**4. Conclusion**

240 Biomarker

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

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

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.

separations.

with pulsed gradient.

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 could be accentuated.

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

Profiling of Endogenous Peptides by Multidimensional Liquid Chromatography 243

Heine, G., Raida, M. and Forssmann, W.-G. (1997). Mapping of peptides and protein

Hille, J.M., Freed, A.L. and Wätzig, H. (2001). Possibilities to improve automation, speed

Hogendoorn, E.A., Dijkman, E., Baumann, B., Hidalgo, C., Sancho, J.-V., Hernandez, F.

Huber, J.F.K. and Lamprecht, G. (1995). Assay of neopterin in serum by means of two-

switching using three retention mechanisms. *J. Chromatogr. B*, 666, 223–232 Issaq, H.J. (2001). The role of separation science in proteomics research. *Electrophoresis*, 22,

Issaq, H.J., Chan, K.C., Janini, G.M., Conrads, T.P. and Veenstra, T.D. (2005).

Kele, M., Guiochon, G. (2002). Repeatability and reproducibility of retention data and band profiles on six batches of monolithic columns. *J. Chromatogr. A*, 960, 19-49. Link, A.J. (2002) Multidimensional peptide separations in proteomics. *Trends Biotechnol.*

Machtejevas, E., John, H., Wagner, K., Standker, L., Marko-Varga, G., Forssmann, W-G.,

Machtejevas, E., Unger, K.K., Ditz, R. (2006). Multidimensional Column Liquid

Machtejevas, E., Denoyel, R., Meneses, J.M., Kudirkaitė, V., Grimes, B.A., Lubda, D., Unger,

Machtejevas, E., Andrecht, S., Lubda, D., Unger, K.K. (2007). Monolithic silica columns of

Machtejevas, E., Unger K.K. (2008). Proteomics Sample Preparation, in: Jörg von Hagen

electrophoresis and alternatives. *Electrophoresis*, 22, 4035–4052

*Enzyme Regul*., 49(1), 121–132

*Chromatogr. A*, 776, 117–124

*Chem*., 71 1111-1118

*Chromatogr. B,* 817, 35–47

*Chromatography B*, 803 121-130

Meyer, 89-111, Wiley-VCH, Weinheim, Germany

materials. *J. Chromatogr. A*, 1123, 38–46

Weinheim, Germany

3629-3638

20(12S), 8-13

targets of Egln1 prolyl hydroxylase knockdown in renal carcinoma cells. *Adv* 

fragments in human urine using liquid chromatography – mass spectrometry*. J.* 

and precision of proteome analysis: A comparison of two-dimensional

(1999). Strategies in using analytical restricted access media columns for the removal of humic acid interferences in the trace analysis of acidic herbicides in water samples by coupled column liquid chromatography with UV detection. *Anal.* 

dimensional high-performance liquid chromatography with automated column

Multidimensional separation of peptides for effective proteomic analysis. *J.* 

Bischoff, R., Unger, K.K. (2004). Automated multidimensional HPLC: sample preparation and identification of peptides from human blood filtrate. *J.* 

Chromatography (LC) in Proteomics – Where Are We Now? in *Proteomics in Drug Research*, ed. M. Hamacher, K. Marcus, K. Stühler, A. van Hall, B. Warscheid, H.E.

K.K. (2006). Sulphonic acid strong cation-exchange restricted access columns in sample cleanup for profiling of endogeneous peptides in multidimensional liquid chromatography Structure and function of strong cation-exchange restricted access

various formats in automated sample clean-up/multidimensional liquid chromatography/mass spectrometry for peptidomics. *J. Chromatogr. A*, 1144 97-101

(Editor), *Sample preparation for HPLC – based proteome analysis*. 245-264, Wiley-VCH,

and mode of action). Operated on a routine basis, MD-LC may provide with the desired data, and after interconnection with the biology outcome could be found.

#### **5. References**


and mode of action). Operated on a routine basis, MD-LC may provide with the desired

Andrecht, S., Anders, J., Hendriks, R., Machtejevas, E., Unger, K. (2004). Effiziente

Boos, K.S. and Rudolphi, A. (1997). The use of restricted-access media in HPLC, Part I –

Boos, K.S. and Grimm, C.H. (1999). High-performance liquid chromatography integrated

Bovanova, L., Brandsteterova, E., (2000). Direct analysis of food samples by high-

Clynen, E., De Loof, A. and Schoofs, L. (2003). The use of peptidomics in endocrine research.

