Comprehensive proteomic analysis of Schizosaccharomyces pombe by two-dimensional HPLC-tandem mass spectrometry - PubMed (original) (raw)

Figure 3

Simplified schematic diagram of the plumbing for liquid flow through the HPLC, autosampler, UV detector flow cell, SCX and RP columns. The flow paths that are used for the separations described in this article are all shown. All tubing is 1/16” outer diameter (OD) PEEK, with varied inner diameters, with the exception that PEEKsil tubing (also 1/16” OD) is used for all sample flow paths. Tubing was obtained primarily from Michrom and also from Upchurch Scientific (Seattle, WA). (A) Flow paths for sample introduction and SCX column stabilization, immediately prior to SCX separation are shown. Abbreviations: ASV, autosampler valve; HPLC V1, HPLC valve 1; mix 2, mixer 2 (for solvents C and D); HPLC V2, HPLC valve 2; UV, UV detector flow cell. Two flow paths, marked by arrows, are active at this stage: 1) Flow of the sample, from the syringe, for sample introduction into the (100 μl) sample loop, and 2) flow from pumps C and D (not shown for clarity), through mixer 2, for stabilization of solvent flow and pressure through the SCX column prior to the SCX separation. (B) Flow path during the SCX separation, which no longer involves sample introduction. To enter this stage following that shown in panel A, HPLC valve 1 switches, resulting in reversal of the sample flow back out of the sample loop and onto the SCX column. Concurrent to the switching of valve 1, the autosampler automatically suspends the fraction collection tool above the vials (Fig. 1B), in order to collect the eluate from the SCX column. The fraction collection tool is automatically moved to a fresh vial every 2.0 min, resulting in collection of 24 × 400 μl fractions. The SCX gradient, consisting of solvents C and D (see the text and Fig. 2A), is applied, resulting in peptide separation throughout the gradient. (C) Flow paths for sample introduction immediately prior to reversed phase (RP) separation; abbreviations: MSV, valve mounted on the mass spectrometer; mix 1, mixer 1 (for solvents A and B); cp tp, capillary peptide trap (polymeric); and AC, C18 reversed-phase, capillary-scale (1/16″ OD) analytical column. Two flow paths, marked by arrows, are active at this stage: 1) Flow of the sample, from the syringe, for sample introduction onto the peptide trap (cp tp) followed by desalting of the captured peptides, and 2) flow from pumps A and B (not shown for clarity), through mixer 1, for stabilization of solvent flow and pressure through the C18 analytical column (AC). The high voltage (1.8 kV) is manually activated at the start of this stage using the tune page, which is part of the control software for the mass spectrometer. (D) Flow path during the reversed-phase separation, which no longer involves sample introduction. To enter this stage following that shown in panel C, the valve mounted on the mass spectrometer switches, resulting in reversal of the solvent flow back through the peptide trap, gradient elution of the peptides off of the peptide trap, their separation in the analytical column, elution, and ionization in the ESI source, just prior to introduction into the mass spectrometer.