict the immunological risk of nanomaterials and implement safe nanoparticle-based applications, This study was supported by the EU FP7 projects NanoTOES (PITN-GA2010-264506), QualityNano (INFRA-2010-262163; TA Project, VITO-TAF-223) and NANoREG (NMP4-LA2013-3105984) and by the H2020 project PANDORA (MSCAITN-2015-671881).

In quantitative proteomics applications, the use of isobaric labels is a very popular concept as they allow for multiplexing, such that peptides from multiple biological samples are quantified simultaneously in one mass spectrometry experiment. Although this multiplexing allows that peptide intensities are affected by the same amount of instrument variability, systematic effects during sample preparation can also introduce a bias in the quantitation measurements. Therefore, normalization methods are required to remove this systematic error. At present, a few dedicated normalization methods for isobaric labeled data are at hand. Most of these normalization methods include a framework for statistical data analysis and rely on ANOVA or linear mixed models. However, for swift quality control of the samples or data visualization a simple normalization technique is sufficient. To this aim, we present a new and easy-to-use data-driven normalization method, named CONSTANd. The CONSTANd method employs constrained optimization and prior information about the labeling strategy to normalize the peptide intensities. Further, it allows maintaining the connection to any biological effect while reducing the systematic and technical errors. As a result, peptides can not only be compared directly within a multiplexed experiment, but are also comparable between other isobaric labeled datasets from multiple experimental designs that are normalized by the CONSTANd method, without the need to include a reference sample in every experimental setup. The latter property is especially useful when more than six, eight or ten (TMT/iTRAQ) biological samples are required to detect differential peptides with sufficient statistical power and to optimally make use of the multiplexing capacity of isobaric labels. This work was supported by SBO grant "InSPECtor" (120025) of the Flemish agency for Innovation by Science and Technology (IWT).

Receptorguanylate cyclases (rGCs) modulate diverse physiological processes including mammalian cardiovascular function and insect eclosion. The Drosophila genome encodes several receptor and receptor-like GCs, but no ligand for any Drosophila rGC has yet been identified. By screening peptide libraries in Drosophila S2 cells, the Drosophila peptide NPLP1-VQQ (NLGALKSSPVHGVQQ) was shown to be a ligand for the rGC, Gyc76C (CG42636, previously CG8742, l(3)76BDl, DrGC-1). In the adult fly, expression of Gyc76C is highest in immune and stress-sensing epithelial tissues, including Malpighian tubules and midgut; and NPLP1-VQQ stimulates fluid transport and increases cGMP content in tubules. cGMP signaling is known to modulate the activity of the IMD innate immune pathway in tubules via activation and nuclear translocation of the NF-kB orthologue, Relish, resulting in increased anti-microbial peptide (AMP) gene expression; and so NPLP1-VQQ might act in immune/stress responses. Indeed, NPLP1-VQQ induces nuclear translocation of Relish in intact tubules and increases expression of the anti-microbial peptide gene, diptericin. Targeted Gyc76C RNAi to tubule principal cells inhibited both NPLP1-VQQ-induced Relish translocation and diptericin expression. Relish translocation and increased AMP gene expression also occurs in tubules in response to dietary salt stress. Gyc76C also modulates organismal survival to salt stress - ablation of Gyc76C expression in only tubule principal cells prevents Relish translocation, reduces diptericin expression, and reduces organismal survival in response to salt stress. Thus, the principal-cell localized NPLP1-VQQ/Gyc76C cGMP pathway acts to signal environmental (salt) stress to the whole organism.

Neuromedin U (NMU) in vertebrates is a structurally highly conserved neuropeptide of which highest levels are found in the pituitary and gastrointestinal tract. In Drosophila, two neuropeptide genes encoding pyrokinins (PKs), capability (capa) and hugin, are possible insect homologs of vertebrate NMU. Here, the ligand for an orphan G protein-coupled receptor in the nematode Caenorhabditis elegans (Ce-PK-R) was found using a bioinformatics approach. After cloning and expressing Ce-PK-R in HEK293T cells, we found that it was activated by a neuropeptide from the C. elegans NLP-44 precursor (EC(50)=18nM). This neuropeptide precursor is reminiscent of insect CAPA precursors since it encodes a PK-like peptide and two periviscerokinin-like peptides (PVKs). Analogous to CAPA peptides in insects and NMUs in vertebrates, whole mount immunostaining in C. elegans revealed that the CAPA precursor is expressed in the nervous system. The present data also suggest that the ancestral CAPA precursor was already present in the common ancestor of Protostomians and Deuterostomians and that it might have been duplicated into CAPA and HUGIN in insects. In vertebrates, NMU is the putative homolog of a protostomian CAPA-PK.

