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. Author manuscript; available in PMC: 2010 Jul 1.
Published in final edited form as: Methods. 2009 Mar 9;48(3):311–319. doi: 10.1016/j.ymeth.2009.02.023

Figure 1.

Figure 1

Instrumentation platform used in the proteomic analyses. (A) Sample injection, from the syringe mounted on the autosampler, into the sample injection port (red arrow). Samples for SCX and RP separations are introduced into the HPLC at this point. (B) Offline collection of SCX fractions using the fraction collection tool (blue and tan), which is magnetically suspended from the autosampler arm (black cylinder). The SCX eluate drips into open vials (red arrow). (C) Syringe drawing up a portion of a SCX fraction (black arrow) for subsequent re-injection into the sample injection port (shown in panel A), for RP HPLC-MS/MS analysis. Scale bars represent 2.5 cm (A–C). (D) Composite instrumentation platform, including the UV detector (UV); solvent bottle holder on top of the module controlling gas flow and choice of 40:40:20 isoproponal:acetonitrile:water (40:40:20 wash solvent) or HPLC solvents (solv.); HPLC pumps/mixers/splitter for solvents A/B and solvents C/D (pumps A/B use a splitter but C/D do not) and 2 valves (HPLC) with the necessary plumbing (PEEK and PEEKsil tubing), SCX and immobilized metal affinity chromatography (IMAC) columns attached (the IMAC application for phosphopeptide enrichment will be described elsewhere); the sample loop mounted on HPLC valve 1 (loop); the sample injection port (port); polymeric trap for peptide capture and desalting (RP-pep. capture) on the valve that is mounted on the mass spectrometer; the RP analytical column (RP column) mounted in the ESI source (ESI); the autosampler/fraction collector containing refrigerated drawers for reagent, sample and SCX fraction storage; and the hybrid linear ion trap- (ion trap) OrbitrapXL (Orbitrap) mass spectrometer; scale bar represents 10 cm.