**2.1 Designing a MD-LC system**

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Fig. 2. Separation of standard protein digest and real biological sample analysis: upper chromatogram - 1 µl BSA digest (1mg/ml), lower chromatogram 8 µl of filtered amniotic fluid. Conditions: column - Chromolith Performance RP-18e 100 mm x 2 mm I.D.; eluents - A: 95% H2O/5% ACN/0.1% TFA (v/v/v), B: 5% H2O/95% ACN/0.085% TFA (v/v/v); gradient - from 5% B to 50% B in 20 min; flow rate - 0.3 ml/min, detection - UV 214 nm.

compounds in a reversed phase mode or in an ion-exchange mode. By regulating the pore size of the particles, the molecular weight exclusion can be varied as well as the molecular weight fractionation range, which allows certain analytes to be trapped at the internal surface. In this case, only proteins and peptides below a certain molecular shape and size have access to the inner pore surface of the RAM, are thus retained while the larger proteins encounter only the hydrophilic, non-adsorptive outer surface, and will be flushed out in the following washing step. Of the RAM, the strong cation exchanger with sulphonic ligands (RAM-SCX) was preferably employed in the sample clean up of proteins, which proved to show an acceptable capacity towards positively charged peptides and proteins. The features described above, when elegantly combined with column switching, become a powerful tool for direct analysis in the profiling of endogeneous peptides in a fully automated,

**Time, min**

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The primary criteria for the choice of a separation phase system are selectivity and orthogonality, mass loadability, and biocompatibility (in case of quantitation). As a rule of thumb, the first dimension should possess a high mass loadability combined with sufficient selectivity and maintenance of bioactivity. Ion exchange chromatography (IEC) therefore is the method of choice offering charge selectivity. In principle, there are two options in IEC, either to employ a cation or anion exchanger, which in return influences the pH working range. Note that either cationic or anionic species are resolved, i.e. only a limited number of species from the whole spectrum. The IEC columns are operated via salt gradients with increasing ionic strength. Consequently, the salt load must be removed before the fractions are transferred to the second dimension column.

It is most common to use reversed phase chromatography as the second dimension. The term RP stands for a number of columns with different degrees of hydrophobicity. The most commonly applied phases are n-octadecyl bonded silicas (RP-18 columns). An intrinsic feature of RP columns is their desalting property. Salts are eluted at the front of the chromatogram, when running a gradient elution with an acidic buffer/acetonitrile mobile phase with increasing acetonitrile content. The hydrophobic surface of the RP packing and the hydrophobic eluent are not favorable with respect to providing a biocompatible environment for proteins: they may change their conformation or denature which may be seen by the appearance of broad peaks, splitting of peaks etc. RP columns possess a much lower mass loadability than IEC columns (10 mg of protein per gram of packing as compared to 100 mg in IEC). An advantage of RP is the fact that the eluents are compatible to MS, provided volatile buffers such as ammonium acetate are employed.

In case of an on-line MD-LC system, the speed of analysis in the second dimension should be as high as possible (Wagner et al., 2002). This, however, conflicts with the requirement of high resolution or high peak capacity. The highest peak capacity in gradient elution RPC is obtained with a shallow gradient at relatively low flow-rate. Thus, a compromise between the desired peak capacity and the gradient time is inevitable. Often gradient times of several hours are applied for the analysis of peptides from protein digests.

A question often arising is: How many dimensions do we need in MD-LC? It becomes obvious that as the number of dimensions increases, the peak capacity will increase. In an ideal case the total peak capacity of the MD-LC system is equal to the product of the individual orthogonal dimensions. At the same time, above two dimensions an on-line MD-LC system becomes very sophisticated in its instrumental setup and may be difficult to control. The major goal in proteomics for the common user is to design a highly efficient, error minimizing and easy-to-handle system. Reduction of the system complexity is the major demand. It is essential to select a minimum number of dimensions to handle complex separations which also should preferably include on-line sample clean-up steps.

### **2.2 Advantages of on-line sample clean-up approaches**

Most sample-preparation procedures are performed manually and are thus time-consuming and laborious. On-line sample clean-up and on-column concentrations avoid this disadvantage. There are a number of important features that is gained by having the liquid phase separation system to be operated on-line. The overall yield in most cases is improved

Profiling of Endogenous Peptides by Multidimensional Liquid Chromatography 233

selective. Dye ligand chromatography, a technique that is extensively used in protein chromatography was surprisingly effective (Andrecht et al., 2004) in particular with regard to high binding capacities and a long column lifetime, however, at the expense of selectivity.

