Project description:The water vole Arvicola terrestris (= Arvicola amphibious L.) is endemic in Europe where its outbreak generates severe economic losses for farmers. Our project aimed at deciphering the modalities of chemical communication in this species, to develop new sustainable methods for populations control. The water vole, as well as other rodents, uses specific urination sites as territorial and sex pheromone markers, the chemical and biochemical identification of which is still unknown. Lateral scent glands and urine samples were collected from wild males and females caught in the field, at different periods of the year. Volatile signals were searched in urine by SPME/GC-MS, and in lateral scent glands by solvent extraction followed by GC-MS. The volatile composition of urine was analysed for each individual and not on pooled samples, showing no significant difference between males and females. Lateral scent glands contained some volatile components (pyrazines, alcohols, terpenes), and mostly long chain fatty acid esters, again without quantitative and qualitative differences between sexes. Conversely, the urinary protein content, analysed by 1D- and 2D-electrophoresis is different, as only males secrete high levels of lipocalins, whatever the reproduction period. The proteins were identified by mass spectrometry and “de novo” analysis (Orbitrap), which provided specific information to design primers for PCR amplification of cDNA sequences. Thus, urinary proteins were characterized by direct amplification from liver RNA. The identified protein sequences are closed to those of Myodes glareolus, a closely related species of Cricetidae, and to hamster Aphrodisin.
Project description:In-depth LC-MS-based proteomic profiling of limited samples has been problematic due, in large part, to the inefficiency of sample preparation and attendant sample losses. To address this issue, we developed On-Micro Solid-phase Extraction Tip-based (OmSET) sample preparation for limited biological samples. OmSET is simple, efficient, reproducible, and scalable, and is a widely accessible method for processing ~200 to 10,000 cells. The developed method benefits from minimal sample processing volumes (1-3 μL) and conducting all sample processing steps on-membrane within a single microreactor. Here, we assessed the feasibility of using micro-SPE tips for nanogram-level amounts of tryptic peptides, minimized the number of required sample handling steps and reduced the hands-on time, and evaluated the capability of OmSET for quantitative analysis of low numbers of human monocytes.
Project description:Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for retrospective studies but protein extraction and subsequent sample processing steps have shown to be challenging for mass spectrometry (MS) analysis. Streamlined high-throughput sample preparation workflows are essential for efficient peptide extraction from complex clinical specimens such as fresh frozen tissues or FFPE. Overall, proteome analysis has gained significant improvements in the instrumentation, acquisition methods, sample preparation workflows and analysis pipelines yet even the most recent FFPE workflows remain complex and are not readily scalable. Here, we present an optimized workflow for Automated Sonication-free Acid-assisted Proteome (ASAP) extraction from FFPE sections. ASAP enables efficient protein extraction from FFPE specimens achieving similar proteome coverage as established methods using time in equipment-heavy sonication-based methods at reduced sample processing time. The broad applicability of ASAP on archived pediatric tumor FFPE specimens resulted in high-quality data with increased proteome coverage and quantitative reproducibility. Our study demonstrates the practicality and superiority of the ASAP workflow as a streamlined, time and cost-effective pipeline for high-throughput FFPE proteomics of clinical specimens.
Project description:We report here that a straightforward change of the standard derivatization procedure for GC–MS metabolomics is leading to a strong increase in metabolite signal intensity. Drying samples between methoxymation and trimethylsilylation significantly increased signals by two- to tenfold in extracts of yeast cells, plant and animal tissue, and human urine. This easy step reduces the cost of sample material and the need for expensive new hardware.
