Principles of Nanoflow Liquid Chromatography and Applications to Proteomics

Abstract

The low levels of endogenous proteins in biological samples and the large dynamic range of the proteome complicate global analysis of gene expression at the protein level. The use of liquid chromatography (LC) in analytical chemistry is well established. However, the relatively low sensitivity associated with conventional LC makes it unsuitable for the analysis of certain biological samples. Furthermore, the flow rates at which it is operated are not compatible with the use of specific detectors, such as electrospray ionization mass spectrometers. Therefore, due to the analytical demands of biological samples, miniaturized LC techniques were developed to allow for the analysis of samples with greater sensitivity than that afforded by conventional LC. In nanoflow LC (nanoLC) chromatographic separations are performed using flow rates in the range of low nanoliter per minute, which result in high analytical sensitivity due to the large concentration efficiency afforded by this type of chromatography. NanoLC, in combination to tandem mass spectrometry, was first used to analyze peptides and as an alternative to other mass spectrometric methods to identify gel-separated proteins. More recently, gel-free analytical approaches based on LC and nanoLC separations have been developed, which are allowing proteomics to be performed in faster and more comprehensive manner than by using strategies based on the classical 2D gel electrophoresis approach.