The iTRAQ-labeled peptides were re-suspended in Buffer A, which contained 10 mM KH2PO4 and 20% ACN (pH 3.0), and was fractioned by SCX using a polysulfoethyl A column (4.6 × 200 mm, 5 μm, 300 Å, Poly LC, Columbia, MD, USA) on a BioCAD™ Perfusion Chromatography System (AB Sciex) at a constant flow rate of 1 mL/min. The composition of Buffer B was 10 mM KH2PO4, 20% ACN, and 600 mM KCl (pH 3.0). The peptides were separated using a 60-min linear gradient. Thirty-five fractions were collected. Following PepClean C18 spin column desalting (Pierce, Rockford, IL, USA), each peptide fraction from SCX was further separated on a reversed-phase C18 capillary column (0.75 × 150 mm, 3 μm, 100 Å, Dionex, Sunnyvale, CA, USA) on an Ultimate LC system coupled with a Probot spotting device (Dionex) as described previously [36 (link)]. The peptides eluted from the reversed-phase LC were mixed with a MALDI matrix (5 mg/mL α-cyano-4-hydroxycinnamic acid in 60% ACN, 0.1% TFA, 5 mM ammonium monobasicphosphate, 50 fmol/μL each of glu-fibrinogen peptide (m/z 1570.677), and adrenocorticotrophin hormone fragments 18–39 (m/z 2465.199) and were spotted onto the stainless steel MALDI plates for MS/MS analysis.
Ultimate lc system
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The Ultimate LC system is a high-performance liquid chromatography (HPLC) instrument designed for analytical and preparative separation of complex mixtures. It features a modular design with interchangeable components, allowing for customization to meet specific application needs. The system provides precise control over flow rates, gradients, and temperature for optimal separation and detection of analytes.
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Lab products found in correlation
3 protocols using ultimate lc system
1
iTRAQ-labeled Peptide Fractionation and Analysis
2
Proteomic Analysis of Platelet Proteins
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Platelets were resuspended at 1 × 109 cells/ml and incubated with TG for 1 min followed by lysis in equal volume of RIPA lysis buffer and immunoprecipitation as described in [39] (link). Extracted samples were run on 5–15% gradient SDS-PAGE gels followed by Colloidal Coomassie staining (see Results ). Segments containing visible bands and clear areas were exposed to in-gel reduction, alkylation and digestion with trypsin using standard protocols. Peptides were extracted from the gel pieces by a series of acetonitrile and aqueous washes. The extract was lyophilised, resuspended in 23 μl of 50 mM ammonium bicarbonate and analysed by LC–MS/MS using an Ultimate LC system (Dionex, UK). Peptides were resolved using 75 μm C18 PepMap column. A 60 minute gradient of acetonitrile in 0.05% formic acid was delivered to elute the peptides at a flow rate of 200 nl/min. Peptides were ionised by electrospray ionisation using a Z-spray source fitted to a QTof-micro (Waters Corp.). The MS/MS analyses were conducted using collision energy profiles based on the mass-to-charge ratio (m/z) and the charge state of the peptide.
The mass spectral data was processed using ProteinLynx Global Server v2.2.5, and the Swiss Prot database using Mascot software v2.2 (http://www.matrixscience.com ). Sequence information was obtained for all the peptides included in the results.
The mass spectral data was processed using ProteinLynx Global Server v2.2.5, and the Swiss Prot database using Mascot software v2.2 (
3
Peptide Separation by Reversed-Phase LC
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Peptide separation by reversed-phase liquid chromatography was performed on an Ultimate LC system (Dionex) complete with Famos autosampler and Switchos II microcolumn switching device for sample clean-up and preconcentration. Sample (30 ml) was loaded at a flow rate of 200 nl/min on a micro-precolumn cartridge (300 mm i.d. x 5mm length, packed with 5 mm C18 100A PepMap). After 5 min, the precolumn was connected with the separating nano-column (75 µm x 15 cm, packed with C18 PepMap100, 3 μm, 100 Å) and the gradient started. Elution gradient varied from 0% to 30% buffer B over 30min, buffer A is 0.1% formic acid in acetonitrile/water 2:98 (vol/vol) and buffer B is 0.1% formic acid in acetonitrile/water 20:80 (vol/vol). The outlet of the LC system was directly connected to the nano electrospray source of an Esquire HCT ion trap mass spectrometer (Bruker Daltonics, Germany). Mass data acquisition was performed in the mass range of 50-1700 m/z using the Standard-Enhanced mode (8100 m/z/s). For each mass scan, a data-dependant scheme picked the 4 most intense doubly or triply charged ions to be selectively isolated and fragmented in the trap and the resulting fragments were mass analyzed using the Ultra Scan mode (50-3000 m/z at 26,000 m/z/s).
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