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Identify more proteins compared to conventional approaches 2D-enabled operation of EASY-nLC Identify ~ 4X more
Identify more proteins compared to conventional approaches 2D-enabled operation of EASY-nLC Identify ~ 4X more

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 ~ 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 split-free nanoscale chromatography system

integrated, split-free nanoscale chromatography system Achieve better analytical performance than regular

Achieve better analytical performance than regular ternary/quaternary solvent delivery systems

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.

highly skilled specialists for successful implementation. Figure 1. Sample: tryptic digest of a protein extract from
highly skilled specialists for successful implementation. Figure 1. Sample: tryptic digest of a protein extract from
highly skilled specialists for successful implementation. Figure 1. Sample: tryptic digest of a protein extract from
highly skilled specialists for successful implementation. Figure 1. Sample: tryptic digest of a protein extract from

Figure 1. Sample: tryptic digest of a protein extract from a human multiple myeloma cell line

a protein extract from a human multiple myeloma cell line Figure 2. Minimal mixing and dilution

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-nLC TM system, 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

2D-LC configuration. 3 simple steps to 2D-LC on an EASY-nLC Original publication: Automated 2D Peptide Separation
2D-LC configuration. 3 simple steps to 2D-LC on an EASY-nLC Original publication: Automated 2D Peptide Separation
2D-LC configuration. 3 simple steps to 2D-LC on an EASY-nLC Original publication: Automated 2D Peptide Separation
Original publication: Automated 2D Peptide Separation on a 1D Nano-LC-MS System, Taylor et al., Journal
Original publication:
Automated 2D
Peptide Separation
on a 1D Nano-LC-MS System,
Taylor et al., Journal of
Proteome Research,
ACS, DOI: 10.1021, 2009

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

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.

www.proxeon.commixing and facilitates precise, low carry-over, step-wise elution of peptides from the SCX column onto the

Recommended materials and methods

Recommended materials and methods Schematics of a 2D EASY-nLC configuration 2D-LC experiments are easily implemented using

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

EASY-nLC method parameters

150 μm ID fused silica column packed with:

Sample pick-up:

8 μL, 20 μL/min

3.5cm C18 material Sample loading: 24 μL, 3 μL/min

cm C18 material

Sample loading:

24 μL, 3 μL/min

3.5cm SCX material Gradient: 400 nL/min 0-35 %B in 120 min 35-100 %B in 10

cm SCX material

Gradient:

400 nL/min 0-35 %B in 120 min 35-100 %B in 10 min 100 % in 10 min

Analytical column

Pre-column re-equilibration:

10 μL, 3 μL/min

75μm ID fused silica column packed with:

Analytical column re-equilibration:

5μL, 0.7 μL/min

10 cm C18 material

10 cm C18 material

Autosampler wash:

100 μL flush volume

Note:

The 2D-LC method can also be implemented as a one-column setup (recommended column specifications:

75 μm ID fused silica, 5 cm SCX material and 5 cm C18 material)

Solvents

Sample/salt plugs

A: 0.1 % formic acid B: 99.9 % acetonitrile, 0.1 % formic acid Important: Desalt
A:
0.1 % formic acid
B:
99.9 % acetonitrile, 0.1 % formic acid
Important: Desalt and
purify samples by
extraction (StageTips™)
solid phase
prior to
2D-LC analysis.

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

Example 1: 9 steps ~ 24 hours analysis time

A1:

Sample (8 μL, < 20 μg 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

A1:

Sample (8 μL, < 20 μg total protein digest)

A2-A4:

25, 100, 500 mM ammonium acetate in 5 % acetonitrile, 0.1 % formic acid

www.proxeon.com< 20 μg total protein digest) A2-A4: 25, 100, 500 mM ammonium acetate in 5 %