Rapid detection of peptide markers for authentication purposes in raw and cooked meat using ambient liquid extraction surface analysis mass spectrometry

In this paper, our previously developed ambient LESA-MS methodology is implemented to analyze five types of thermally treated meat species, namely beef, pork, horse, chicken, and turkey meat, in order to select and identify heat-stable and species-specific peptide markers. In-solution tryptic digests of cooked meats were deposited onto a polymer surface, followed by LESA-MS analysis and evaluation using multivariate data analysis and tandem electrospray MS. The five types of cooked meat were clearly discriminated using principal component analysis and orthogonal partial least squares discriminant analysis. A number of 23 heat stable peptide markers unique to species and muscle protein were identified following data-dependent tandem LESA-MS analysis. Surface extraction and direct ambient MS analysis of mixtures of cooked meat species was performed for the first time and enabled detection of 10% (w/w) of pork, horse, and turkey meat, and 5% (w/w) of chicken meat in beef, using the developed LESA-MS/MS analysis. The study shows, for the first time, that ambient LESA-MS methodology displays specificity sufficient to be implemented effectively for the analysis of processed and complex peptide digests. The proposed approach is much faster and simpler than other measurement tools for meat speciation; it has potential for application in other areas of meat science or food production

Parafilm-assisted microdissection: a sampling method for mass spectrometry-based identification of differentially expressed prostate cancer protein biomarkers

International audienceMass spectrometry-based methods for prostate cancer biomarker discovery are hampered by their low-throughput capabilities because of extensive sample preparation. We present the parafilm-assisted microdissection technique coupled with label-free quantification and bioinformatics analysis as a means to evaluate directly protein expression changes on benign and tumor regions

In Utero Exposure to Metformin Reduces the Fertility of Male Offspring in Adulthood.

peer reviewedMetformin is a drug used for the treatment of type 2 diabetes and disorders associated with insulin resistance. Metformin is also used in the treatment of pregnancy disorders such as gestational diabetes. However, the consequences of foetal exposure to metformin on the fertility of exposed offspring remain poorly documented. In this study, we investigated the effect of in utero metformin exposure on the fertility of female and male offspring. We observed that metformin is detectable in the blood of the mother and in amniotic fluid and blood of the umbilical cord. Metformin was not measurable in any tissues of the embryo, including the gonads. The effect of metformin exposure on offspring was sex specific. The adult females that had been exposed to metformin in utero presented no clear reduction in fertility. However, the adult males that had been exposed to metformin during foetal life exhibited a 30% reduction in litter size compared with controls. The lower fertility was not due to a change in sperm production or the motility of sperm. Rather, the phenotype was due to lower sperm head quality - significantly increased spermatozoa head abnormality with greater DNA damage - and hypermethylation of the genomic DNA in the spermatozoa associated with lower expression of the ten-eleven translocation methylcytosine dioxygenase 1 (TET1) protein. In conclusion, while foetal metformin exposure did not dramatically alter gonad development, these results suggest that metabolic modification by metformin during the foetal period could change the expression of epigenetic regulators such as Tet1 and perturb the genomic DNA in germ cells, changes that might contribute to a reduced fertility

Real-Time Molecular Diagnosis of Tumors Using Atmospheric Pressure Infrared Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry

International audienceTissue diagnosis is critical in the clinical management of cancer patients. Mass Spectrometry (MS) has long been of interest for discriminating cells using tissue sections of patient biopsies. However, the main challenge arises in the context of intraoperative tissue analyses, where the MS instrument must operate within a surgical environment. To date, only the Intelligent Knife (iKnife) system allowed real-time monitoring during surgery by collecting aerosol released during tissue excision with an electric scalpel or bipolar forceps [1]. Recently we have demonstrated that in vivo real-time analyses can be less invasive using laser ablation. In our prototype, a fibered IR Optical Parametric Oscillator (OPO) is tuned at 2.94 µm to excite the most intense vibrational band (O-H stretching mode) of water molecules found abundantly in all biological tissues. Water acts as a natural endogenous Matrix-Assisted Laser Desorption-Ionization (MALDI) matrix leading to the production of ions that can be transported over a few meters to a MS instrument for analysis without requiring further post-ionization handling. The molecular patterns (metabolites, lipids and proteins) thus retrieved are specific to cell phenotypes and benign versus cancer regions can easily be differentiated [2]. We present the first assessment of our prototype in a veterinary surgery room [3]. For this purpose, a series of benchmark studies have been initiated with the aim of building up an extensive databank able to relate distinct molecular profiles to a specific type of pathology, namely the dog sarcoma. These studies are accompanied by the development of a real-time query interface. Classification models based on tumor grade (cancer/normal/necrotic) and cancer subtype developed in this work showed a minimum of ~98% correct classification when put to use. The system demonstrated clear-cut margin detection capabilities that have been validated in correlation with histology. Finally, this instrument enables real-time diagnostics by the immediate interrogation of classification models established ahead of time