Cortes, H.J. (1990). *Multidimensional Chromatography. Techniques and Applications*, Marcel

Desilets, C.P., Rounds, M.A., Regnier, F.E. (1991). Semipermeable-surface reversed-phase media for high-performance liquid chromatography. *J. Chromatogr*., 544, 25-39. Fort, P.E., Freeman, W.M., Losiewicz, M.K., Sigh, R.S.J., Gardner, T.W. (2009). The Retinal

Giddings, J.C. (1984). Two-dimensional separations: concept and promise. *Anal. Chem*., 56,

Giddings, J. C. (1995). Sample dimensionality: a predictor of order-disorder in component peak distribution in multidimensional separation. *J. Chromatogr. A*, 703, 3–15 Gevaert, K., Van Damme, J., Goethals, M., Thomas, G.R., Hoorelbeke, B., Demol, H.,

Govorukhina, N.I., Keizer-Gunnink, A., van der Zee, A.G.J., de Jong, S., de Bruijn, H.W.A.,

Guryča, V., Kieffer-Jaquinod, S., Garin, J., Masselon, C.D. (2008). Prospects for monolithic nano-LC columns in shotgun proteomics. *Anal. Bioanal. Chem*., 392, 1291-1297. Griffin, T.J., Gygi, S.P., Ideker, T., Rist, B., Eng, J., Hood, L. and Aebersold, R. (2002).

Hagestam, I.H., Pinkerton, T.C. (1985). Internal surface reversed-phase silica supports for

Haffey, W.D., Mikhaylova, O., Meller, J., Yi, Y., Greis, K.D., and Czyzyk-Krzeska M.F.

levels in Saccharomyces cerevisiae. *Mol. Cell. Proteomics*, 1, 323-333

Proteome in Experimental Diabetic Retinopathy: Up-regulation of Crystallins and Reversal by Systemic and Periocular Insulin. *Molecular & Cellular Proteomics*, 8,

Martens, L., Puype, M., Staes, A. and Vandekerckhove, J. (2002). Chromatographic isolation of methionine-containing peptides for gel-free proteome analysis. *Mol.* 

Bischoff, R. (2003). Sample preparation of human serum for the analysis of tumor markers. Comparison of different approaches for albumin and γ-globulin

Complementary profiling of gene expression at the transcriptome and proteome

(2009). iTRAQ Proteomic identification of pVHL-dependent and -independent

performance liquid chromatography. *J. Chromatogr. A*, 880 149-168

Probenvorbereitung fűr die Proteomanalyse von Kőrperflűssigkeiten. *Laborwelt*,

solid-phase exctraction in bioanalysis using restricted access precolumn packings.

data, and after interconnection with the biology outcome could be found.

Classification and review. *LC-GC Int*., 15, 602-611

*Trends Anal. Chem*., 18, 175-180

*Gen. Comp. Endocr*., 132, 1-9

*Cell. Proteomics*, 1, 896-903

depletion. *J. Chromatogr. A*, 1009, 171-178

liquid chromatography. *Anal. Chem*., 57 1757-1763

Dekker, New York

767-779

1258–1270

**5. References** 

5/2, 4–7

targets of Egln1 prolyl hydroxylase knockdown in renal carcinoma cells. *Adv Enzyme Regul*., 49(1), 121–132


Profiling of Endogenous Peptides by Multidimensional Liquid Chromatography 245

Richter, R., Schulz-Knappe, P., Schrader, M., Standker, L., Jurgens, M., Tammen, H. and

plasma: database of circulating human peptides. *J. Chromatogr. B*, 726, 25–35 Rieux, L., Niederlander, H., Verpoorte, E., Bischoff, R. (2005), Silica monolithic columns:

Schulz-Knappe, P., Zucht, H-D., Heine, G., Jürgens, M., Hess, R. and Schrader, M. (2001).

mixtures. *Combinatorial Chemistry & High Throughput Screening*, 4, 207–217 Souverain, S., Rudaz, S., Veuthey, J.-L., (2004), Restricted access materials and large particle

Svec, F., Tennikova, T., Deyl, Z. (editors) (2003). *Monolithic materials: preparation, properties* 

Szabolcs, F., Jeno, F., Katalin, G. (2009). Characterisation of new types of stationary phases for fast liquid chromatographic applications. *J. Pharm. Biomed. Anal*., 50(5), 703-712 Unger, K.K., Machtejevas, E. (2007). Mehr als nur eine Säule – die Möglichkeiten der

Unger, K.K., Machtejevas, E. (2009). Peptid-Profilanalyse aus Körperflüssigkeiten.

