( of 50:1) for 2–24 h. The trypsin ...)2D-LC analysis of phosphopeptides in brain tissue using Ettan MDLC and Finnigan LTQ

Liquid chromatography
Using the offline 2D-LC configuration of the Ettan MDLC, 40 µg of trypsin-digested mouse brain sample was injected onto a 2.1 x 250-mm BioBasic SCX column and eluted at 100 µl/min with a linear salt gradient of 0–30% B over 40 min (A: 20 mM citric acid, pH 2.5, 25% CH3CN; B: A + 1 M NH4Cl). Fractions were collected twice every minute (Fig 1). The fractions were injected onto a 0.3 x 5-mm Zorbax RPC trap column, where they were desalted. RPC separation was performed on a Zorbax 0.075 x 150-mm analytical column at 250 nl/min with a linear gradient of 0–60% B over 50 min (A: 0.1% formic acid; B: 84% CH3CN and 0.1% formic acid), a step up to 100% B, and then holding at 100% B for 10 min. To increase throughput, one pair of columns was equilibrated while the other pair was used for analysis, a standard preprogrammed method on the Ettan MDLC.

Mass spectrometry
A Finnigan LTQ linear ion trap was used. The MS method consisted of a cycle combining one full MS scan with three MS/MS events (25% collision energy) followed by an MS³ event (35% collision energy) that was triggered upon detection of -98, -49, or -32.7 Da from the precursor (neutral loss of phosphoric acid, charge states 1⁺, 2⁺, and 3⁺). Dynamic exclusion duration was set to 30 s. The MS/MS and MS³ spectra from all the runs were searched using TurboSEQUEST protein identification software. Modifications were set to allow for the detection of oxidized Met (+16); carboxyamidomethylated Cys (+57); phosphorylated Ser, Thr, and Tyr (+80); and dehydrated Ser and Thr (-18).

Results and discussion
By injecting a large amount of sample and separating it on an analytical scale SCX column, colle
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