This paper describes the sensitivity study and performance evaluation of high-performance liquid chromatography-laser-induced fluorescence detection (HPLC-LIF) system assembled in our laboratory for proteomics applications. The limits of Detection (LOD) of several serum proteins have been estimated with this instrument and are found to be much lower compared to other commonly used proteomics techniques like SELDI, MALDI, 2-D-SDS-PAGE, and so forth. Techniques for improving the LOD still further with similar setup are briefly discussed. Using the system, protein profiles of serum in normal, malignant, and premalignant conditions were recorded for different malignancy situations. 1. Introduction High-performance liquid chromatography (HPLC) is a versatile tool for the separation and analysis of biological and pharmaceutical compounds. The analysis of many trace analytes even in complex systems can be carried out with the effective utilization of HPLC and proper selection of a highly sensitive detection technique. Currently, various detection techniques are in use with HPLC, namely, UV-visible absorption, fluorescence, refractive index, nuclear magnetic resonance (NMR), mass spectrometry (MS), pulsed electrochemical detection (PED), and so forth [1–3]. Laser-induced fluorescence (LIF) is one of the most sensitive techniques for qualitative and quantitative analysis [4–13]. Improving the detection limit of HPLC still further is one of the main challenges in analytical chemistry. The usual methods for this are (i) increasing the amount of analyte either by injection of larger volumes of sample or by analyte preconcentration and (ii) improving the signal-to-noise ratio (S/N) through instrument optimization [11]. Extensive research and development made for improving the sensitivity/detection limits have led to various new methodologies and significant modifications in the detection schemes. Derivatization is a widely used technique, where efficient fluorescent molecules are mixed or tagged with the sample which in turn helps to detect and identify the analyte with higher sensitivity. Since the number of standard fluorescent molecules available for this application is small, the derivatization methods have got limited popularity. Even though this method has achieved detection limits for amino acids at subfemtomole levels [12], the existing methods have several drawbacks like derivative instability, reagent interferences, long preparation time, and so forth [2]. There is an urgent need for determination of extremely small quantities of proteins in physiological
References
[1]
W. W. Christie, “Detectors for high-performance liquid chromatography of lipids with special reference to evaporative light scattering detection,” Advances in Lipid Methodology, pp. 239–271, 1992.
[2]
K. Petritis, C. Elfakir, and M. Dreux, “A comparative study of commercial liquid chromatographic detectors for the analysis of underivatized amino acids,” Journal of Chromatography A, vol. 961, no. 1, pp. 9–21, 2002.
[3]
G. D. Reed, “An evaluation of electrochemical detection in reverse-phase HPLC,” Journal of High Resolution Chromatography & Chromatography Communications, vol. 11, pp. 675–677, 1988.
[4]
S. A. Soper, S. M. Lunte, and T. Kuwana, “High sensitivity fluorescence detection of naphthalenedialdehyde derivatized amino acids with a low power helium-cadmium laser for high performance liquid chromatographic analysis,” Analytical Sciences, vol. 5, pp. 23–29, 1989.
[5]
V. B. Kartha, S. S. Nayak, J. Kurien, J. Jacob, and M. Valiathan, “Investigation of laser spectroscopy methods for early diagnosis of neoplasia: development of instrumentation and analytical techniques,” DST No SP/S2/L01/98, Department of Science and Technology, Government of India, 1998.
[6]
K. Venkatakrishna, K. M. Pai, C. M. Krishna et al., “Protein profiles in oral pre-malignancies: a laser spectroscopy study,” in Proceedings of the 4th DAE-BRNS National Laser Symposium, vol. 345, Allied Publishers, New Delhi, India, 2001.
[7]
K. Venkatakrishna, V. B. Kartha, K. M. Pai et al., “HPLC-LIF for early detection of oral cancer,” Current Science, vol. 84, no. 4, pp. 551–557, 2003.
