Mayfly and fish species identification and sex determination in bleak (Alburnus alburnus) by MALDI-TOF mass spectrometry.

Besides food quality control of fish or cephalopods, the novel mass spectrometry (MS) approaches could be effective and beneficial methods for the investigation of biodiversity in ecological research. Our aims were to verify the applicability of MALDI-TOF MS in the rapid identification of closely related species, and to further develop it for sex determination in phenotypically similar fish focusing on the low mass range. For MALDI-TOF MS spectra analysis, ClinProTools software was applied, but our observed classification was also confirmed by Self Organizing Map. For verifying the wide applicability of the method, brains from invertebrate and vertebrate species were used in order to detect the species related markers from two mayflies and eight fish as well as sex-related markers within bleak. Seven Ephemera larvae and sixty-one fish species related markers were observed and nineteen sex-related markers were identified in bleak. Similar patterns were observed between the individuals within one species. In contrast, there were markedly diverse patterns between the different species and sexes visualized by SOMs. Two different Ephemera species and male or female fish were identified with 100% accuracy. The various fish species were classified into 8 species with a high level of accuracy (96.2%). Based on MS data, dendrogram was generated from different fish species by using ClinProTools software. This MS-based dendrogram shows relatively high correspondence with the phylogenetic relationships of both the studied species and orders. In summary, MALDI-TOF MS provides a cheap, reliable, sensitive and fast identification tool for researchers in the case of closely related species using mass spectra acquired in a low mass range to define specific molecular profiles. Moreover, we presented evidence for the first time for determination of sex within one fish species by using this method. We conclude that it is a powerful tool that can revolutionize ecological and environmental research.

HPLC-MS/MS analysis of steroid hormones in environmental water samples.

Today, freshwaters, such as lakes and rivers, are subject to controlled pollution. Steroid hormones are chemically very stable highly lipophilic molecules. Their biological properties have a strong impact on the endocrine regulation of species. Steroids have estrogenic, androgenic, thyroidogenic or progestogenic effects and based on them, they could disturb the physiological mechanisms of freshwater species. We focused on progestins as they are the main active ingredients of contraceptive pharmaceuticals. Progestins have been shown to impair reproduction in fish, amphibians, and mollusks at low ng/L concentrations. Certain progestins, such as levonorgestrel (LNG) have androgenic properties also. We selected the most used active substances drospirenone (DRO), LNG, and progesterone (PRG) and then developed and optimized a liquid chromatographic-mass spectrometric method with solid-phase extraction to measure them. Using our sensitive method (LOQ 0.03-0.11 ng/L) we could measure steroids even between 0.1 and 1 ng/L. Analyzing freshwater samples from the Lake Balaton catchment area, we found influents where the concentration of these hormones was 0.26-4.30 (DRO), 0.85-3.40 (LNG), and 0.23-13.67 (PRG) ng/L. Out of 53 collecting places, 21 contained measurable progestin levels, which clearly demonstrates the applicability of our method, legitimates toxicology experiments with effected species, and indicates monitoring efforts.

Comparative protein composition of the brains of PACAP-deficient mice using mass spectrometry-based proteomic analysis.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a widespread neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. The diverse biological actions provide the background for the variety of deficits observed in mice lacking endogenous PACAP. PACAP-deficient mice display several abnormalities, such as sudden infant death syndrome (SIDS)-like phenotype, decreased cell protection, and increased risk of Parkinson's disease. However, the molecular and proteomic background is still unclear. Therefore, our aim was to investigate the differences in peptide and protein composition in the brains of PACAP-deficient and wild-type mice using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric (MS)-based proteomic analysis. Brains from PACAP-deficient mice were removed, and different brain areas (cortex, hippocampus, diencephalon, mesencephalon, brainstem, and cerebellum) were separated. Brain pieces were weighed, homogenized, and further processed for electrophoretic analysis. Our results revealed several differences in diencephalon and mesencephalon. The protein bands of interest were cut from the gel, samples were digested with trypsin, and the tryptic peptides were measured by matrix-assisted laser desorption ionization time of flight (MALDI TOF) MS. Results were analyzed by MASCOT Search Engine. Among the altered proteins, several are involved in metabolic processes, energy homeostasis, and structural integrity. ATP-synthase and tubulin beta-2A were expressed more strongly in PACAP-knockout mice. In contrast, the expression of more peptides/proteins markedly decreased in knockout mice, like pyruvate kinase, fructose biphosphate aldolase-A, glutathione S-transferase, peptidyl propyl cis-trans isomerase-A, gamma enolase, and aspartate amino transferase. The altered expression of these enzymes might partially account for the decreased antioxidant and detoxifying capacity of PACAP-deficient mice accompanying the increased vulnerability of these animals. Our results provide novel insight into the altered biochemical processes in mice lacking endogenous PACAP.

