Waters - nanoACQUITY UPLC System with 2D Technology
Employs reversed-phase chromatography at pH 10 in the first dimension, followed by reversed-phase chromatography at pH 2 in the second dimension.
Conventional 2D-LC uses ion exchange (IEX) followed by reversed- phase (RP). Any IEX approach will use salt-containing buffers that can cause ionization background and fouling problems if they enter a mass spectrometer (MS). Since IEX separations are based solely on the charge of the peptide, the IEX dimension often results in poor chromatographic resolution with peptides appearing in multiple fractions, making data interpretation difficult.
This improved 2D approach uses RP at pH 10 in the first dimension, followed by RP at pH 2 in the second dimension for results that far exceeds those of conventional IEX methodolgies.
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Features of nanoACQUITY UPLC System with 2D Technology
- High-resolution in both dimensions by exploiting the wide-ranging ionic and hydrophobic structure of peptides
- Better protein identifications, quantification, and sequence coverage
- Improved separation, method generation wizards, and enhanced data algorithms
High Resolution Separations in Both Dimensions
When ion exchange is used as the first separation dimension, poor resolution and bleed between fractions could occur. Using a highly resolving support in the first step provides you with better information in the last step. A reversed phase support at pH 10, followed by a sub-2-micron separation at lower pH, useful, orthogonal separations that yield high quality data is obtained.
Gilar M. ET AL. J.Sep.Sci. 2005,28,1694-1703.
A salt-free approach was used to change the selectivity of the separation. A new selectivity was achieved and maintained chromatographic resolution whole using MS-friendly, volatile mobile phase modifiers.
Secure Identifications for Best-In-Class Reproducibility
An increased number of secure identifications is shown as a function of 2D-LC resolution. Both reproducible and incremental identifications of proteins based on 2D-LC can be seen.
Increased Resolution Equals Better Protein Identifications
In this separation, the power of 2D-LC for resolution of complex proteomic samples is demonstrated by this tryptic digest of E. coli. Using nanoACQUITY® in one dimension, 0.5 µg were loaded on-column to resolve and identify 365 proteins from 2874 peptides. Identification of 7661 and 9415 peptides from the 5-step and 10-step separations respectively were obtained by adding 2.5 µg to the 2D-LC System.
The 2D-LC enabled
- Identification of twice as many proteins more securely based
- Recovery of more peptides
- Greater protein coverage with high confidence in results
nanoACQUITY 2D-LC Configuration
To load the sample, inject from the sample manager onto an RP column packed with 3 µm particles (RP1). RP1 provides loading capacity without sacrificing resolution. Then, elute fractions from RP1 to the trapping column and on to RP2, the more highly resolving sub-2-mircon column, for higher peak capacity and better data quality.
The nanoACQUITY UPLC® System is equipped with two valves: one for injection in the sample manager and a second valve in the heating and trapping module (HTM). The entire method is driven from MassLynx™ Application Manager through method generators or wizards.
General Specifications
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