Separation methods in cancer research, real samples

Cancer is one of the biggest threats of current population because it is the most common cause of death [1]. In total, about 200 cancer forms have been recognized, however lung cancer, prostate cancer, breast cancer, and colon cancer cause more than 50% of all deaths [1]. The high mortality is caused not only by effectivity of the treatment, which may be in numerous cases quite low, but also by the diagnostics of the disease in the late stage [2]. Therefore, it is obvious that an early diagnosis of cancer is playing a key role in the successful treatment and biomarkers are sought in genome, proteome and metabolome. The main disadvantage connected to modern gene arrays, which are nowadays one of the most commonly used tools for genomic analysis is the fact that they require mRNA as starting material. Therefore, even though one can survey the expression of all genes in cells or tissues, the sample preparation is a drawback. In proteomic research there are in general two strategies used. One is the ‘bottom-up’ approach and the other is the ‘top-down’ method. The bottom-up technique is based on a tryptic digestion of the protein mixture followed by separation of the fragments and analysis by MS, which can be done either on-line by electrospray ionization of off-line by matrix-assisted laser desorption and ionization. The disadvantage of this type of analysis is that limited information about the intact protein is provided. On the other hand, during the “top-down” method, the intact proteins and protein complexes are separated first and then analyzed by MS. Therefore, this approach can be used to obtain molecular information about the intact protein and may be advantageous for the detection of proteins’ post-translational modifications [3]. The main problem is the high complexity and wide dynamic range of peptides in body fluids (i.e. blood serum, saliva, sputum, cerebrospinal fluid, etc.). Too many peptides are present spread over a range of concentrations exceeding 1012 in the case of serum. To overcome this obstacle, it is crucial to simplify the complexity and remove the major components of the matrix, which are usually masking the signal of components of interest by some kind of separation method, which offers high resolution and can cope with a wide dynamic range of peptide concentrations [4]. In both, top-down and bottom-up methods, a powerful separation technique is required to obtain the information of interest. Despite the development of improved analytical tools for analysis of clinical and biochemical samples, gel electrophoresis is still the gold standard used up-to-date. Its two-dimensional variant – 2D gel electrophoresis – is taking advantage of combination with isoelectric focusing. The 2D sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) approach to protein profiling are accessible and economical method that enables the detection of hundreds of proteins on a single gel plate. Resolution has been enhanced by the introduction of immobilized pH gradients, which enable the analyst to tailor the pH gradient for maximum resolution using ultrazoom gels with a narrow pH gradient range. With modern 2D-PAGE, it is not unusual to resolve two proteins that differ in pI by 0.001 U [5]. Another limitation of 2D-PAGE include the labor-intensive and time-consuming nature of the technique, poor reproducibility, limited dynamic range of detection (undetectable for the mass range <20,000 m/z), and under-representation of certain classes of proteins, so that truly comprehensive analysis is impossible. Furthermore, it cannot provide accurate Mr information and it still remains difficult to interface 2-DE directly to MS analysis [4,6].

1. Kaluzna-Czaplinska, J.; Jozwik, J. Current applications of chromatographic methods for diagnosis and identification of potential biomarkers in cancer. Trac-Trends Anal. Chem. 2014, 56, 1-12.
2. Yang, Z.Y.; Sweedler, J.V. Application of capillary electrophoresis for the early diagnosis of cancer. Anal. Bioanal. Chem. 2014, 406, 4013-4031.
3. Gao, M.X.; Qi, D.W.; Zhang, P.; Deng, C.H.; Zhang, X.M. Development of multidimensional liquid chromatography and application in proteomic analysis. Expert Rev. Proteomics 2010, 7, 665-678.
4. Kolch, W.; Neususs, C.; Peizing, M.; Mischak, H. Capillary electrophoresis - mass spectrometry as a powerful tool in clinical diagnosis and biomarker discovery. Mass Spectrom. Rev. 2005, 24, 959-977.
5. Issaq, H.J.; Veenstra, T.D. Two-dimensional polyacrylamide gel electrophoresis (2d-page): Advances and perspectives. Biotechniques 2008, 44, 697-700.
6. Yoo, C.; Pal, M.; Miller, F.R.; Barder, T.J.; Huber, C.; Lubman, D.M. Toward high sequence coverage of proteins in human breast cancer cells using on-line monolith-based hplc-esi-tof ms compared to ce ms. Electrophoresis 2006, 27, 2126-2138.
7. Chen, Y.R.; Ma, Z.H.; Li, A.Y.; Li, H.W.; Wang, B.; Zhong, J.; Min, L.S.; Dai, L.C. Metabolomic profiling of human serum in lung cancer patients using liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry and gas chromatography/mass spectrometry. J. Cancer Res. Clin. Oncol. 2015, 141, 705-718.
8. Musharraf, S.G.; Mazhar, S.; Choudhary, M.I.; Rizi, N.; Atta ur, R. Plasma metabolite profiling and chemometric analyses of lung cancer along with three controls through gas chromatography-mass spectrometry. Sci Rep 2015, 5.
9. Ma, W.; Gao, P.; Fan, J.; Hashi, Y.; Chen, Z.L. Determination of breath gas composition of lung cancer patients using gas chromatography/mass spectrometry with monolithic material sorptive extraction. Biomed. Chromatogr. 2015, 29, 961-965.
10. Ma, H.Y.; Li, X.; Chen, J.M.; Wang, H.J.; Cheng, T.T.; Chen, K.; Xu, S.F. Analysis of human breath samples of lung cancer patients and healthy controls with solid-phase microextraction (spme) and flow-modulated comprehensive two-dimensional gas chromatography (gc x gc). Anal. Methods 2014, 6, 6841-6849.
11. Premstaller, A.; Oberacher, H.; Walcher, W.; Timperio, A.M.; Zolla, L.; Chervet, J.P.; Cavusoglu, N.; van Dorsselaer, A.; Huber, C.G. High-performance liquid chromatography-electrospray ionization mass spectrometry using monolithic capillary columns for proteomic studies. Anal. Chem. 2001, 73, 2390-2396.
12. Qian, W.J.; Jacobs, J.M.; Liu, T.; Camp, D.G.; Smith, R.D. Advances and challenges in liquid chromatography-mass spectrometry-based proteomics profiling for clinical applications. Mol. Cell. Proteomics 2006, 5, 1727-1744.
13. Zhao, Y.Y.; Cheng, X.L.; Vaziri, N.D.; Liu, S.M.; Lin, R.C. Uplc-based metabonomic applications for discovering biomarkers of diseases in clinical chemistry. Clin. Biochem. 2014, 47, 16-26.
14. Dakna, M.; He, Z.Y.; Yu, W.C.; Mischak, H.; Kolch, W. Technical, bioinformatical and statistical aspects of liquid chromatography-mass spectrometry (lc-ms) and capillary electrophoresis-mass spectrometry (ce-ms) based clinical proteomics: A critical assessment. J. Chromatogr. B 2009, 877, 1250-1258.
15. Zhang, X.W.; Wei, D.; Yap, Y.; Li, L.; Guo, S.Y.; Chen, F. Mass spectrometry-based „omics“ technologies in cancer diagnostics. Mass Spectrom. Rev. 2007, 26, 403-431.

PDF