High resolution spatial distribution of neuropeptides by MALDI imaging mass spectrometry in the terrestrial snail, Helix pomatia.

Imaging mass spectrometry (IMS) is a powerful technique that combines the chemical and spatial analysis of surface materials. It allows spatial localization of peptides, proteins or lipids that are recorded in parallel without the need of a label. It is currently one of the most rapidly developing techniques in the proteomics toolbox. In the present study, accurate mass matrix-assisted laser desorption/ionization imaging mass spectrometry (MALD IMS) was used for direct molecular mapping of nervous tissue at micrometer spatial resolution. Cryosections of the whole brain of the terrestrial snail, Helix pomatia, were placed on indium-tin-oxide (ITO)-coated conductive glass slides and covered with a thin layer of α-cyano-4-hydroxycinnamic acid (CHCA) matrix by electro spray deposition. High-resolution molecular ion maps of well-known neuropeptides, such as FMRFamide were constructed. FMRFamide is known to exert powerful modulatory effect on synaptic transmission in molluscs. FMRFamide was predominantly localized in the cluster of neurons in the pro-, meso- and postcerebral regions of cerebral ganglia, pedal ganglia and right parietal ganglia of the central nervous system. Our present study, using MALDI IMS confirmed the distribution of FMRFamide containing cells in the Helix central nervous system previously detected by antibody dependent immunohistochemistry.

Effects of pituitary adenylate cyclase activating polypeptide on human sperm motility.

Pituitary adenylate cyclase activating polypeptide (PACAP), a neuropeptide with diverse effects, was originally isolated as a hypothalamo-hypophyseal peptide. Subsequent studies showed highest levels of PACAP in the testis after the brain, suggesting that it influences the development and functioning of spermatozoa. Indeed, it has been proven that PACAP has an effect on spermatogenesis, both locally and via influencing the hypothalamo-hypophyseal-gonadal axis. The aim of the present study was to determine whether PACAP has an effect on human sperm motility and whether it is present in the human seminal fluid. Furthermore, the sperm head morphology was studied in mice lacking endogenous PACAP. Human samples were obtained from healthy adult volunteers and andrological patients. The effects of PACAP on the motility of human sperm cells were investigated using a computer aided sperm analysis system. In cases where the motility was lower, addition of PACAP to the samples increased the motility and the ratio of rapid progressive and medium progressive sperm motility groups. The presence of PACAP could not be detected in human seminal fluid samples by means of mass spectrometry. Investigating sperm head morphology with routine histology in PACAP deficient mice revealed that both the longitudinal and transverse diameters were significantly lower in PACAP deficient mice, without marked difference in the shape, as revealed by scanning electron microscopy.

High-throughput screening of saliva for early detection of oral cancer: a pilot study.

The success of tumour therapy depends considerably on early diagnosis. Therefore, we aimed to develop a widely available, cheap, non-invasive, high-throughput method suitable for screening high-risk populations, at least, for early signs of malignant transformation in the oral cavity. First, in order to identify suitable tumour marker candidates, we compared the protein patterns of five selected saliva samples obtained from healthy controls and tumour patients after electrophoretic separation, excised the bands that were consistently up-regulated in the tumour patients only, and performed matrix-assisted laser-desorption ionisation (MALDI)-time of flight (TOF) tandem mass spectrometry (MS/MS) analysis of the proteins in these bands after in-gel tryptic digestion. From the panel of proteins identified, we chose annexin 1 and peroxiredoxin 2 for further studies based on their presence in the saliva of all five oral cancer patients only. Then, we performed a homology search of protein databases using the primary sequence of each in silico tryptic fragment peptide of these two proteins as bait, and selected a unique peptide for each. Finally, we performed targeted MALDI-TOF MS peptide analysis in a blinded fashion on all samples obtained from 20 healthy controls and 22 tumour patients for the presence of these peptides. We found both peptides present in the saliva samples of all cancer patients only. Even though these tumour markers should be validated in a wider population, our results indicate that targeted MALDI-TOF MS analysis of unique peptides of putative saliva protein tumour biomarkers could be the method of choice for cost-efficient, high-throughput screening for the early detection of oral cancer.