Invited Speaker

New Analytical Technologies for Applications in Proteomics
G. K. Bonn
Austria


The idea of tracing potential biomarkers through proteomics is getting deeper in its approaches and becoming fast a reality. The whole sequence of human proteome is getting obvious with the passage of time. However, it is still far away to pinpoint biomarker proteins out of complex biofluids and adopt them for clinical diagnosis, prognosis and therapeutic applications. Major focus is placed on the development of novel innovative analytical techniques in the field of life sciences, using high performance single- and multi-dimensional separation and enrichment methods, such as solid-phase extraction (SPE), liquid chromatography (HPLC) or capillary electrophoresis (CE) coupled to mass spectrometry (MS). A newly emerged technology, termed as Material-Enhanced Laser Desorption/Ionization (MELDI) suffices the basic requirements and is applied to reduce the complexity of proteomic samples while liquid chromatography (LC) is used for fractionation, followed by MALDI-MS/MS including database searching analysis [1]. Different MELDI carriers are employed as support materials to specifically bind peptides and proteins from biofluids like serum or urine. Novel monolithic stationary phases such as poly(p-methylstyrene-co-1,2-bis-p-(vinylphenyl)ethane (MSt/BVPE), are applied to achieve separations at high speed and high resolution [2]. MS/BVPE based capillaries provide a reliable and robust separation platform due to high mechanical stability and low swelling. Due to the possibility to adjust the meso- and macroporosity of these monolithic stationary phases, studies in proteomics as well as metabolomics can be performed. Moreover, the potential of organic monoliths with incorporated TiO2 and ZrO2 nanoparticles in shape of pipette tips is employed for the selective enrichment of phosphorylated peptides from tryptic digests [3]. When compared to conventional immobilized metal-ion affinity chromatography (IMAC) and commercial phosphorylation-enrichment kits, higher selectivity can be reached.

An additional proteomic strategy is based on the analysis of cancer tissue sections by MALDI-MS imaging to obtain the spatial distribution of the expressed peptides and proteins. In a further step these results can be linked and compared with the final output of serum profiling experiments. The serum and tissue mass fingerprints are analyzed by bioinformatic tools to trace the mass peaks that discriminate between cancer and non-cancer for further identification by LC-MS/MS. Advanced bioinformatic tools are used for quantification and comparison of individual proteins and up and down-regulation domains between serum samples from prostate cancer patients and control. In parallel non-invasive infrared-imaging of tissue slices from prostate glands is carried out to trace potential markers very close at the tumour region.

References

[1] Rainer, M.; Huck, C.W.; Gjerde, D.; Bonn, G.K. Advanced profiling method based on MELDI-MS for high-throughput pattern analysis in proteomics. (2009), LCGC Europe 22(9), 448 - 457.
[2] Trojer, L; Lubbad, S.H; Bisjak, C.P; Wieder, W; Bonn, G.K: Comparison between monolithic conventional size, microbore and capillary poly(p-methylstyrene-co-1,2-bis(p-vinylphenyl)ethane) high-performance liquid chromatography columns. Journal of Chromatography A (2007), 2(1146) 216 - 224.
[3] Rainer, M.; Sonderegger, H.; Bakry, R.; Huck, C. W.; Morandell, S.; Huber, L. A.; Gjerde, D. T.; Bonn, G. K. Analysis of protein phosphorylation by monolithic extraction columns based on poly(divinylbenzene) containing embedded titanium dioxide and zirconium dioxide nano-powders. Proteomics (2008), 8(21), 4593-4602.