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2D-LC analysis of phosphopeptides in brain tissue using Ettan MDLC and Finnigan LTQ

> Ettan MDLC 18-1176-44, 11-0008-41

NAP™ 10 Columns 17-0854-01

PlusOne™ DTT 17-1318-01

PlusOne Tris 17-1321-01

Trypsin, sequencing grade 17-6002-75


Other products required
Finnigan LTQ mass spectrometer (Thermo Electron)

TurboSEQUEST™ protein identification software (Thermo Electron)

BioBasic™ SCX, 2.1 x 250 mm (Thermo Electron)

Zorbax™ 300-SB C18 trap column, 300 µm i.d. x 5 mm, 3 µm (Agilent)

Zorbax 300-SB C18 analytical column, 75 µm i.d. x 150 mm, 3 µm (Agilent)

Ammonium bicarbonate (Merck)

Citric acid (Fluka)

Formic acid, ultrapure (Merck Suprapur™)

Iodoacetic acid (Merck)

Acetonitrile, HPLC grade

Water, HPLC grade


Methods
Sample preparation
Approximately 0.5 mg of mouse brain tissue was prepared according to the following procedure. The proteins were dissolved by adding 1 ml of 9 M urea with 50 mM DTT to the tissue and allowing it to incubate for 60 min at 20 °C. A 1-ml aliquot consisting of 8 M urea, 250 mM TrisHCl, and 125 mM iodoacetamide, pH 8.8, was added. The mixture was allowed to incubate for 60 min at 20 °C. A 1-ml aliquot of this mixture was buffer exchanged with 20 mM ammonium bicarbonate, pH 7.8, on a NAP-10 desalting column. The protein sample was digested with trypsin (concentration ratio
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