To date, no great interest has been shown in the clinical implementation of recent Image-guided radiation therapy (IGRT) modalities in rectal cancer since only a few studies have been published on this issue. This may be explained by the fact that with current treatment modalities locoregional recurrences are already very low (around 10%). However, there is still room for improvement in treatment of high risk patients (cT3 CRM+, cT4, N+). In these patients better results may be obtained improving radiation technique from 2D to 3D, which showed to be more reliable in terms of target coverage. Also, when higher doses are delivered, Intensity Modulated Radiation Therapy (IMRT) may be used to spare small bowel. But before employing 3D irradiation or IMRT, a proper definition of our clinical target volume (CTV) and planning target volume (PTV) is needed. The CTV should encompass the tumour site, the mesorectum and the lateral nodes, recognized as the most likely sites of local recurrence, with different incidence according to tumour stage. Recent studies discussed the correct delineation of these target volumes in respect of tumour site and stage. From the preliminary results of a study conducted in Rome University 2D planning seemed insufficient to cover the different target volumes especially in T4 patients compared to 3D planning. Also an appropriate PTV margin is necessary in order to manage set-up errors and organ motion. Particularly in these patients, the knowledge of mesorectal movement is required to avoid target missing. Large mesorectal displacements were observed in a study carried out in Leuven University in collaboration with Rome University. A systematic review of the literature together with the data from these first experiences led to the awareness that IGRT could help us to follow the target volume and organs at risk during the treatment, allowing adjustments to improve accuracy in dose delivery, especially when dose escalation studies are planned in the treatment of rectal cancer.

Endogenous circadian clocks are inherent to all living organisms. They are needed to guarantee successful life since they regulate very important biological processes such as behavior and reproduction. Secretin-like G-protein coupled receptors are very important factors in the signal transduction pathways of circadian clocks. In this review, we will focus on the role of two secretin-like signaling pathways that play an important role in the regulation of the mammalian and the insect clock, respectively: the pituitary adenylate cyclase-activating polypeptide (PACAP) and pigment dispersing factor (PDF) signaling pathways. Both pathways are most likely related although their function in the biological clock differs.

In quantitative shotgun proteomic analyses by liquid chromatography and mass spectrometry, a rigid study design is necessary in order to obtain statistically relevant results. Hypothesis testing, sample size calculation and power estimation are fundamental concepts that require consideration upon designing an experiment. For this reason, the reproducibility and variability of the proteomic platform needs to be assessed. In this study, we evaluate the technical (sample preparation), labeling (isobaric labels), and total (biological + technical + labeling + experimental) variability and reproducibility of a workflow that employs a shotgun LC-MS/MS approach in combination with TMT peptide labeling for the quantification of peripheral blood mononuclear cell (PBMC) proteome. We illustrate that the variability induced by TMT labeling is small when compared to the technical variation. The latter is also responsible for a substantial part of the total variation. Prior knowledge about the experimental variability allows for a correct design, a prerequisite for the detection of biologically significant disease-specific differential proteins in clinical proteomics experiments. Evelyne Maes is funded by an Emmanuel Van der Schueren grant of the Vlaamse Liga tegen Kanker (VLK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The free-living nematode Caenorhabditis elegans is one of the most studied models in a wide variety of research fields with applications in agro- or pharmaceutical industries. It has been used for the development of new anthelminthic drugs and was proven to yield key insights in neurodegenerative diseases and metabolic syndromes. Due to its suitability for high-throughput genetic screens, efficiency for RNA interference approaches and the availability of thousands of mutants, most studies were carried out at the genetic level. However, determining the cellular function of each gene product remains an unfinished goal in this post-genomic era. A systems biology approach focusing on the actual gene products (i.e. proteins) can help unraveling this puzzle. A fundamental pillar in this research is mass spectrometry-based proteomics. We here provide an in-depth overview of proteomics-related studies in C. elegans research, with special emphasis on the methodologies and biological applications.

Although genomics has delivered major advances in cancer prognostics, treatment and diagnostics, it still only provides a static image of the situation. To study more dynamic molecular entities, proteomics has been introduced into the cancer research field more than a decade ago. Currently, however, the impact of clinical proteomics on patient management and clinical decision-making is low and the implementations of scientific results in the clinic appear to be scarce. The search for cancer-related biomarkers with proteomics however, has major potential to improve risk assessment, early detection, diagnosis, prognosis, treatment selection and monitoring. In this review, we provide an overview of the transition of oncoproteomics towards translational oncology. We describe which lessons are learned from currently approved protein biomarkers and previous proteomic studies, what the pitfalls and challenges are in clinical proteomics applications, and how proteomic research can be successfully translated into medical practice.

