This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Top-down sequencing and protein structural characterization has shown great utility on electrospray FTMS instruments. It is, however, entirely possible to perform top-down mass spectrometry using vibrationally cooled (VC) MALDI FTMS instrument. The vibrationally cooled MALDI built at the NCRR Mass Spectrometry Resource at Boston University School of Medicine is capable of cooling the vibrational motion of the ions and is a critical parameter for the generation of intact proteins in MALDI FTMS. The model protein for testing this technique was ubiquitin. The intact species was desorbed from appropriate matrices using both UV (355 nm) and IR (2940 nm) laser. The SORI CAD excitation of molecular ions produced several sequence fragments from which a protein sequence tag can be formed which is more than sufficient for identification via database searching. The sequence coverage was approximately 50%. Further studies have to be done to optimize the fragment generation and detection procedure, and the method must be tested for biologically derived (non-synthetic) samples. More experiment are underway concering sample preparation and the modification of ion source of HP MALDI to allow use of liquid matrices such as glycerol or even water. Generation of multiply charged species of intact proteins from liquid matrices will likely generate improved sequence coverage. Thus, top down sequencing of (small) proteins by VC MALDI FTMS is a promising technique for protein identification and structural studies. The top down data generated from ubiquitin was obtained for the first time for vibrationally cooled MALDI FTMS. These data have been submitted for publication in a peer-reviewed journ
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