Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 17, Num. 2, 2000, pp. 117

Biotecnología Aplicada 2000;17:117

New Technical Developments of Automates for High Sensitive Analytical Biotechnology

Christian Wurzel, Brigitte Wittmann-Liebold, WITA GmbH

Warthestr. 21, D-14513 Teltow, Germany. E-mail: wurzel@wita.de

Papers from Biotecnología Habana`99 Congress.
November 28-December 3, 1999. Code Number: ba00030

The research field of proteomics shifts the focus of biochemical interest to functional analysis. Whereas genome analysis reflects only the static state of the inherited information, the visualization and protein identification of entire cell extracts at different cell stages (proteomics) yields information on dynamic situations and hence, on the function of proteins. At least in parts protein separation and analysis is highly sensitive and automated, and concepts of high throughtput proteomics become visible due to new technical developments. Applications of the micro system technology make these systems smaller and faster. In opposite to DNA analysis, where PCR technique enables sample reproduction, protein analysis has to fight sensitivity problems. This is especially the case when limited material obtained by cell culturing or from biopsies and body fluids shall be analyzed. In the past, due to many technical innovations, the sensitivity of the Edman degradation of proteins and peptides could be increased from micromolar amounts requested in the first amino acid sequencer constructed by Edman and Begg [1] to low pico molar protein quantities necessary in the present day’s automates. However, yet, the sensitivity has to be further enhanced in order to enable the analysis of proteins and peptides which are expressed in extremely low concentrations. With the use of high resolution 2-dimensional gel electrophoresis, which allows to resolve up to 10,000 proteins of a complex protein mixture from entire cells or tissue extracts [2], differences in the protein expression during differentiation and embryonal development can be traced [3]. Other proteome studies are concerned with antibiotic resistance, drug screening, and tumor development when apoptosis is inhibited [4]. In the last few years, new technical developments in mass spectrometry facilitated high sensitive measurements of peptides and allowed to obtain peptide masses and partial sequences for identification of the protein spots by PSD-MALDI-MS or nanospray-ESI-techniques [5, 6]. On the other hand, some disadvantages of these techniques are:

While identification and confirmation of known proteins are mostly straightforward by these techniques, the interpretation of the data for unknown or modified proteins is much more difficult. Hence, alternative approaches, e.g. by direct amino acid sequencing employing the Edman chemistry, serve as an additional method that allows more extended sequence results and, therefore, an unambiguous assignment of the investigated proteins. A high sensitive Edman type protein sequencer has to cover at least three innovations:

Liquid handling

In present day’s sequencers, the consumption of reagents and solvents is relatively high; typically microliter volumes in the range of 20 to 200 are delivered due to the general construction of the machines and of the reactor, converter and the valves and their dimensions. The amounts of solvents are high, mainly in order to clean up and wash the delivery lines. The reagents adhere to the inner surface of the dosing system and represent liquid and dried remainders which may disturb the chemistry of the degradation and reduce the yields appropriately. We present an integrated approach to overcome these drawbacks. In the micro reaction and analysis system, all liquid handling devices as valves and reaction chambers necessary to perform the Edman degradation are fully integrated. The dimension of the valves and reactor and converter are considerably reduced. Therefore, the switching time of a valve (the response time) in the microreaction system is much faster than in common valves or valve systems. The smallest amounts of a reagent in sub micro liter amounts can be delivered. Due to the complete integration, no external connecting lines between the parts are necessary. The inner surface is much decreased, no hidden edges due to ferrules or fittings occur. Reagent delivery, washings and dryings of the lines can be substantially reduced [7, 8].

Modifications in chemistry
and the detection system

Commercial sequencers are equipped with 1-IPLC and UV-detection. This limits the sensitivity to the upper femtomolar range. These systems cannot overcome the Lambert-Beer’s Law. To gain higher sensitivity in protein sequencing, changes to the detection system are necessary, but require also changes in the chemistry. Generally two options are given:

b) a post degradation fluorescent labelling, or an additional post-cleavage reaction which couples a high sensitive marker to the released amino acid derivative [10]. These methods allow to connect a capillary electrophoresis with laser induced fluorescence detection (CE-LIF) or a mass spectrometric (MS) detection system directly to the Edman sequencer. These innovations are under development.

References

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3. Brockstedt E, Peters-Kottig M, Badock V, Hegele-Hartung C, Lessl M. Identification of a new LI-1/CG-regulated protein and the corresponding gene in the mouse ovary: Mouse vas deferens protein (manuscripts submitted).

4. Brockstedt E, Rickers A, Kostka S, Laubersheimer A, Dörken B, Wittmann-Liebold B, et al. Identification of apoptosis-associated proteins in a human Burkitt lymphoma cell line, cleavage of hnRNP Al by caspase 3. J Biol Chem 1998;273: 28057–64.

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6. Wilm M, Shevchenko A, Houtheave T, Breit S, Schweigerer L, Fotsis T, et al. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 1996;379(6564): 466–9.

7. Wurzel C, Wittmann-Liebold B. A new design of a wafer based micro Reaction System. In: Microreaction technology. W. Ehrfeld (editor) Springer;1998.p.219–24.

8. Wurzel C, Wittmann-Liebold B. New Approaches for Innovations in Sensitive Edman Sequencing. The Design of a Wafer Based Chip Sequencer. In P Jolles, H Jörnvall (editors.). Protein Sequences. Novel strategies and proteome analysis. Birkhäuser Verlag, Basel, in press.

9. Hess D, Nika H, Chow DT, Bures EJ, Morrison HD, Aebersold R. Liquid chromatography-electrospray ionization mass spectrometry of 4-(3-pyridinylmethylaminocarboxypropyl) phenylthiohydantoins. Anal Biochem 1995;224:373–81.

10. Tsugita A, Kamo M, Shikama N. Sensitization of Edman amino acid derivatives using the fluorescent reagent 4-amino-fluorescein. J Biochem 1989;106:60–5.