| A great deal of success has been achieved in the understanding of cytokine structures, functions, and synthesis. However, the understanding of the sophisticated cytokine network and how it is regulated physiologically is still limited. In order to improve this understanding, the development of rapid and convenient methods to separate and characterize the cytokines is necessary. The goal of this research is to establish such a method using high performance liquid chromatography (HPLC) coupled to electrospray ionization mass spectrometry (ESI-MS).; In the research described here, first, conventional electrospray mass spectrometry and nanospray ionization mass spectrometry methods were compared for the analysis of various small proteins, including several cytokines. The detection limits achieved for the various proteins using nanospray ionization were several orders of magnitude lower than those achieved with conventional electrospray ionization mass spectrometry techniques. Second, the use of a heparin column for concentration of proteins prior to analysis was evaluated. Finally, separation methods for different cytokines using capillary HPLC coupled to nanospray ionization mass spectrometry were developed. Separation of the cytokines TNF-alpha and IL-1 was achieved using this technique with a resolution of approximately 3. |
