%0 Journal Article
%T Highly Sensitive High Performance Liquid Chromatography-Laser Induced Fluorescence for Proteomics Applications
%A Ajeetkumar Patil
%A K. S. Choudhari
%A Vijendra Prabhu
%A V. K. Unnikrishnan
%A Sujatha Bhat
%A Keerthilatha M. Pai
%A V. B. Kartha
%A C. Santhosh
%J ISRN Spectroscopy
%D 2012
%R 10.5402/2012/643979
%X 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¨C3]. Laser-induced fluorescence (LIF) is one of the most sensitive techniques for qualitative and quantitative analysis [4¨C13]. 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
%U http://www.hindawi.com/journals/isrn.spectroscopy/2012/643979/