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Electrospray (ESP) MS and Edman sequencing are used to identify 21 heterogeneous yellow-brown coloured peptides repurified by RP-HPLC from reduced, carboxymethylated and trypsin digested α1-microglobulin (α1m). The peptides are identified by MS/MS and all peptides in a tryptic digest of α1m are analyzed by peptide mapping using ESP LC/MS. Fluorescence spectra are also measured.

Infusion1: 50 μl inj., 50% aq. acetonitrile contg. 0.1% trifluoroacetic acid (TFA), 10 μl/min.

MS1: Micromass Bio-Q, ESP, pos. mode, m/z up to 1600.

Infusion2: samples in 50% aq. MeOH contg. 0.1% trichloroacetic acid, Au-plated glass capillary.

MS2: Bruker Esquire LC, ESP, pos. mode.

LC: Isco system, 5 μl inj., 15 cm x 1 mm i.d. (Deltabond ODS), 90% aq. acetonitrile contg. 0.025% TFA, 30 μl/min split 1:1 to MS and UV (350 nm).

MS3: Micromass Quattro-BQ, ESP, pos. mode, continuum mode, m/z 230-1450, 5 s/scan.

MS/MS spectra of the doubly charged ions indicate the presence of chromophores with masses 122-282 Da, assigned to diff. coloured peptides, covalently attached to Lys-92, Lys-118 and Lys-130 of human α1m. The Lys are semiburied in the lipocalin pocket of the protein. There is also an uncoloured lipophilic, hydroxylated and aminated substance noncovalently associated with the chromophores.

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Glycosylated proteins are in-gel deglycosylated with PNGase F in H 2 18O-contg. medium in order to label partially (50%) the glycosylated Asn residues prior to tryptic digestion and peptide mass mapping by MALDI MS with database searching. Peptides extd. from the gel pieces and present in the in-gel proteolysis supernatant mixt. are also analyzed by nano-electrospray (ESP) MS/MS. For MALDI, a satd. matrix soln. in acetone is mixed 4:1 (v/v) with nitrocellulose in 50% acetone-isopropanol and prepd. by the fast evapn. method. A drop of 10% formic acid is deposited and 0.3-0.5 μl of the digest supernatant is added and allowed to dry.

MS1: modified Bruker Reflex, MALDI, TOF, pos. mode, N2 laser, 337 nm.

Infusion: desalted peptides in 5% formic acid.

MS2: Perkin-Elmer Sciex QqTOF prototype, nano-ESP, pos. mode, CAD: N2, 20-60 eV.

Due to isotope labelling, more peptides are detected by MALDI MS to give better database search specificity. Detection of a labelled peptide helps partial sequencing because N-linked carbohydrates are attached solely to Asn residues that form part of the NXS/T sequence. This increases search specificity 100-fold. For all proteins studied (bovine fetuin, human α1-acid glycoprotein, HIVrgp120) glycosylation sites are identified by MALDI MS when 10 pmol glycoprotein is subjected to SDS-PAGE. ESP MS/MS may be used to verify glycosylation site assignment and give detailed sequence information by peptide sequencing of the labelled peptides. An example is given with a peptide from fetuin.

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We performed a 2-DE analysis of proteins of the newly established spontaneously immortalized clonal cell line EM-G3 derived from a primary lesion of infiltrating ductal breast carcinoma. EM-G3 cells may represent progenitors of the mammary epithelial cells spontaneously immortalized in early phase of cancerogenesis. We compared the protein profile of EM-G3 line with proteins from populations of normal mammary epithelial cells (NME), and determined the phenotype of both types of cells. NME cells are a mixture of both main cell types in breast epithelia, myoepithelial and luminal cells. The EM-G3 breast cancer cell line has a unique basal-like phenotype. We identified proteins that are differently expressed in these cells. Cytokeratin 16, cytokeratin 19, squamous cell carcinoma antigen 1, caphepsin B and caspase 14 were predominantly expressed by NME cells. Cytokeratin 13, isoelectric variant of annexin 5, isoelectric variant of chloride intracellular channel protein 1, glyoxalase 1 and glutamine synthetase were predominantly expressed by EM-G3 cells. The proteins up-regulated in EM-G3 cells may represent potential protein markers of mammary epithelial cells progenitors and may be important in early phase of carcinogenesis.

