We offer different possibilities helping you to find your desired documents. First you can select literature by certain criteria and refine results by selecting more and more of them. Alternatively, you can use our powerful full-text search to browse our database. But there is still a third option: if you use the full-text search and get lots of results, you can use the criteria ADDITIONALLY to reduce obsolete matches.
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.
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.
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.
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.
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.
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.
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).
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.
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.
Although the most important application of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is “proteomics,” there is growing evidence that this soft ionization method is also useful for phospholipid (PL) analysis. Although all PLs are detectable by MALDI-TOF MS, some lipid classes, particularly those with quaternary amines such as phosphatidylcholines (PCs), are more sensitively detected than others, and these suppress the signals of less sensitively detected PLs when complex mixtures are analyzed. Therefore, a separation of the total organic extract into individual lipid classes is necessary. As MALDI uses a solid sample, the direct evaluation of thin-layer chromatography (TLC) plates is possible. We report here on a method of directly coupling MALDI-TOF MS and TLC that can be easily implemented on commercially available MALDI-TOF devices. A total extract of hen egg yolk is used as a simple PL mixture to demonstrate the capabilities of this method. It will be shown that “clean” spectra without any major contributions from fragmentation products and matrix peaks can be obtained, and that this approach is even sensitive enough to detect the presence of PLs at levels of less than 1% of the total extract.
link to article
link to article
