Data reliability through high retention time repeatability
The high basic performance of the LC-20ADnano equipped with the RFC system is demonstrated by a retention time repeatability of less than 0.20 RSD% (n=6) at a flow rate of 300 nL/min. Proteome analysis requires the separation and identification of many enzyme digest peptides that have very similar characteristics. Also very important is retention time repeatability for peak comparison of samples in differential analysis. The high repeatability of the Prominence nano robustly supports the acquisition of the high-precision data required for effective proteome analysis.
High resolution in 2-dimensional LC
In analysis of complex samples such as protein enzyme digests, reversed-phase 1-dimensional separation does not offer sufficient peak capacity. Conversely, 2-dimensional separation provides larger peak capacity, enabling powerful separation of complex samples. Therefore, 2-dimensional separation is conducted using a combination of separation modes which are not subject to mutual interaction. The Prominence nano system allows easy construction of a 2-dimensional LC combining the cation exchange with reversed-phase modes*, providing a core system with all the performance needed for effective proteome analysis. The figure below shows an analysis of 200 fmol protein from yeast. Unseparated peak components in the 1st dimension are separated into different fractions in the 2nd dimensional analysis, yielding a larger number of peaks than can be achieved in a single 1D system.
|Mobile phase||Ammonium formate buffer
Salt concentration step gradient
|Trap column||L-column Micro (5 mmL.×300mm I.D.)|
|Trapping period||5 min|
|Desalting solvent||Water/Formic acid = 100/0.1|
|Desalting flow rate||40mL/min|
|Desalting period||5 min|
|Column||PicoFrit (100 mmL.×75mm I.D.)|
|Mobile phase||Gradient elution
A) Water/Acetonitrile/Formic acid = 95/5/0.1 (v/v)
B) Water/Acetonitrile/Formic acid = 5/95/0.1 (v/v)
|Sample||Proteins in Yeast Protein Digest
(equivalent to 200 fmol)
* Other combinations of separation modes are possible using different type of columns in the 1st and 2nd dimensions, respectively. In this case, there may be some limitation in the separation or detection method due to the interaction between separation modes or the types of mobile phase used.
Effortless Coordination with MALDI-TOFMS
The Prominence nano system can be used not only connected on-line to an MS nano-ESI interface, but also to a MALDI-TOFMS with the Accuspot, a MALDI plate spotter. For accurate spotting of each chromatographic peak onto the MALDI plate, an LC with nano flow-rate control optimized for the range of hundreds nL to 1 µL is required. The Prominence nano system is perfectly suited for this purpose.
|Column||MonoCap for Fast-flow (250 mmL. × 100 µm I.D.)|
|Mobile phase||A) Water/Acetonitrile/TFA = 95/ 5/0.1 (v/v)
B) Water/Acetonitrile/TFA = 10/90/0.1 (v/v)
|Flow rate||1 µL/min|
|Trap column||ODS (1 mmL. × 0.5 mm I.D.)|
|Spot interval||12-second intervals
(Spot volume per spot : 200 nL excluding matrix solution)