Project description:<p>Clinical metabolic phenotyping employs metabolomics and lipidomics to detect and measure thousands of metabolites and lipids within human samples. This approach aims to identify metabolite and lipid changes between phenotypes (e.g. disease status) that aid understanding of biochemical mechanisms driving the phenotype. Sample preparation is a critical step in clinical metabolic phenotyping: it must be reproducible and give a high extraction yield of metabolites and lipids. Here, we assessed the extraction of polar metabolites from human urine and polar metabolites and lipids from human plasma for analysis by ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) metabolomics and lipidomics. We evaluated several monophasic (urine and plasma) and biphasic (plasma) extractions, and we also tested alterations to (a) solvent-biofluid incubation time and temperature during monophasic extraction, and (b) phase partitioning time during biphasic extraction. Extracts were analysed by three UHPLC-MS assays: (i) HILIC for urine and plasma, (ii) C18 aqueous reversed phase for urine, and (iii) C18 reversed phase for plasma lipids, and the yield and reproducibility of each method was assessed. For HILIC UHPLC-MS plasma and urine analysis, monophasic 50:50 methanol:acetonitrile had the most detected putatively-identified polar metabolites. If lipid removal from the plasma polar HILIC extract is required, then the biphasic methanol/chloroform/water method is recommended. For C18 (aqueous) UHPLC-MS urine analysis, 50:50 methanol:water had high reproducibility and yield. For C18 UHPLC-MS plasma lipidomics, monophasic 100% isopropanol had the highest detection response of all annotated lipid classes. Increasing monophasic incubation time and temperature had little benefit on metabolite and lipid yield and reproducibility.</p>
Project description:Urine provides a diverse source of information related to health status and is ideal for clinical proteomics because of its ease of collection. To date, there is no standard operating procedure for reproducible and robust urine sample processing for mass spectrometry-based clinical proteomics. To address this need, a novel workflow was developed based on an on-bead protein capture, clean up, and digestion without the requirement from pre-processing steps such as precipitation. The workflow was applied to an acute kidney injury (AKI) pilot study. Urine from clinical samples and a pooled sample were subjected to automated sample preparation in a KingFisher™ Flex magnetic handling station using a novel urine-HILIC (uHLC) approach based on MagReSyn® HILIC microspheres. For benchmarking, the pooled sample was also prepared using a published protocol based on an on-membrane (OM) protein capture and digestion workflow. Peptides were analysed by LCMS in DIA mode using a Dionex Ultimate 3000 UPLC coupled to a Sciex 5600 mass spectrometer. Data was searched in Spectronaut™ 17. Following statistical analysis, candidate protein markers were filtered at ≥ 2-fold change in abundance, ≥ 2 uniques peptides and ≤ 1% false discovery rate. Both workflows showed similar peptide and protein identifications in the pooled sample. The uHLC workflow was easier to set up and complete, having less hands-on time than the OM method. With fewer manuel processiing steps, a lower peptide and protein CV was observed in the uHLC technical replicates. Analysis of clinical samples revealed many significant, differentially abundant kidney injury-associated urinary proteins. The pilot data derived using this novel workflow provides information on the urinary proteome of patients with AKI. Further exploration in a larger cohort using this novel high-throughput method is warranted.
Project description:Development of a modified solid phase extraction (SPE) approach for improved peptide yield and analysis sensitivity with LC-MS based peptidomics of urine.
Project description:GC-MS is a commonly used metabolomic platform for the analysis of urine. A key step in the preparation of samples for GC-MS is derivatisation, in particular, methoximation and trimethylsilylation. This paper presents an assessment of automated derivatisation protocols for GC-MS-based untargeted metabolomic analysis of rat urine. Automated batch and in-time (a sample ready for injection every 70 minutes) derivatisation protocols were tested using BSTFA and MSTFA. Principal component analysis determined differences based upon protocol tested (PC-1; 19%) and silylation reagent (PC-2; 17%) used. Of 249 compounds, 40 compounds were significantly different (P<0.05) based upon reagent and 154 compounds were significantly different (P<0.05) based upon protocol. A key outcome of this study was the demonstrated effects of derivatisation including reagent and protocol (i.e. reaction duration, temperature and mixing speed) on individual urinary metabolites. It is hoped that the current work will provide a reference on which to base future GC-MS-based untargeted and targeted metabolomic analyses of urine.