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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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The Bruker 300-MS is an easy-to-use mass spectrometer providing a choice of configurations to match your budget and your needs. Saving you time, money and headaches, it offers:

• High throughput
• Reliable quantification
• Excellent sensitivity

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High Sensitivity with Unmatched Flexibility

The Bruker 320-MS provides femtogram sensitivity, a 2000 m/z mass range and a choice of chromatographic and ionization configurations to match your budget and your needs – all in less than 72 cm (28 in.) of linear bench space.

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New Bruker 400-GC Series gas chromatographs now provide faster separations, increased confidence in analytical results and an outstanding range of complete solutions.

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Bruker continues to find new and novel ways to meet your changing needs. As a leader in elemental analysis you can be assured that when you buy a Bruker ICP-MS, you’re buying more than just an instrument. You’re buying a relationship with one of the most respected and experienced instrument companies in the world. 

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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).