Basic Sciences: Proteomics

 

Prof. Dr. Dr. F. Grus, PhD, MD

Dr. N. Perumal, PhD

J. Heyne, M.Sc.

K. Träger, M. Sc., 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.

In current projects we are using this technology to find biomarkers in other diseases than dry-eye like glaucoma, AMD etc., 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.

 

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.