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. Author manuscript; available in PMC: 2010 Jul 15.
Published in final edited form as: Electrophoresis. 2008 Apr;29(8):1604–1611. doi: 10.1002/elps.200700741

Figure 3.

Figure 3

On-line 2D RP/SCX/PLOT/MS/MS analyses of an in-gel tryptic digest sample of a gel section (>40 kDa) of 1200 SiHa cells: (A) PLOT/MS analysis of the peptides that were unretained on the SCX column; (B–F) PLOT/MS analyses of eluted sample after each salt step for ammonium acetate concentrations of 5, 10, 15, 20, and 250 mM, respectively. 2 μL of ammonium acetate solution was delivered to the SCX column for each salt step elution. RP gradient: 0% mobile phase B (0.1% (v/v) formic acid, 10% (v/v) water in acetonitrile), 0 min; 45% B, 60 min; 80% B, 65 min; 100% A (0.1% (v/v) formic acid in water), 70 min. Data collection was initiated at the start of the gradient. Flow rate: ~20 nL/min with a column inlet pressure of ~2300 psi. The sample was first loaded onto the C18 desalting column, then gradient eluted onto the SCX column, and finally stepwise eluted and trapped onto the monolithic micro-SPE column. The trapped sample was switched on-line to the PLOT column for separation.