G-protein coupled receptors (GPCRs) are ancient molecules that can sense environmental and physiological signals. Currently, the majority of the predicted Caenorhabditis elegans GPCRs are orphan. Here, we describe the characterization of such an orphan C. elegans GPCR, which is categorized in the tachykinin-like group of receptors. Since the C. elegans genome predicts only one tachykinin-like peptide (SFDRMGGTEFGLM), which could not activate the receptor, we hypothesized that one or some of the numerous FMRFamide related peptides (FaRPs) could be the cognate ligands for this receptor. This hypothesis was based on the suggestion that RFamides may be ancestral neuropeptides, from which a lot of the amidated neuropeptides, including tachykinins, derived. Indeed, we found that the orphan receptor encoded by the Y59H11AL.1 gene is activated by several C. elegans neuropeptides, including SPMERSAMVRFamide. These peptides activate the receptor in a concentration-dependent way.

SummaryThe neuropeptide Pigment-Dispersing Factor (PDF) is a principle transmitter regulating circadian locomotor rhythms in Drosophila. We have identified a Class II (secretin-related) G protein-coupled receptor (GPCR) that is specifically responsive to PDF and also to calcitonin-like peptides and to PACAP. In response to PDF, the PDF receptor (PDFR) elevates cAMP levels when expressed in HEK293 cells. As predicted by in vivo studies, cotransfection of Neurofibromatosis Factor 1 significantly improves coupling of PDFR to adenylate cyclase. pdfr mutant flies display increased circadian arrhythmicity, and also display altered geotaxis that is epistatic to that of pdf mutants. PDFR immunosignals are expressed by diverse neurons, but only by a small subset of circadian pacemakers. These data establish the first synapse within the Drosophila circadian neural circuit and underscore the importance of Class II peptide GPCR signaling in circadian neural systems.

G-protein-coupled receptors (GPCRs) constitute one of the largest families of membrane-spanning proteins. Their importance in drug development has been proven over and over again. Therefore, they remain one of the most significant groups of molecules to be characterized. In the postgenomic era, the methods used for the characterization of GPCRs have dramatically changed: the predicted orphan receptors are now often used to ascertain the ligands (reverse pharmacology), whereas, in the past, the bioactive ligand was used to identify the receptor (classic approach). In this review, we will give an overview of the recent postgenomic functional assays that are frequently used to link the orphan GPCR of both vertebrate and invertebrate organisms with their ligands.

We report the cloning of the complete coding sequence of the putative chicken type 2 corticotropin-releasing hormone receptor (CRH-R2) by rapid amplification of cDNA ends (RACE). The chicken CRH-R2 is a 412-amino acid 7-transmembrane G protein-coupled receptor, showing 87% identity to the Xenopus laevis and Oncorhynchus keta CRH-R2s, and 78-80% to mammalian CRH-R2s. The distribution of CRH-R2 mRNA was studied by RT-PCR analysis and compared to CRH-R1 distribution. Both CRH-R1 and CRH-R2 mRNA are expressed in the main chicken brain parts. In peripheral organs, CRH-R1 mRNA shows a more restricted distribution, whereas CRH-R2 mRNA is expressed in every tissue investigated, indicating that a number of actions of CRH and/or CRH-like peptides remain to be discovered in the chicken as well as in other vertebrates.

In this study, we describe the cloning and the characterization of the third FMRFamide-related peptide (FaRP) receptor in Caenorhabditis elegans, the VRFa receptor 1. Numerous structurally different FaRPs were synthesized and used to screen the orphan C26F1.6 receptor for activation. Two peptides ending in M(orL)VRFamide elicited a calcium response in receptor expressing mammalian cells. The response is dose-dependent and appeared to be very specific, since very closely related FaRPs were less active, even the other peptides ending in M(orL)VRFamide. Pharmacological profiling of the most active peptide suggests that SMVRFa is the most active binding core. N-terminal extension decreases peptide activity.

A seven transmembrane G-protein coupled receptor has been cloned from Drosophila melanogaster. This receptor shows structural similarities to vertebrate Neuropeptide Y(2) receptors and is activated by endogenous Drosophila peptides, recently designated as short neuropeptide Fs (sNPFs). sNPFs have so far been found in neuroendocrine tissues of four other insect species and of the horseshoe crab. In locusts, they accelerate ovarian maturation, and in mosquitoes, they inhibit host-seeking behavior. Expression analysis by RT-PCR shows that the sNPF receptor (Drm-sNPF-R) is present in several tissues (brain, gut, Malpighian tubules and fat body) from Drosophila larvae as well as in ovaries of adult females. All 4 Drosophila sNPFs clearly elicited a calcium response in receptor expressing mammalian Chinese hamster ovary cells. The response is dose-dependent and appeared to be very specific. The short NPF receptor was not activated by any of the other tested arthropod peptides, not even by FMRFamide-related peptides (also ending in RFamide), indicating that the Arg residue at position 4 from the amidated C-terminus appears to be crucial for the response elicited by the sNPFs.