The direct injection of biological samples onto the chromatographic column without any sample preparation is in most cases highly problematic and may lead to an irreversible contamination of the separation columns, which deteriorate selectivity and column performance. A powerful asset to circumvent all the named problems is the implementation of restricted access material for sample preparation. Special Solid Phase Extraction (SPE) supports possessing restricted access properties have been developed (Hagestam and Pinkerton, 1985, Yu et al., 1997, Boos and Rudolphi, 1997). In 1991, Desilets et al. (Desilets, et al., 1991) introduced the restricted access term. Silica based Restricted Access Materials have been developed for the clean-up in bioanalysis; first for low molecular weight compounds in biofluids (Rbeida, et al. 2005) and subsequently for biopolymers such as peptides (Wagner et al. 2001). Those supports were able to withstand several hundred plasma or serum injections (total volume of 5–7 ml) without losing performance. The concept and the methodology were successfully used for the sample clean-up of peptides and proteins out of biofluids by extending the range of available materials employing cation and anion-exchanger RAM (Machtejevas et al., 2004)). Specific non-silica based RAM were also developed for the investigation of the food (Bovanova & Brandsteterova 2000) and environmental matrices (Hogendoorn et al., 1999). Vijayalakshmi and co-workers (Pitiot et al., 2004) have presented a new RAM called a bi-dimensional chromatographic support operating on a size exclusion mode and an affinity or pseudoaffinty mode. A survey on the current state-of-art of RAMcolumns in sample pre-treatment is given Souverain et. al. (Souverain et al., 2004). A RAM support developed by Boos and Grimm (Boos & Grimm, 1999) is based on SCX-diol modification to improve performances in terms of efficiency, retention and reproducibility. Račaitytė et al. (Račaitytė et al. 2000) have shown that this type of RAMs is highly suitable for the on-line extraction and analysis of neuropeptides in plasma. Machtejevas et al. (Machtejevas et al. 2006) analyzed the pore structural parameters and size exclusion properties of LiChrospher strong cation-exchange and reverse phase restricted access materials. For peptide analysis out of the biofluids, the strong cation-exchange functionality seems to be particularly suitable mainly because of the high loadability of the strong cationexchange restricted access material (SCX-RAM) and the fact that one can work under nondenaturing conditions to perform effective chromatographic separations. The proper column operating conditions leads to the total effective working time of the RAM column to

be equal to approximately 500 injections (depending on the type of sample).

The principle of the restricted access support is based upon the presence of two chemically different surface properties of porous silica particles (Figure 3). The outer surface of the particles (25 – 40 mm O.D.) is highly biocompatible: it possesses diol modification and hence is hydrophilic, while the pore surface chemistry is tailored as a hydrophobic dispersion phase with C-18 functionality or as a strong cation exchanger with SO3 functionality. An advantage of these adsorbents relies on the simultaneous occurrence of two chromatographic separation mechanisms: selective reversed-phase interaction or ion exchange chromatography of lower molecular mass analytes and size exclusion

**2.3 Restricted access material columns** 

compared to off-line approaches and methodologies. Exposed surfaces are kept to a minimum that usually is the main cause of sample losses. Overall precision can also be controlled by having yields above 50 %. It is possible to handle yields that are lower; however, it generally is a real analytical challenge to obtain operational stability within such analytical processes. Direct injection of samples onto HPLC columns is substantially advantageous in the clinical laboratories in terms of its time- and labor-saving capabilities, in addition to other advantages given below. General direct injection methods have been devised which deal with the problem of many different proteins being present in the sample. The methods include the pre-column technique, restricted access materials, and chromatography in mobile phases containing surfactant. High performance affinity chromatography is also a direct injection technique will demonstrate its power in near feature (Figure 1 d). The characteristic and performance of each direct injection technique are comprehensively discussed below for the analysis of biological samples.

The pre-column technique is the direct injection technique that is mostly reported. The precolumn technique utilizes two columns in series (pre-column and analytical) connected by a switching valve. The most common pre-column technique employs a reversed phase precolumn and a reversed-phase analytical column: the sample is injected into an aqueous mobile phase flowing through a pre-column (1-4 cm in length, 3 - 4.6 mm I.D.) which retains lipophilic compounds, passing non-retained hydrophilic compounds to waste. The switch in valve is then changed and components retained on the pre-column are eluted onto the analytical column by increasing the solvent strength of the mobile phase. This technique serves the dual function of concentration of analyte and removal of hydrophilic substances. There are many advantages of the pre-column injection technique in comparison to traditional sample preparation techniques: time saving in comparison to the labor-intensive liquid-liquid extraction and precipitation techniques, high reproducibility and high, also a superior detection limit capabilities due to its allowance for injection of large sample volumes.

Several types of column-switching designs have been applied. The back flush design is most often used because it reduces band broadening (Yamashita et al., 1992). However some prefer the forward-flush mode to protect the analytical column from possible impurities at the head of the column. Use of an on-line ~0.5µm filter is recommended, which needs periodic replacement. A design that incorporates a second pre-column parallel to the first has been employed which increases sample throughput, by alternating injection on one precolumn and back flushing of retained compounds on the other pre-column.

Most biofluids contain large amounts of well-known proteins such as albumin and IgGs, which overwhelm the separation system and make the detection of the low abundant proteins and peptides very difficult. It is thus advantageous to remove these proteins prior to digestion or direct separation. There are alternative ways of reducing the overall protein load by specific adsorption of albumin and IgG to affinity matrices (Nakamura et al., 2002, Wang et al., 2003, Govorukhina et al., 2003). While usually an affinity matrix is generally highly specific, in high content samples the affinity ligand is limited to exhibit its specificity. There are degrees of specificity between highly selective immunoaffinity matrices and less selective but more robust affinity supports using synthetic ligands. In an effort to reduce the amount of albumin from human serum, a number of affinity matrices has been evaluated based on antibodies or dye ligands. Antibody-mediated albumin removal was efficient and selective. Dye ligand chromatography, a technique that is extensively used in protein chromatography was surprisingly effective (Andrecht et al., 2004) in particular with regard to high binding capacities and a long column lifetime, however, at the expense of selectivity.
