http://repub.eur.nl/res/aut/15749/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/30305/ 2008-03-01T00:00:00Z Background: A Java™ application is presented, which compares large numbers (n > 100) of raw FTICR mass spectra from patients and controls. Two peptide profile matrices can be produced simultaneously, one with occurrences of peptide masses in samples and another with the intensity of common peak masses in all the measured samples, using the peak- and background intensities of the raw data. In latter way, more significantly differentially expressed peptides are found between groups than just using the presence or absence in samples of common peak masses. The software application is tested by searching angiogenesis related proteins in glioma by comparing laser capture micro dissected- and enzymatic by trypsin digested tissue sections. Results: By hierarchical clustering of the presence-absence matrix, it appears that proteins, such as hemoglobin alpha and delta subunit, fibrinogen beta and gamma chain precursor, tubulin specific chaperone A, epidermal fatty acid binding protein, neutrophil gelatinase-associated lipocalin precursor, peptidyl tRNA hydrolase 2 mitochondrial precursor, placenta specific growth hormone, and zinc finger CCHC domain containing protein 13 are significantly different expressed in glioma vessels. The up-regulated proteins in the glioma vessels with respect to the normal vessels determined by the Wilcoxon-Mann-Whitney test on the intensity matrix are vimentin, glial fibrillary acidic protein, serum albumin precursor, annexin A5, alpha cardiac and beta actin, type I cytoskeletal 10 keratin, calcium binding protein p22, and desmin. Peptide masses of calcium binding protein p22, Cdc42 effector protein 3, fibronectin precursor, and myosin-9 are exclusively present in glioma vessels. Some peptide fragments of non-muscular myosin-9 at the C-terminus are strongly up-regulated in the glioma vessels with respect to the normal vessels. Conclusion: The less rigorous than in general used commercial propriety software de-isotope algorithm results in more mono-isotopic peptide masses and consequently more proteins. Centroiding of peptide masses takes place by taking the average over more spectra in the profile matrix. Cytoskeleton proteins and proteins involved in the calcium signaling pathway seem to be most up-regulated in glioma vessels. The finding that peptides at the C-terminus of myosin-9 are up-regulated could be ascribed to splicing or fragmentation by proteases. http://repub.eur.nl/res/pub/35367/ 2007-06-15T00:00:00Z Aberrant splice variants are involved in the initiation and/or progression of glial brain tumors. We therefore set out to identify splice variants that are differentially expressed between histologic subgroups of gliomas. Splice variants were identified using a novel platform that profiles the expression of virtually all known and predicted exons present in the human genome. Exon-level expression profiling was done on 26 glioblas tomas, 22 oligodendrogliomas, and 6 control brain samples. Our results show that Human Exon arrays can identify subgroups of gliomas based on their histologic appearance and genetic aberrations. We next used our expression data to identify differentially expressed splice variants. In two independent approaches, we identified 49 and up to 459 exons that are differentially spliced between glioblastomas and oligodendrogliomas, a subset of which (47% and 33%) were confirmed by reverse transcription-PCR (RT-PCR). In addition, exon level expression profiling also identified >700 novel exons. Expression of ∼67% of these candidate novel exons was confirmed by RT-PCR. Our results indicate that exon level expression profiling can be used to molecularly classify brain tumor subgroups, can identify differentially regulated splice variants, and can identify novel exons. The splice variants identified by exon level expression profiling may help to detect the genetic changes that cause or maintain gliomas and may serve as novel treatment targets. http://repub.eur.nl/res/pub/14086/ 2006-09-05T00:00:00Z BACKGROUND: Statistical comparison of peptide profiles in biomarker discovery requires fast, user-friendly software for high throughput data analysis. Important features are flexibility in changing input variables and statistical analysis of peptides that are differentially expressed between patient and control groups. In addition, integration the mass spectrometry data with the results of other experiments, such as microarray analysis, and information from other databases requires a central storage of the profile matrix, where protein id's can be added to peptide masses of interest. RESULTS: A new database application is presented, to detect and identify significantly differentially expressed peptides in peptide profiles obtained from body fluids of patient and control groups. The presented modular software is capable of central storage of mass spectra and results in fast analysis. The software architecture consists of 4 pillars, 1) a Graphical User Interface written in Java, 2) a MySQL database, which contains all metadata, such as experiment numbers and sample codes, 3) a FTP (File Transport Protocol) server to store all raw mass spectrometry files and processed data, and 4) the software package R, which is used for modular statistical calculations, such as the Wilcoxon-Mann-Whitney rank sum test. Statistic analysis by the Wilcoxon-Mann-Whitney test in R demonstrates that peptide-profiles of two patient groups 1) breast cancer patients with leptomeningeal metastases and 2) prostate cancer patients in end stage disease can be distinguished from those of control groups. CONCLUSION: The database application is capable to distinguish patient Matrix Assisted Laser Desorption Ionization (MALDI-TOF) peptide profiles from control groups using large size datasets. The modular architecture of the application makes it possible to adapt the application to handle also large sized data from MS/MS- and Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometry experiments. It is expected that the higher resolution and mass accuracy of the FT-ICR mass spectrometry prevents the clustering of peaks of different peptides and allows the identification of differentially expressed proteins from the peptide profiles. http://repub.eur.nl/res/pub/13830/ 2005-09-01T00:00:00Z Leptomeningeal metastasis (LM) is a devastating complication that occurs in 5% of patients with breast cancer. Early diagnosis and initiation of treatment are essential to prevent neurological deterioration. However, early diagnosis of LM remains challenging because 25% of cerebrospinal fluid (CSF) samples produce false-negative results at first cytological examination. We developed a new, MS-based method to investigate the protein expression patterns present in the CSF from patients with breast cancer with and without LM. CSF samples from 106 patients with active breast cancer (54 with LM and 52 without LM) and 45 control subjects were digested with trypsin. The resulting peptides were measured by MALDI-TOF MS. Then, the mass spectra were analyzed and compared between patient groups using newly developed bioinformatics tools. A total of 895 possible peak positions was detected, and 164 of these peaks discriminated between the patient groups (Kruskal-Wallis, p<0.01). The discriminatory masses were clustered, and a classifier was built to distinguish patients with breast cancer with and without LM. After bootstrap validation, the classifier had a maximum accuracy of 77% with a sensitivity of 79% and a specificity of 76%. Direct MALDI-TOF analysis of tryptic digests of CSF gives reproducible peptide profiles that can assist in diagnosing LM in patients with breast cancer. The same method can be used to develop diagnostic assays for other neurological disorders.