[8]
S. K. Singh, R. L. Martis, Sujatha et al., “Protein profile study of clinical samples of ovarian cancer using High Performance Liquid Chromatography-Laser Induced Fluorescence (HPLC-LIF),” in Proceedings of the Ultrasensitive and Single-Molecule Detection Technologies, San Jose, Calif, USA, January 2006.
[9]
Sujatha, L. Rai, B. R. Krishnanand, K. K. Mahato, V. B. Kartha, and C. Santhosh, “Protein profile study of the cervical cancer using HPLC- LIF,” in Proceedings of the Ultrasensitive and Single-Molecule Detection Technologies, San Jose, Calif, USA, January 2006.
[10]
Sujatha, Protein profile and laser fluorescence spectroscopic studies of clinical samples for early detection of cervical cancer [Ph.D. thesis], Manipal University, 2009.
[11]
E. S. Yeung and M. J. Sepaniak, “Laser fluorometric detection in liquid chromatography,” Analytical Chemistry, vol. 52, no. 13, pp. 1465A–1471A, 1980.
[12]
E. S. Yeung and R. E. Synovec, “Detectors for liquid chomatography,” Analytical Chemistry, vol. 58, no. 12, 1986.
[13]
K. C. Chan, T. D. Veenstra, and H. J. Issaq, “Comparison of fluorescence, laser-induced fluorescence, and ultraviolet absorbance detection for measuring HPLC fractionated protein/peptide mixtures,” Analytical Chemistry, vol. 83, no. 6, pp. 2394–2396, 2011.
[14]
M. J. Sepaniak, “The clinical use of laser-excited fluorometry,” Clinical Chemistry, vol. 31, no. 5, pp. 671–678, 1985.
[15]
C. A. Burtis and E. R. Ashwood, Tiets Fundamentals of Clinical Chemistry, W.B.Saunders Company, Philadelphia, Pa, USA, 5 edition, 2001.
[16]
J. N. Adkins, S. M. Varnum, K. J. Auberry et al., “Toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry,” Molecular & Cellular Proteomics, vol. 1, no. 12, pp. 947–955, 2002.
[17]
C. Wrotnowski, “The future of plasma proteins,” Genetic Engineering News, vol. 18, p. 14, 1998.
[18]
M. W. Turner and B. Hulme, The Plasma Proteins: An Introduction, Pitman Medical & Scientific Publishing, London, UK, 1970.
[19]
N. L. Anderson and N. G. Anderson, “The human plasma proteome: history, character, and diagnostic prospects,” Molecular & Cellular Proteomics, vol. 1, no. 11, pp. 845–867, 2002.
[20]
P. D. Griffiths, “Serum levels of creatine phosphokinase,” Journal of Clinical Pathology, vol. 17, pp. 56–57, 1964.
[21]
D. Neumeier, M. Knedel, U. Wurzburg, N. Hennrich, and H. Lang, “Immunologischer nachweis von creatinkinase-MB im serum beim myokardinfarkt,” Klin Wochenschr, vol. 53, pp. 329–333, 1975.
[22]
F. Sanger, “Chemistry of insulin,” Science, vol. 129, no. 3359, pp. 1340–1344, 1959.
[23]
K. Pyorala, “Relationship of glucose tolerance and plasma insulin to the incidence of coronary heart disease: results from two population studies in Finland,” Diabetes Care, vol. 2, no. 2, pp. 131–141, 1979.
[24]
P. Ducimetiere, E. Eschwege, and L. Papoz, “Relationship of plasma insulin levels to the incidence of myocardial infarction and coronary heart disease mortality in a middle-aged population,” Diabetologia, vol. 19, no. 3, pp. 205–210, 1980.
[25]
T. A. Welborn and K. Wearne, “Coronary heart disease incidence and cardiovascular mortality in Busselton with reference to glucose and insulin concentrations,” Diabetes Care, vol. 2, no. 2, pp. 154–160, 1979.
[26]
B. Alberts, A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter, Molecular Biology of the Cell, Garland Science, New York, NY, USA, 4th edition, 2007.