Unger K.K., Tanaka, N. and Machtejevas, E. (2011). *Monolithic silicas in separation science:* 

Wagner, K., Miliotis, T., Marko-Varga, G., Bischoff, R. and Unger, K.K. (2002). An automated

Wang Y.Y., Cheng, P., Chan, D.W. (2003). A simple affinity spin tube filter method for

Westermeier, R., Naven, T., and Hēpker, H.-R. (2008). *Proteomics in Practice. A Guide to* 

Willemsen, O., Machtejevas, E., Unger, K.K. (2004). Enrichment of proteinaceous materials

Xiong, L., Zhang, R., Regnier F.E. (2004). Potential of silica monolithic columns in peptide

Yamashita, K,, Motohashi, M., Yashiki, T. (1992). Automated high-perfomance liquid

*Concepts, syntheses, characterization, modeling and applications*. Wiley-VCH,

on-line multidimensional HPLC system for protein and peptide mapping with

removing high-abundant common proteins or enriching low-adundant biomarkers

*Successful Experimental Design* 2nd Ed. WILEY-VCH Verlag GmbH & Co. KGaA,

on a strong cation-exchange diol silica restricted access material (RAM): proteinprotein displacement and interaction effects. *J. Chromatography A*, 1025, 209-216. Wolters, D.A., Washburn, M.P. and Yates III, J.R. (2001). An automated multidimensional

protein identification technology for shotgun proteomics. *Anal. Chem*., 73, 5683-

chromatographic method for the simultaneous determination of cefotiam and delta 3-cefotiam in human plasma using column switching. *J. Chromatogr*., 577,

*and applications*. Elsevier Amsterdam, The Netherlands. P. 800.

mehrdimensionalen LC. *Nachrichten aus der Chemie*, 6 661-664

integrated sample preparation. *Anal. Chem*., 74, 809–820

for serum proteomic analysis. *Proteomics,* 3, 243-248

separations. *J. Chromatogr. A*, 1030, 187–194

*J. Sep. Sci*., 28, 1628–1641

*ChromChat*, 4 44 -47

Weinheim, Germany, P. 374

Weinheim, Germany, P. 482

5690

174-179

analysis. *J. Chromatogr. B*, 801, 141-156

Forssmann, W.-G. (1999). Composition of the peptide fraction in human blood

Synthesis, characterisation and applications to the analysis of biological molecules.

Peptidomics: the comprehensive analysis of peptides in complex biological

supports for on-line sample preparation: an attractive approach for biological fluids


Machtejevas, E., Unger, K.K. (2008). Multidimensional Liquid Chromatography: Theory and

Machtejevas, E., Marko-Varga. G., Lindberg, C., Lubda, D., Hendriks, R., Unger, K.K. (2009).

Mazsaroff, I., Regnier, F.E. (1988). Phase ratio determination in an ion-exchange column having pores partially accessible to proteins. *J. Chromatogr.*, 442: 15-28 Meyers, J.J., Liapis, A.I. (1999). Network modeling of the convective flow and diffusion of

Nakamura, K., Suzuki, T., Kamichika, T., Hasegawa, M., Kato, Y., Sasaki, H., Inouye, K.

Pandey, A. and Mann, M. (2000). Proteomics to study genes and genomes. *Nature*, 405,

Pang, J.X., Ginnanni, N., Donge, A.R., Hefta, S.A. and Opiteck, G.J. (2002). Biomarker

Pinkerton, T.C. (1991). High-performance liquid chromatography packing materials for the

Pitiot, O., Poraht, J., Guzmann, R., Vijayalakshmi, M.A. (2004). 9th International Symposium

Premstaller, A., Oberacher, H., Walcher, W., Timperio, A.M., Zolla, L., Chervet, J-P.,

Račaitytė, K., Lutz, E.S.M., Unger, K.K., Lubda, D., Boos, K.S. (2000). Analysis of

Rbeida, O., Christiaens, B., Hubert, Ph., Lubda, D., Boos, K.-S., Crommen, J. and Chiap, P.