Laser Ablation with Vacuum Capture for MALDI Mass Spectrometry of Tissue

© 2015 American Society for Mass Spectrometry. We have developed a laser ablation sampling technique for matrix-assisted laser desorption ionization (MALDI) mass spectrometry and tandem mass spectrometry (MS/MS) analyses of in-situ digested tissue proteins. Infrared laser ablation was used to remove biomolecules from tissue sections for collection by vacuum capture and analysis by MALDI. Ablation and transfer of compounds from tissue removes biomolecules from the tissue and allows further analysis of the collected material to facilitate their identification. Laser ablated material was captured in a vacuum aspirated pipette-tip packed with C18 stationary phase and the captured material was dissolved, eluted, and analyzed by MALDI. Rat brain and lung tissue sections 10 μm thick were processed by in-situ trypsin digestion after lipid and salt removal. The tryptic peptides were ablated with a focused mid-infrared laser, vacuum captured, and eluted with an acetonitrile/water mixture. Eluted components were deposited on a MALDI target and mixed with matrix for mass spectrometry analysis. Initial experiments were conducted with peptide and protein standards for evaluation of transfer efficiency: a transfer efficiency of 16% was obtained using seven different standards. Laser ablation vacuum capture was applied to freshly digested tissue sections and compared with sections processed with conventional MALDI imaging. A greater signal intensity and lower background was observed in comparison with the conventional MALDI analysis. Tandem time-of-flight MALDI mass spectrometry was used for compound identification in the tissue

Quantitative urinary proteomics using stable isotope labelling by peptide dimethylation in patients with prostate cancer

Prostate cancer (PCa) is the most commonly diagnosed malignancy in men. The current prevalent diagnosis method, prostate-specific antigen (PSA) screening test, has low sensitivity, specificity and is poor at predicting the grade of disease. Thus, new biomarkers are urgently needed to improve the PCa diagnosis and staging for the management of patients. The aim of this study is to investigate the first voided urinary sample after massage for biomarker discovery for PCa. In this work, untargeted metabolomic profiling of the first voided urinary sample after massage from 28 confirmed prostate cancer patients, 20 benign enlarged prostate patients and 6 healthy volunteers was performed using liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/MS). Single and multiple peptide protein and cross-linking molecules were identified using PEAKS software. Analytical and diagnostic performance was tested using the Student's t test, Benjamini Hochberg correction and the receiver operating characteristic (ROC) curves. Using differential display analysis to compare peptides and cross-linking molecules of urinary samples between patients with benign, enlarged prostate and malignant cancer, we identified multiple peptides derived from osteopontin (SPP1) and prothrombin (F2) that are lower in PCa patients than in benign and enlarged prostate. The diagnosis accuracies of SPP1 and F2 peptides are 0.65-0.77 and 0.68-0.72, respectively. In addition to this, there are significant differences between PCa and benign/enlarged prostate patients in pyridinoline (PYD) and deoxypyridinoline (DPD) (p value = 0.001). Differences also, as shown in the excretion of these molecules for different stages of PCa (p value = 0.04) as the level of DPD and DPD/PYD ratio, were high in patients with locally advanced tumours. The study underscores the importance of proteomics analysis, and our results demonstrate that a urinary-based in depth proteomic approach allows the potential identification of dysregulated pathways and diagnostic biomarkers. </p