[27]
S. Menon, Sujatha, K. K. Kumar et al., “Protein profile study of breast cancer tissues using HPLC-LIF: a pilot study,” in Proceedings of the Advanced Biomedical and Clinical Diagnostic Systems V (SPIE '07), San Jose, Calif, USA, January 2007.
[28]
A. Salimi, M. Roushani, S. Soltanian, and R. Hallaj, “Picomolar detection of insulin at renewable nickel powder-doped carbon composite electrode,” Analytical Chemistry, vol. 79, no. 19, pp. 7431–7438, 2007.
[29]
S. D. Lidofsky, W. D. Hinsberg, and R. N. Zare, “Enzyme-linked sandwich immunoassay for insulin using laser fluorimetric detection,” Proceedings of the National Academy of Sciences of the United States of America, vol. 78, no. 3, pp. 1901–1905, 1981.
[30]
M. R. Beamish, P. Llewellin, and A. Jacobs, “A rapid method for the detection of ferritin in serum,” Journal of Clinical Pathology, vol. 24, no. 6, pp. 581–582, 1971.
[31]
S. Zhang, K. Jiao, H. Chen, and M. Wang, “Detection of ferritin in human serum with a MAP-H2O2-HRP voltammetric enzyme-linked immunoassay system,” Talanta, vol. 50, no. 1, pp. 95–101, 1999.
[32]
T. Yin, W. Wei, L. Yang, X. Gao, and Y. Gao, “A novel capacitive immunosensor for transferrin detection based on ultrathin alumina sol-gel-derived films and gold nanoparticles,” Sensors and Actuators B, vol. 117, no. 1, pp. 286–294, 2006.
[33]
Q. Z. Zhu, Y. Y. Lin, D. H. Li, J. G. Xu, and X. Q. Guo, “Detection of human serum albumin by a photoinduced fluorogenic reaction,” Analytical Letters, vol. 32, no. 9, pp. 1775–1786, 1999.
[34]
W. Xu, Y. Wei, D. Xing, Q. Chen, and S. Luo, “Quantification of human serum albumin by highly sensitive chemiluminescence method,” in Proceedings of the IEEE/ICME International Conference on Complex Medical Engineering (CME '07), pp. 1693–1697, May 2007.
[35]
J. M. Fujima and N. D. Danielson, “Determination of creatine kinase activity and phosphocreatine in off-line and on-line modes with capillary electrophoresis,” Analytica Chimica Acta, vol. 375, no. 3, pp. 233–241, 1998.
[36]
M. S. Chidananda, Laser spectroscopic and biochemical investigations on clinical samples in cancer of human uterine cervix [Ph.D. thesis], Manipal University, 2007.
[37]
D. T. Chiu, S. J. Lillard, R. H. Scheller et al., “Probing single secretory vesicles with capillary electrophoresis,” Science, vol. 279, no. 5354, pp. 1190–1193, 1998.
[38]
P. C. D. Hobbs, “Ultrasensitive laser measurements without tears,” Applied Optics, vol. 36, no. 4, pp. 903–920, 1997.
[39]
A. J. Rai and D. W. Chan, “Cancer proteomics: serum diagnostics for tumor marker discovery,” Annals of the New York Academy of Sciences, vol. 1022, pp. 286–294, 2004.
[40]
V. Kumar, R. S. Cotran, and S. L. Robbins, Basic Pathology, Prism Books, Bangalore, India, 5th edition, 1992.
[41]
Sujatha, L. Rai, P. Kumar, K. K. Mahato, V. B. Kartha, and C. Santhosh, “Serum protein profile study of normal and cervical cancer subjects by high performance liquid chromatography with laser-induced fluorescence,” Journal of Biomedical Optics, vol. 13, no. 5, Article ID 054062, 2009.
[42]
A. Patil, V. Prabhu, K. S. Choudhari et al., “Evaluation of high-performance liquid chromatography laser-induced fluorescence for serum protein profiling for early diagnosis of oral cancer,” Journal of Biomedical Optics, vol. 15, no. 6, Article ID 067007, 2010.
