Figure 1. Sample: tryptic digest of a protein extract from a human multiple myeloma cell line
Figure 2. Minimal mixing and dilution of salt solutions during stepwise elution from SCX column ensures optimal separation. Spray current profiles measured at MS inlet during salt injections using Thermo Fisher LTQ OrbiTrap XL.
Figure 1 demonstrates that a simple nanoscale chromatography system set-up, using an EASY-nLCTMsystem, enables more proteins to be identified compared to conventional 2D (quaternary split-flow solvent delivery) or 1D configurations. Figure 2 shows the effectiveness of the stepwise elution achieved using the EASY-nLC system in a 2D-LC configuration.
tion tomated 2D Peptide Separa Original publication: Au , Taylor et al., Journal of on a 1D Nano-LC-MS System DOI: 10.1021, 2009 Proteome Research, ACS,
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Analytical column 75m ID fused silica column packed with: 10 cm C18 material
Note: The 2D-LC method can also be implemented as a one-column setup (recommended column specifications: 75 m ID fused silica, 5 cm SCX material and 5 cm C18 material)
Sample/salt plugs Example 1: 9 steps ~ 24 hours analysis time A1: Sample (8 L, < 20 g total protein digest) A2-B3: 25, 50, 75, 100, 125, 150, 200, 500 mM ammonium acetate in 5 % acetonitrile, 0.1 % formic acid Example 2: 3 salt steps ~ 8 hours analysis time
rify samples by Important: Desalt and pu ageTips) solid phase extraction (St prior to 2D-LC analysis.
A1: A2-A4:
Sample (8 L, < 20 g total protein digest) 25, 100, 500 mM ammonium acetate in 5 % acetonitrile, 0.1 % formic acid
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