Identify more proteins compared to conventional approaches

2D-enabled operation of EASY-nLC

Identify ~4X more proteins than 1D and ~20% more proteins than conventional 2D-LC Follow a simple method using a fully integrated, split-free nanoscale chromatography system Automated two-dimensional liquid chromatography (2D-LC) coupled on-line with MS/MS instrumentation offers exceptional performance when analyzing complex protein/peptide samples. The 2D-LC step is usually based an on-line strong cation-exchange (SCX) separation followed by a reverse phase (RP) separation. Configurations using ternary or quaternary gradient generation systems to step-elute peptides from the SCX column, perform a gradient elution from the RP column, followed by MS/MS analysis are inherently complex, requiring highly skilled specialists for successful implementation. Achieve better analytical performance than regular ternary/quaternary solvent delivery systems

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.

3 simple steps to 2D-LC on an EASY-nLC

In this set-up, salt solutions are drawn from vials in the autosampler of the EASY-nLC system and pumped through the biphasic column. This eliminates the requirement for ternary or quaternary gradient systems thus greatly simplifying the experimental set-up. Split-free injection of salt solutions from the autosampler minimizes the risk of dilution and mixing and facilitates precise, low carry-over, step-wise elution of peptides from the SCX column onto the RP column.

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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Recommended materials and methods

Schematics of a 2D EASY-nLC configuration

2D-LC experiments are easily implemented using a biphasic pre-column combined with a RP analytical column. A 2D-LC experiment consists of a batch of normal EASY-nLC injections, starting with an injection of the actual sample and followed by a number of salt plug injections of increasing ionic strength. Biphasic pre-column 150 m ID fused silica column packed with: 3.5 cm C18 material 3.5 cm SCX material EASY-nLC method parameters Sample pick-up: Sample loading: Gradient: 8 L, 20 L/min 24 L, 3 L/min 400 nL/min 0-35 %B in 120 min 35-100 %B in 10 min 100 % in 10 min 10 L, 3 L/min 5L, 0.7 L/min 100 L flush volume

Analytical column 75m ID fused silica column packed with: 10 cm C18 material

Pre-column re-equilibration: Analytical column re-equilibration: Autosampler wash:

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)

Solvents A: B: 0.1 % formic acid 99.9 % acetonitrile, 0.1 % formic acid

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

EASY-nLC and StageTips are trademarks of Proxeon A/S. 2010

www.proxeon.com