Hplc nanoeasy proxeon

Spots protein of interest were excised from the preparative gel with Screen Picker (Proteomics Consult, Kampenhout, Belgium), and peptides were extracted with trifluoroacetic acid after destained and trypsin-digestion [55 (link)]. Resulting peptides were analysed by mass spectrometry (MS) using either an LC/MSD XCT Ultra Ion Trap equipped with a 1100 HPLC system and a chip cube (Agilent Technologies, Santa Clara, California, USA) or a LTQ XL-Orbitrap ETD equipped with a HPLC NanoEasy-PROXEON (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Database searches were done with the MASCOT search engine version 2.3 against SwissProt and NCBInr (Matrix Science, London, UK). In those spots resulting to contain more then one proteins, possible post-translational phosphorylations were searched in STY aminoacids. For each identified protein, subcellular location was controlled in UniProtKB as Gene Ontology (GO) annotation (http://www.uniprot.org/uniprot).

Pooled protein T extracts (10 µg per lane) were separated by 1DE, and images of blue-stained gel were acquired with the Chemidoc system. A total of 10 gel portions in the MW range between 75 and 37 kDa containing proteins cross-reacting with the FGB antibody (Figure 2, rectangle and numbered lanes) were excised, reduced by incubation with 10 mM dithiothreitol (1 h at 57 °C), and alkylated with 55 mM iodoacetamide (45 min at room temperature). Samples were further washed with NH₄HCO₃, dehydrated, trypsin digested and processed for LC-MS/MS analyses using a LTQ XL-Orbitrap ETD equipped with a HPLC NanoEasy-PROXEON (Thermo Fisher Scientific, Waltham, MA, USA). Database searches were done with the MASCOT search engine version 2.3 against SwissProt and NCBInr (Matrix Science, London, UK), and the presence of FGB was searched among first 15 report hits.