**3. Application example: The case study**

The analysis concept is based on an on-line sample preparation and a two-dimensional LC (see Figure 7) system: pre-separating the majority of the matrix components from the analytes which are retained on a RAM-SCX (LiChrospher 60 XDS (SO3/Diol), two 25 x 4mm

Fig. 7. Multidimensional chromatographic separation platform with integrated on-line sample clean-up.

Profiling of Endogenous Peptides by Multidimensional Liquid Chromatography 241

This usually reflects redundancy (peptides that elute in more than one fraction), peptide species with and without oxidative states, and a small number of mass spectrometric

**1000 1500 2000 2500 3000 3500 4000**

**Mass, Da**

The fully automated 2D-LC system performing an effective fractionation combined with offline MALDI TOF MS offers an enormous potential for human peptidomics screening on a daily basis. The system offers a high flexibility to be optimized for effective analysis of other biofluids such as amniotic fluid, sputum, urine, etc. The systems are completely automated and perform a high number of analysis cycles with low cost per analysis. Fast and comprehensive mapping of bio peptides and protein fragments will open possibilities to recognize novel and specific biomarkers that will help to diagnose disease and possibly

Novel restricted access materials have shown high efficiency in sample clean-up after direct on-line biofluid injections. Benefits of monolithic silica columns such as: super eminent low backpressure compared to particulate packed columns, therefore high variability of flow rates adjustments is possible; superior long term stability and data reproducibility analyzing various proteinaceous samples; much higher flow rates allows speeding up the overall analysis: fast separation, washing and re-equilibration. When those two novel developments are combined in a elegant multidimensional and fully automated way proteomic analysis

In the future, proteomics will play the major role in drug discovery, accelerating the various steps involved – target identification, target validation, drug discovery (efficacy, selectivity

Fig. 8. Example of a human plasma peptide map. Injection volume is 48µl.

provide valuable information for new drug development.

derivatives, such as fragment ions.

**0**

**4. Conclusion** 

could be accentuated.

**20**

**40**

**60**

**Fraction number**

**80**

**100**

id) column followed by a solvent switch and transfer of the trapped peptides using five salt steps by mixing 20 mM phosphate buffer (pH 2.5) (eluent A1) and 20 mM phosphate buffer with 1.5 M sodium chloride (eluent B1) at following proportions: 85/15; 70/30; 65/45; 45/55; 0/100 at the constant 0.1 ml/min flow rate was performed after the switching for the second dimensional strong cation exchange analytical separation and trapped onto the RP column by means of column switching in a way to perform two-dimensional orthogonal separations.

Desorption of the adsorbed species from the RAM-SCX column could be accomplished by employing an eluent with a higher solvent strength or pH than the eluent at the loading. We preferred the salt steps as the pH needed double time for re-equilibration. The desorption step was repeated several times to eliminate memory effects. In order to avoid sample to sample cross contamination, two blank gradients were typically applied (with specific analytes or higher loadings it could reach up to five blank gradients). The further steps of the analysis e.g. the transfer from the RAM-SCX column to the next (analytical cation exchange column) is heavily dependant on the way this transfer is performed. Three different modes could be chosen to elute the trapped sample from the RAM-SCX column: isocratic, one step elution with a strong solvent, elution with a linear gradient and elution with pulsed gradient.

A desalting and preconcentration of the fractions containing proteinaceous components were performed on two identical trap columns Zorbax 300 SB-C18, 5 μm particles, 5 x 0.3 mm I.D. obtained from Agilent (Agilent, Waldbronn, Germany). As a final column a monolithic fused silica RP-18 endcapped capillary column of dimensions 150 x 0.1 mm I.D. (Chromolith CapRod, Merck KGaA, Darmstadt, Germany) was used. We preferred the monolithic type of column over particulate capillary column for the following reasons: (a) monolithic silica columns offers high variability of flow rates adjustments, which is particularly useful in the set up of multidimensional LC MS system to adjust for different column sizes; (b) the monolithic silica columns implemented in the multidimensional LC MS system meets the requirement of high reproducibility as with particulate columns; (c) in terms of column robustness and usage flexibility monolithic silica columns are superior that packed particulate columns eg.: one could cut the top end column when damaged, furthermore, there is no change in the permeability as a result of pressure fluctuation; the end of the capillary directly connected to the MS; no frits are required etc. Standard acetonitrile gradient with 0.1 % formic acid at constant 2 µl/min flow rate separated trapped peptides in 40 min. The end of reverse phase capillary column directly inserted in an in house made robotic spotting apparatus so that the droplets are accumulated above MALDI plate and directed consequently from spot to spot with 2 minute intervals filling 100 spot MALDI plate per sample (5 fractions from the RAM-SCX column (salt steps), 20 fractions from the monolithic capillary RP 18e column, 5x20=100). After all plate positions were filled and dried out properly matrix material, consisting of -cyano-4-hydroxycinamonic acid in 50 % acetonitrile / 4 % formic acid / water (v/v/v) of a volume of 0.5 µl was spotted on the top. The MALDI plate was kept in dark place and analysed within the 12 hours.

As already mentioned the system performs an on-line directly injected human plasma, cerebrospinal fluid and urine sample separation in a fully automated way, by a scale down strategy, gaining in sensitivity. In all peptide displays (Figure 8), between 1,000 and 4,000 mass spectrometric signals appeared, which correspond to 500 – 2,000 individual peptides. This usually reflects redundancy (peptides that elute in more than one fraction), peptide species with and without oxidative states, and a small number of mass spectrometric derivatives, such as fragment ions.

Fig. 8. Example of a human plasma peptide map. Injection volume is 48µl.

The fully automated 2D-LC system performing an effective fractionation combined with offline MALDI TOF MS offers an enormous potential for human peptidomics screening on a daily basis. The system offers a high flexibility to be optimized for effective analysis of other biofluids such as amniotic fluid, sputum, urine, etc. The systems are completely automated and perform a high number of analysis cycles with low cost per analysis. Fast and comprehensive mapping of bio peptides and protein fragments will open possibilities to recognize novel and specific biomarkers that will help to diagnose disease and possibly provide valuable information for new drug development.
