Chemosensory attention, habituation and detection in women and men.

The aim of this study was to investigate whether there are differences between women and men in how chemosensory stimuli are processed. Event-related potentials from 36 participants (18 men) showed that women had larger P3 amplitudes when attending, but not when ignoring CO(2) and amyl acetate stimuli compared with men. Conversely, auditory P3 was reduced by the same degree in women and men. No gender differences were found in magnitude estimations over time. Women had lower detection thresholds for CO(2), but not for n-butanol, compared with men. The main finding was that women and men differ in cognitive measures of chemosensory processing.

Immunoaffinity enrichments followed by mass spectrometric detection for studying global protein tyrosine phosphorylation.

Phosphorylation of protein tyrosine residues regulates important cell functions and is, when dysregulated, often crucially involved in oncogenesis. It is therefore important to develop and evaluate methods for identifying and studying tyrosine phosphorylated (P-Tyr) proteins. P-Tyr proteins are present at very low concentrations within cells, requiring highly selective enrichment methods to be detected. In this study, we applied immunoaffinity as enrichment step for P-Tyr proteins. Five selected anti-phosphotyrosine antibodies (monoclonal antibodies 4G10, PY100, PYKD1, 13F9 and one polyclonal antiserum) were evaluated with respect to their capability to enrich P-Tyr proteins from cell extracts of the K562 leukemia cell line. The enrichment resulted in the detection of a group of proteins that potentially were tyrosine-phosphorylated (putative P-Tyr proteins). High accuracy identification of actual P-Tyr sites were performed using a highly selective and sensitive liquid chromatography Fourier transform mass spectrometer (LC-FTMS) setup with complementary collision activated dissociation (CAD) and electron capture dissociation (ECD) fragmentations. 4G10 and PY100 antibodies recognized the greatest number of putative P-Tyr proteins in initial screening experiments and were therefore further evaluated and compared in immunoaffinity enrichment of both P-Tyr proteins and peptides. Using the 4G10 antibody for enrichment of proteins, we identified 459 putative P-Tyr proteins by MS. Out of these proteins, 12 were directly verified as P-Tyr proteins by MS analysis of the actual site. Using the PY100 antibody for enrichment of peptides, we detected 67 P-Tyr peptides (sites) and 89 putative P-Tyr proteins. Generally, enrichment at the peptide level made it difficult to reliably determine the identity of the proteins. In contrast, protein identification following immunoaffinity enrichment at the protein level gave greater sequence coverage and thus a higher confidence in the protein identification. By combining all available information, 40 proteins were identified as true P-Tyr proteins from the K562 cell line. In conclusion, this study showed that a combination of immunoaffinity enrichment using multiple antibodies of both intact and digested proteins in parallel experiments is required for best possible coverage of all possible P-Tyr proteins in a sample.

Serum protein pattern in sarcoidosis analysed by a proteomics approach.

BACKGROUND: An altered protein expression in bronchoalveolar lavage fluid (BALF) of sarcoidosis patients has previously been reported. As this disease is systemic, involving not only the lungs, a change in the serum components is also to be expected. In the present study we therefore analysed the total serum protein profile of the patients earlier reported, with active sarcoidosis. METHODS: We used a proteomics approach to assess overall changes in the protein content of serum in patients with acute sarcoidosis (n = 6) compared to healthy controls (n = 4). RESULTS: Our results show a significantly higher number of protein-spots in serum of the patients compared to the healthy individuals in the pH range 4.5-6.7 (median 886 vs. 742, p < 0.05). The total protein concentrations of the patients' sera were also significantly higher compared to the controls (median 62 vs. 56 mg/mL, p < 0.05). Measurement of the optical densities of the protein-spots from two-dimensional electrophoresis gels, covering pH interval 4.5-6.7, showed varying levels of expression of 22 different serum proteins in the patients compared to the controls. These proteins are involved in immune responses and some are known markers of inflammation. We found three proteins, which were changed concomitantly in the BALF as described in a previous report, and sera of the same patients. CONCLUSIONS: Our results are of importance in order to identify biochemical markers in blood of sarcoidosis patients. Ideally, a combination of markers in BALF and serum may turn out to be disease specific.

A neuroproteomic approach to targeting neuropeptides in the brain.

A novel universal neuropeptide display approach in the mass range of 300-5000 Da was developed to complement two-dimensional gel electrophoresis in the analysis of peptides and small proteins from brain tissue samples. For the analysis of neuropeptides we utilized on-line nanoscale capillary reversed phase liquid chromatography and electrospray ionization quadrupole-time of flight mass spectrometry. The method was employed for the analysis of a large number of peptides from three specific rat brain regions. Approximately 1500 peptides from each brain region were detected in the same analysis. Several of these peptides were sequenced using collision-induced dissociation and identified by database search tools. In addition, a method for comparing peptide elution profiles between samples was developed, to provide two- and three-dimensional computer graphics of the profiles and to pinpoint differences for statistical measurements. Among the characterized peptides were fragments from proteins such as hemoglobin, alpha-synuclein, stathmin, cyclophilin, actin, NADH dehydrogenase, cytochrome c oxidase and prosomatostatin, as well as the bioactive neuropeptides W-hemorphin-4, and LW-hemorphin-7. The present study showed that the combination of nanoscale reversed phase liquid chromatography and high-resolution tandem mass spectrometry provides a novel and powerful approach to investigate a large number of peptides and protein fragments in the brain.