eye-research

Universitäts-Augenklinik Mainz
Experimentelle Ophthalmologie
Leitung: PD Dr. Dr. F. Grus
Langenbeckstr. 1, 55131 Mainz

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Basic Sciences: Proteomics

Dr. S. Funke, PhD

Dr. N. Perumal, PhD

N. Wehrwein (MTA)

M. Kegel (MTA)

Protein-Profiling

The up- and down regulation of proteins can provide important informations about disease processes and can lead to a better understanding of the pathogenesis. This process might identify important biomarkers of the disease and thus will lead to predictive medicine.

Beside conventional two-dimensional electrophoresis, our group is using the Seldi-TOF (surface enhanced laser desorption and ionization in time-of-flight mass spectrometry) ProteinChip. Furthermore, we have state-of-the technologies in our proteomics unit in our lab. This includes a Maldi-TOFTOF (Bruker Ultraflex), different robots for sample preparation, HPLC and nano-HPLC systems coupled to automated Maldi-Target spotting, and Protein Microarrayer.

Recently, we could use this technology to find biomarkers for dry-eye disease (s. News section).

In current projects we are using this technology to find biomarkers in other diseases than dry-eye, and to assess the side-effects of local eye medications e.g. lumbrificants, the effects of contact lenses on the tear film, and to test cosmetics for possible side effects on the tear film and ocular surface.

Technology

Previous studies demonstrated that the electrophoretic analysis of tear proteins is able to detect differences in the tear protein profiles between patients suffering from dry-eye syndrome and healthy subjects. The electrophoretic gels were digitized, the peak volumes quantified and furthermore analyzed by multivariate statistics and artificial neural networks. In these studies, the detection of dry-eye syndrome based on the specific electrophoretic patterns was demonstrated to be superior to detection by clinical standard tests e.g. Schirmer test (BST) and break-up time (BUT). Furthermore, altered protein profiles in tear can not only be found in dry-eye, but in some other ocular and systemic diseases e.g. diabetes mellitus and other factors that might interfere with the ocular surface e.g. contact lenses and smoking.
All these studies provide evidence that specific proteins or peptides in tear film can be used as diagnostic biomarkers for dry-eye, ocular surface diseases, and even systemic disease states such as diabetes mellitus.
The investigation of tear film proteins was not only limited to one-dimensional electrophoretic techniques. In other studies, the tear film proteins were extensively analyzed by high-performance liquid chromatography, two-dimensional electrophoretic approaches, or other chromatographic tests . 2D electrophoresis is a big step forward in proteome research because it offers a much better protein separation compared to 1D electrophoresis. In the first dimension the proteins migrate to their isoelectric point that is specific for each protein. In the second dimension the isoelectrically focused proteins are segregated according to their molecular weights. Using this technology, it was possible to analyze and quantify single proteins in tear film and to establish a tear film protein map.
However, the two-dimensional electrophoretic analysis of proteins is very time-consuming and often reveals problems in reproducibility. This is particularly important when this technique is used for mass screening of samples in clinical routine. To eliminate gel-to-gel variations, very complex and tedious anti-smiling and alignment procedures must be performed to ensure the quality of the spot matching process. Furthermore, the sensitivity of this method limits the analysis to proteins being larger than approximately 8 to 10 kDa.
Considering the increasing demand for mass-screening of protein profiles in tear fluid, a method is needed that overcomes the problems of two-dimensional electrophoretic separations.
The aim of this study was to analyze the usefulness of a recently developed proteomics technology, the ProteinChip system (SELDI= surface enhanced laser desorption/ionization) for the protein profiling of tears. This method was first described by Hutchens and Yip and utilizes affinity surfaces to retain proteins based on their physico-chemical characteristics, followed by direct analysis by time-of-flight (TOF) mass spectrometry (MS). This technique can be used for a rapid and efficient analysis of body fluids such as sera and cell culture samples. Proteins can be separated on different chip surfaces e.g. cationic and anionic exchangers, hydrophobic surfaces, and metal-ion affinity-chromatographic surfaces. Proteins that are retained on the chromatographic surfaces due to the chosen washing and binding conditions can be easily purified from contaminants such as buffer salts or detergents prior to the analysis by mass spectrometry (figure 1 and 2). Furthermore, the very high sensitivity of this instrument is ideal for the analysis of small sample volumes, such as those typically available from tear fluid. This ProteinChip system is widely used in other proteomic approaches. An additional advantage of this technique in comparison to electrophoretic techniques is its ability to screen down to the molecular size of peptides (approx. 200 Da).