Regnier, F., Amini, A., Chakraborty, A., Geng, M., Ji, J., Riggs, L., Sioma, C., Wang, S. and

Capillary Columns for Proteomic Studies. *Anal. Chem*., 73: 2390-2396 Quaglia, M., Machtejevas, E., Hennessy, T. and Unger, K.K. (2006). Size Exclusion

*Optimization*,- Wiley-VCH, Weinheim, Germany, 383-403

detection. *J. Pharm. and Biomed. Anal*., 36/5: 947-954

approach to proteomics. *LC-GC*, 19, 200–213

discovery in urine by proteomics. *J. Proteome Res*., 1, 161-169

New Jersey, USA

*A*, 972, 21-25

837-846

544: 13-23

135-144

Human Urine. *J. Sep. Sci*. 32, 2223 – 2232

chromatographic column. *J. Chromatogr. A*, 852, 3-23

Applications in Industrial Chemistry and the Life Sciences. Steven A. Cohen and Mark R. Schure (Editors), *Peptidomics*. 207-220. John Wiley & Sons Inc., Hoboken,

Profiling of Endogenous Peptides by Multidimensional Liquid Chromatography: On-line Automated Sample Cleanup Procedures for Biomarker Discovery in

molecules adsorbing in monoliths and in porous particles packed in a

(2002). Evaluation and applications of a new dye affinity adsorbent. *J. Chromatogr.* 

analysis of small molecules in biological matrices by direct injection. *J. Chromatogr.,*

on Biochromatography, 5–7 May, Bordeaux, France, Programme & Abstracts, p. 34

Cavusoglu, N., van Dorsselaer, A., Huber, Ch.G. (2001). High-Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry Using Monolithic

Chromatography (SEC) of biopolymers) Optimization strategies and trouble shouting. S. Kromidas (Editor), *HPLC Made to Measure – A Practical Handbook for* 

neuropeptide Y and its metabolites by high-performance liquid chromatography– electrospray ionization mass spectrometry and integrated sample clean-up with a novel restricted-access sulphonic acid cation exchanger. *J. Chromatogr. A*, 890:

(2005). Integrated on-line sample clean-up using cation exchange restricted access sorbent for the LC determination of atropine in human plasma coupled to UV

Zhang, X. (2001). Multidimensional chromatography and the signature peptide


**1. Introduction** 

the mental stresses.

indicate both PNE and PNI).

biomarkers in responding to stress.

modelling approach are introduced.

**12** 

*Japan* 

Shusaku Nomura

*Nagaoka University of Technology,* 

**Salivary Hormones, Immunes and** 

In the modern society, the issues on mental health have always been pressing and global problem, and unfortunately it remains today unsolved. It does not apply only to a personal matter, but also having a huge impact on economics as it has estimated a decade ago of that the social cost concerning mental health problem in European Union was 3 to 4% of GNP (Gabriel, 2000). However generally speaking, the mental stress is quite difficult to be aware of personally. It is hard for anyone to manage mental stress by on his/her own. Therefore it is an urgent task to figure out a "practical" methodology to evaluate, manage and control

On the other hand, recent developments of the molecular analysis techniques has been revealed that particular hormones and immune substances secreted within human body change its level in responding to human mental state. For an instance, salivary Immunoglobulin-A shows a transient increase against short-term psychological stressors such as mental arithmetic task, stroop task, academic presentation (Valdimarsdottir, 1994). "All illnesses come from the mind" is no longer a folk story. These particular hormones and immune substances can potentially be a practical biomarker for human mental stress. Number of hormones and other substances in our body has been studied as a possible stress biomarker (Izawa, 2004; Wakida, 2004), and the number of academic research has been increasing as well, as shown in Figure 1. Currently it forms an interdisciplinary research field called psychoneuroendocrinology (PNE) and/or psychoneuroimmunology (PNI) (Ader, 2001) (hereafter, we use the term psychoneuroendocrine-immunology (PNEI) to

PNEI must be a contributory research field which should possibly establish "practical" criteria for objectively evaluating human mental state. However it is a relatively new field of study still developing, there are a lot of stuffs to be investigated, e.g. the precise stress response of these biomarkers in the time series, the sensitivity of the response against other than acute stressors, physiological mechanism dynamically regulating the release of these

In this chapter, PNEI research of its background, method, experiments, and mathematical

**Other Secretory Substances as** 

**Possible Stress Biomarker** 

Yu, Z., Westerlund, D. and Boos, K.S. (1997). Determination of methotrexate and its metabolite 7-hydroxymethotrexate by direct injection of human plasma into a column-switching liquid chromatographic system using post-column photochemical reaction with fluorimetric detection. *J. Chromatogr. B*, 689, 379-386