The phosphorylation of proteins is one of the most important post-translational modifications in nature. Knowledge of the quantity or degree of protein phosphorylation in biological samples is extremely important. A combination of liquid chromatography (LC) and inductively coupled plasma mass spectrometry (ICP-MS) allows the absolute and relative quantification of the phosphorus signal.A comparison between dynamic reaction cell quadrupole ICP-MS (DRC-Q-ICP-MS) and high-resolution sector field ICP-MS (SF-ICP-MS) in detecting signals of phosphorus-containing species using identical capillary LC (reversed-phase technology) and nebulizer settings was performed.A method to diminish the reversed-phase gradient-related signal instability in phosphorus detection with LC/ICP-MS applications was developed. Bis(4-nitrophenyl)phosphate (BNPP) was used as a standard to compare signal-to-noise ratios and limits of detection (LODs) between the two instrumental setups. The LOD reaches a value of 0.8 µg L(-1) when applying the DRC technology in Q-ICP-MS and an LOD of 0.09 µg L(-1) was found with the SF-ICP-MS setup. This BNPP standard was further used to compare the absolute quantification possibilities of phosphopeptides in these two setups.This one-to-one comparison of two interference-reducing ICP-MS instruments demonstrates that absolute quantification of individual LC-separated phosphopeptides is possible. However, based on the LOD values, SF-ICP-MS has a higher sensitivity in detecting phosphorus signals and thus is preferred in phosphopeptide analysis.

Globally, colorectal cancer (CRC) is the third most common malignant neoplasm. However, highly sensitive, specific, noninvasive tests that allow CRC diagnosis at an early stage are still needed. As circulatory blood reflects the physiological status of an individual and/or the disease status for several disorders, efforts have been undertaken to identify candidate diagnostic CRC markers in plasma and serum. In this review, the challenges, bottlenecks and promising properties of mass spectrometry (MS)-based proteomics in blood are discussed. More specifically, important aspects in clinical design, sample retrieval, sample preparation, and MS analysis are presented. The recent developments in targeted MS approaches in plasma or serum are highlighted as well.

Shotgun proteomics is a powerful technology to study the protein population of a biological system. This approach employs tandem mass spectrometry for amino acid sequencing. Fragmented ion masses can be used in correlative database-searching, like SEQUEST or Mascot, to identify peptides. The database-search method depends upon a score function that evaluates matches between the predicted ions and the ions observed in the tandem mass spectrum. Principally, peptide identification based on tandem MS and database-search algorithms does not take into account information about isotope distributions of the precursor ions. To determine the effectiveness of these search algorithms in terms of their ability to distinguish between correct and incorrect peptide assignments, we propose an additional metric that quantifies the similarity between the theoretical isotopic distribution for the precursor ions selected for tandem MS and the experimental mass spectra by using Pearson's χ2 statistic. The observed association between Pearson's χ2 statistic and the score function indicates that good scores can be obtained for molecules which exhibit atypical isotope profiles, while low scores can be obtained for fragment spectra which have a clear peptide-like isotope pattern. These results demonstrate that Pearson's χ2 statistic can be used in conjunction with the score of database-search algorithms to increase the sensitivity and specificity of peptide identification. Biological significance In this manuscript, we present a workflow that provides a new perspective on the quality of peptide-to-spectrum matches (PSM) employed in database-searching strategies for peptide identification. Additional views on a dataset can facilitate a more hypothesis-driven interpretation of the mass spectrometry signals. The similarity metric on the PSM scores contemplates the isotopic profile and results in a measure that conveys a degree of biomolecular similarity observed from the precursor of the selected tandem MS spectra. A close agreement between the PSM score and the similarity metric will result in a higher confidence for the identification of the selected precursor ion.

Reversible phosphorylation is one of the most important post-translational modifications in mammalian cells. Because this molecular switch is an important mechanism that diversifies and regulates proteins in cellular processes, knowledge about the extent and quantity of phosphorylation is very important to understand the complex cellular interplay. Although phosphoproteomics strategies are applied worldwide, they mainly include only molecular mass spectrometry (like MALDI or ESI)-based experiments. Although identification and relative quantification of phosphopeptides is straightforward with these techniques, absolute quantification is more complex and usually requires for specific isotopically phosphopeptide standards. However, the use of elemental mass spectrometry, and in particular inductively coupled plasma mass spectrometry (ICP-MS), in phosphoproteomics-based experiments, allow one to absolutely quantify phosphopeptides. Here, these phosphoproteomic applications with ICP-MS as elemental detector are reviewed. Pioneering work and recent developments in the field are both described. Additionally, the advantage of the parallel use of molecular and elemental mass spectrometry is stressed. ispartof: Mass Spectrometry Reviews vol:35 issue:3 pages:350-360 ispartof: location:United States status: published