r. The supernatant was removed and the pellet resuspended in 1 M NaCl PBS by sonication and then pelleted again at 100,000 x_g for 1 hour.

The resulting cell membranes were solubilized by sonication in 500 L of buffer containing 6 M urea and 0.1% SDS in 50 mM TRIS pH 8.0. Figure 1. Fluidic path configuration of the orthogonal 2D LC-MS/MS system used for analysis of complex protein mixtures. ICAT Labeling and Proteolysis The cell membrane suspension was reduced with TCEP (5 mM) for 30 minutes at room temperature. The sample was diluted to 3 mL to reduce the urea concentration and then split into two fractions prior to alkylation with ICAT. Fraction A (2 mL) was labeled with d₀-ICAT and Fraction B (1 mL) with d₈-ICAT. Fractions A and B were alkylated with ICAT reagent for 1 hour in the dark at room temperature. After alkylation, both fractions were recombined to yield a theoretical ratio of 1: 0.5 for d₀: d₈. A total of 90 g of trypsin was added to the mixture followed by overnight incubation at 37C.

After digestion, the samples were acidified with 40 L of formic acid and 5 L of TFA to a pH < 2. A cloudy precipitate was formed which was removed by centrifugation. The supernatant was removed and examined by RP-HPLC to verify the presence of peptides prior to analysis by 2D LC-MS/MS. Figure 2. Graphical user interface for programming of the 2D LC gradients (SCX and RP) and MS acquisition parameters using ProteomeX. Orthogonal 2D LC- MS/MS
Orthogonal 2D LC-MS/MS was performed
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