Identification of biologically relevant peptides by nanoflow HPLC microelectrospray ionization mass spectrometry

Mass Spectrometry (MS) is powerful tool for studying a variety of biological problems, especially when coupled to nano-flow chromatography. The advances in ion trap mass spectrometry, along with powerful computer operating systems, have allowed for a dramatic increase in the amount of information, MS and MS/MS, that can be obtained from a single sample. Even with these advances, lower detection limits, with less off-line sample handling is a goal.; Efforts were made to reduce the limits of detection, in antigen identification projects, by utilizing advanced scanning techniques on the TSQ 7000 and employing the Fourier transform mass spectrometer (FTMS) for high resolution mass determination. These projects allowed for the determination of immunologically relevant peptides from samples containing approximately 10,000 peptides eluted from MHC class I proteins on the surface of the cells. Known antigens found in the sample by utilizing Selected Reaction Monitoring scans on the TSQ-7000. The limit of detection for this experiment was approximately 100 amol, or less than one copy per cell presentation of the antigens. Similar studies in 1999 had a published limit of detection of 5 fmol. Determination of an unknown antigen was determined using an on-line column effluent splitting device coupled to a FTMS. Utilization of the FTMS allowed for a 6 fold reduction in sample.; In another project, efforts were made to identify post-translational modifications on histone core proteins. DNA wraps around histone proteins to be packed into nucleosomes and chromosomes. Modifications on histones are thought to control the transcriptional activity of the DNA that is wrapped around the protein. Through the use of enzymatic digests and the Finnigan LCQ ion trap we were able to identify several new sites of acetylation and several new sites of phosphorylation.; These are simply examples of the projects that can be analyzed using the techniques described. As genomes continue to be sequenced, efforts will shift to identifying the proteins that are derived from these genes. Mass spectrometry is already becoming the method of choice to determine these proteins and efforts to lower limits of detection will make it an even more valuable resource for protein identification.