Multidimensional Liquid Chromatography of Proteins Using Monolithic IEX and RP Columns

摘要

The development of monolithic columns during the past years has brought a great improvement in the field of biopolymer analysis. Monolithic columns offer several advantages over particulate columns due to their macroporous structure leading to fast mass transfer, low backpressure and high resolution. These characteristics are ideal for designing two-dimensional LC methods for the separation of intact protein. Here we discuss the performance of a 2D-LC protein separation method based on monolithic columns for proteomics research. Proteins are separated on a new polymethacrylate based monolithic IEX column in the first dimension using a salt gradient. Fractions are collected and re-injected onto a poly-styrene divinylbenzene (PS-DVB) based reversed phase monolithic column. The RP separation is followed by a second fraction collection step that allows the analysis of proteins of interest by means of tandem MS sequencing after tryptic digestion. The performance and robustness of the 2D-LC method are studied with a set of protein standards and a bacterial protein lysate.