This peptide belongs to the F IX heavy chain (see Figure 4 and References [36, 37]). disease filtration, is definitely also an efficient step for the removal of residual impurities, primarily high molecular excess weight proteins, such as vitronectin and inter-alpha inhibitor proteins. In each production step, the active component, pd F IX and contaminating proteins are monitored by biochemical and immunochemical methods and by LC-MS/MS and their removal documentedOur strategy is very helpful for further process optimization, quick recognition of target proteins with relatively low large quantity, and for the design of subsequent methods for his or her removal or purification. fraction, collected during the isolation process, about 15C25 g protein of each sample were solubilized in NuPAGE sample buffer (Invitrogen, Carlsbad, CA, U.S.A.), and SDS-PAGE was performed as explained previously . SDS-PAGE was performed in two self-employed experiments. 2.6. In-gel digestion process The gel bands of interest were excised by extracting 6C10 gel particles with clean glass Pasteur pipettes and digested with trypsin as explained previously [11, 12]. 2.7. In-solution digestion process 50 g of the acetone-precipitated and denatured protein pellet was resolubilized in 100 L of NH4HCO3 (pH 8.0)/8M urea. The resolubilized proteins were reduced with 20 mM dithiotreitol (37 C, 45 min) and then alkylated with 50 mM iodoacetamide at space temp S1PR2 for 30 min in the dark. Before tryptic digestion, 100 mM NH4HCO3, pH 8.0, was added to reduce the concentration of urea. Trypsin was added to the protein combination at an enzyme to substrate percentage of 1 1:60 w/w, and the digestion was performed as explained previously . The producing tryptic peptides were dried and subject to the LC-MS/MS analysis after becoming redissolved in formic acid:water:ACN:trifluoroacetic acid combination (0.1:95:5:0.01 v/v). 2.8. Recognition of proteins with LC-MS/MS Tryptic peptides were separated on a 12 cm (75 m I.D.) analytical column having a 5 m Monitor C18 resin (Column Executive, Ontario, CA, U.S.A) and containing a ~4 m ESI emitter tip. Solvent A was 0.1 M acetic acid in water, and solvent B was 0.1 M acetic acid in ACN. Peptides were eluted using a linear ACN gradient (0C70%) solvent B over 30 min (Agilent Systems, Paolo Alto, CA, U.S.A.). Maximum Fasudil parking during the time when peptides were expected to elute was accomplished by reducing the circulation rate from 200 nL/min to ~20 nL/min. Eluting peptides were launched onto an LTQ linear ion capture mass spectrometer (Thermo Electron Corporation, San Jose, CA, U.S.A.) having a 1.9 kV electrospray voltage. Full MS scans in the range of 400C1800 were followed by data-dependent acquisition of MS/MS spectra for the five most abundant ions, using a 30-second dynamic exclusion time. Protein recognition was performed in, at least, two self-employed experiments as explained previously . Database searching was performed using the maximum lists in the SEQUEST system . The precursor-ion tolerance was 2.0 Daltons and the fragment-ion tolerance was 0.8 Daltons. Enzymatic digestion was specified as trypsin, with up to 2 missed cleavages allowed. The search contained sequences identified as human Fasudil being in NCBIs nr database (November, 2006), which was created using the FASTA filtering tools found in BioWorks (Thermo). A list of reversed-sequences was created from these entries and appended to them for database searching so that false positive rates could be estimated . This composite database contained approximately 490,000 Fasudil entries. For parallel LC-MS/MS analysis of samples taken for the isobaric tag for relative and complete quantification (iTRAQ) analyses (observe below), a nano LC-MS/MS system was used. Tryptic digest were separated having a nano RP column (C-18 PrepMap 100, LC Packings/Dionex, Sunnyvale, CA, USA) as previously explained, with the column eluate launched directly onto QStar XL mass spectrometer (Applied Biosystems, Foster City, CA, USA and Sciex, Concord, Ontario, Canada) via slectrospray ionization . Half second scans (300C1500 Thompson (Th)) were used to identify candidate ions for fragmentation during MS/MS scans. Up to five 1.5 s MS/MS scans.
This peptide belongs to the F IX heavy chain (see Figure 4 and References [36, 37])