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Separation and Identification of Proteins by LC/LC-MS

In an effort to better understand cellular processes, the analysis of proteins has received continual interest since the inception of proteomics in the early 1990s. Proteomics presents a significant challenge to separation scientists due to the complexity and diversity of proteins in biological systems. Liquid chromatography has been an invaluable tool in the separation of complex proteins allowing high speed, resolution, and sensitivity for limited sample quantities. The focus of the proposed research is to improve the separation quality of complex protein mixtures spanning from a liquid chromatographic (LC) separation of monoclonal antibodies to a multidimensional LC approach for differential protein samples. To maximize resolution and chromatographic efficiency, several experimental parameters can be modified. Such conditions evaluated in this study to date consist of column chemistries, organic modifiers, ion-exchangers, and temperature. The field of proteomics can generally be divided into two approaches: top-down and bottom-up. With a bottom-up approach, proteins are digested, separated, and analyzed by mass spectrometry. Databases are then compared for matches among the peptide sequences and identification made based on those peptides. Top-down analysis requires the separation and analysis of proteins in their intact form. Certain modes of chromatography are more amenable to the analysis of intact proteins than others, although not without tradeoffs in performance. Size-exclusion and ion-exchange chromatography generally allow for better recovery than reversed-phase separations and with little or no denaturation of proteins. As a result, these methods of separation are noted as good choices for first dimension separation in multidimensional LC. For ion-exchange and reversed-phase separation, gradient elution is required. A mixture of proteins will exhibit a large range in retention factor, k’, that will rapidly change as the pH or salt concentration is adjusted in ion exchange chromatography or the percent organic modifier increased in reversed phase separation. It is not uncommon to see a 20% change in k’ with an increase of 0.1% organic modifier for a 30-kDa protein1. This strong relationship between k’ and modifier concentration is a powerful tool in protein separations though frequently it is insufficient for resolution of all components in a complex mixture. A significant challenge is posed by the general reliance of reversed-phase chromatography on silica-based particles – especially at high pressure. While endcapping to cover exposed silanol groups is routinely done to prevent unwanted interactions, this process is not complete. Lower recovery and ghosting in subsequent runs are the result of proteins interacting with unreacted silanols. [1,2]