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Targeted differentiation of neural progenitor cells (NPCs) is a challenge for treatment of neurodegenerative diseases by cell replacement therapy and cell signalling manipulation. Here, we applied a proteome profiling approach to the rat striatal progenitor model cell line ST14A in order to elucidate cellular differentiation processes. Native cells and cells transfected with the glial cell line-derived neurotrophic factor (GDNF) gene were investigated at the proliferative state and at seven time points up to 72 h after induction of differentiation. 2-DE combined with MALDI-MS was used to create a reference 2-DE-map of 652 spots of which 164 were identified and assigned to 155 unique proteins. For identification of protein expression changes during cell differentiation, spot patterns of triplicate gels were matched to the 2-DE-map. Besides proteins that display expression changes in native cells, we also noted 43 protein-spots that were differentially regulated by GDNF overexpression in more than four time points of the experiment. The expression patterns of putative differentiation markers such as annexin 5 (ANXA5), glucosidase II beta subunit (GLU2B), phosphatidylethanolamine-binding protein 1 (PEBP1), myosin regulatory light chain 2-A (MLRA), NASCENT polypeptide-associated complex alpha (NACA), elongation factor 2 (EF2), peroxiredoxin-1 (PRDX1) and proliferating cell nuclear antigen (PCNA) were verified by Western blotting. The results reflect the large rearrangements of the proteome during the differentiation process of NPCs and their strong modification by neurotrophic factors like GDNF.

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Commercial FT/ICR mass spectrometers installed in both labs. are described and their performances illustrated by the anal. of peptides, proteins, oligonucleotides, fullerenes and synthetic polymers. The instrument features easily interchangeable ion sources: MALDI, SIMS, EI/CI and electrospray (ESP).

MS: Bruker Daltonics BioApex 94e, FT/ICR, 9.4-T, ESP: Analytica source, N2 nebulizing gas, heated drying gas, or lab.-built ESP: 0.1 mm i.d. stainless steel needle etched to conical shape, no nebulizing or drying gas, shutter to permit multipole storage-assisted dissociation, MALDI: N2 laser, 337 nm.

The lower mass limit is m/z 29 and the highest obsd. single charged m/z value is 8565 (by MALDI). Resolution is > 4,000,000 for m/z 130.9916 from PFTBA and is > 1,200,000 for ubiquitin ([M+10H]10+) by ESP. There is clear resolution between C59N+ and C58 13C2 + (mass diff. 3.6 mDa).

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Corynebacterium jeikeium is a lipid-requiring pathogen that is considered as part of the normal microflora of the human skin and associated with severe nosocomial infections. Systematic reference maps of the cytoplasmic, cell surface-associated, and extracellular proteome fractions of the clinical isolate C. jeikeium K411 were examined by 2-DE coupled with MALDI-TOF MS. A sum total of 555 protein spots were identified by PMF, corresponding to 358 different proteins that were classified into functional categories and integrated into metabolic pathways. The majority of the proteins were linked to housekeeping functions in energy production and translation and to physiological processes in amino acid, carbohydrate, nucleotide, and lipid metabolism. A complete enzymatic machinery necessary to utilize exogenous fatty acids by -oxidation was detected in the cytoplasmic proteome fraction. In addition, several predicted virulence factors of C. jeikeium K411 were identified in the cell surface-associated and extracellular subproteome, including the cell surface proteins SurA and SurB, the surface-anchored pilus subunits SapA and SapB, the surface-anchored collagen adhesin CbpA, the cholesterol esterase Che, and the acid phosphatase AcpA.

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Background: Statistical comparison of peptide profiles in biomarker discovery requires fast, userfriendly 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 FTICR mass spectrometry prevents the clustering of peaks of different peptides and allows the identification of differentially expressed proteins from the peptide profiles.

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Detecting DNA mismatches by using selfassembled monolayers (SAMs) which include regions of DNA-capture probes: The hybridization of complementary target DNA versus mismatched DNA is distinguished by MALDI-TOF mass spectrometry. The extent to which mismatched DNA can hybridize to capture probes depends on the type and position of the mismatch. Spots of higher wettability show the locations of DNA
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A general screening method based on solid phase extraction (SPE) and liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) was developed and investigated with 124 different doping agents, including stimulants, -blockers, narcotics, 2-adrenergic agonists, agents with anti-estrogenic activity, diuretics and cannabinoids. Mixed mode cation exchange/C8 cartridges were applied to SPE, and chromatography was based on gradient elution on a C18 column. Ionization of the analytes was achieved with electrospray ionization in the positive mode. Identification by LC/TOFMS was based on retention time, accurate mass and isotopic pattern. Validation of the method consisted of analysis of specificity, analytical recovery, limit of detection and repeatability. The minimum required performance limit (MRPL), established by World Anti-Doping Agency (WADA), was attained to 97 doping agents. The extraction recoveries varied between 33 and 98% and the median was 58%. Mass accuracy was always better than 5 ppm, corresponding to a maximum mass error of 0.7 mDa. The repeatability of the method for spiked urine samples, expressed as median of relative standard deviations (RSD%) at concentrations of MRPL and 10 times MRPL, were 14% and 9%, respectively. The suitability of the LC/TOFMS method for doping control was demonstrated with authentic urine samples.

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