Figure.1: ProteinChip system (Ciphergen, Fremont USA). The sample is applied to a „chip“ surface. After separation of proteins due to different wash-binding-conditions on the chromatographic chip surfaces, the chips are analyzed by means of a time-of-flight mass spectrometer.

Figure 2: Seldi-Chip-surfaces. There are different chip surfaces available: hydrophobic, hydrophilic, cationic-exchanger, anionic, exchanger, and metall-ion affinity chromatography surfaces.

This slide shows individual SELDI spectra in the molecular weight region up to 30 kDa.

OnChip - Autoantibody analysis

Normal sera contain a large number of naturally occurring auto-antibodies which can mask important disease-associated ones. Western blotting has evolved as the most important tool to demonstrate auto-antibodies in autoimmune diseases, because its possibility of simultaneous screening for a wide spectrum of different antigens. In previous studies we could show the diagnostic potential of the analysis of auto-antibodies in autoimmune diseases by means of multivariate statistics and artificial neural networks. However, the Western blotting procedure remains very time-consuming and is also limited in sensitivity.
Therefore, we used an on-chip approach for the analysis of auto-antibodies. This ProteinChip system uses ProteinChip arrays and SELDI-TOF (surface-enhanced laser desorption/ionisation in time-of-flight mass spectrometry) technology (Ciphergen, Fremont, USA) for capturing, detection, and analysis of proteins without labelling or without the need of chemical modification. The micro-scale design of the arrays allows the analysis of very small quantities of proteins. In the present study, we used arrays with biological activated surfaces that permit antibody capture studies. Protein-A-Chips were incubated with sera of patients (n=12). After washing, the chips were incubated with a complex solution of auto-antigens and subsequently washed again. If the Protein-A bound auto-antibodies recognized their antigens, these proteins could be separated by their molecular masses and were be detected by mass spectrometry. Previous studies using monoclonal antibodies could demonstrate that the detection limit is in the attomole level.
Furthermore all sera were analyzed by conventional Western blotting for direct comparison.

In the present study, we could show complex on-chip antibody-antigen reactions. At higher molecular weights (>30kDa) the detection sensitivity of this on-chip method was comparable to conventional Western blotting. At lower molecular weights, the Western blot technique is easily exceeded by the on-chip method.

Considering that this on-chip procedure is quite easy to use, is much less time consuming than Western blotting, and is much more sensitive at least in the low molecular weight range, the SELDI-TOF technology is a very promising approach for the screening of auto-antibodies in autoimmune diseases. Due to its versatility, this on-chip technology could allow the large-scale screening for complex auto-antibody distributions for diagnostic purposes and early detection of autoimmune diseases could be made possible.

Literature:

Funke S, Azimi D, Wolters D, Grus FH, Pfeiffer N (2012) Longitudinal analysis of taurine induced effects on the tear proteome of contact lens wearers and dry eye patients using a RP-RP-Capillary-HPLC-MALDI TOF/TOF MS approach. J Proteomics 75:3177-3190.

Grus FH, Joachim SC, Pfeiffer N (2007) Proteomics in ocular fluids. Proteomics Clin Appl 1:876-888.

 Grus FH, Podust VN, Bruns K, Lackner K, Fu S, Dalmasso EA, Wirthlin A, Pfeiffer N (2005) SELDI-TOF-MS ProteinChip array profiling of tears from patients with dry eye. Invest Ophthalmol Vis Sci 46:863-876.

Grus FH, Joachim SC, Pfeiffer N. Analysis of complex autoantibody repertoires by surface-enhanced laser desorption/ionization-time of flight mass spectrometry. Proteomics. 2003;3:957-61.

 

 

Important :

Our group could develop a novel method for the screening of complex autoantibody profiles based on mass spectrometry!

Proteomics :

Today, projects to map the entire genome of several organisms have been successfully completed.
The goal of researchers now is to determine the function of the protein repertoire; to determine if proteins are up- or downregulated correlated to disease processes (proteomics). Beside the use of conventional proteomics methods, our groups is developing specialized methods for the analysis of proteins in microscale approachs that are needed for the investigation of ocular fluids e.g. tear fludis and aqueous humor.

Proteognostics:

Cooperation with Proteognostics for the rapid microscale analysis of tearfluid using Seldi-TOF MS

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