This proposal will involve the development of new methodology and instrumentation for sequencing of protein digests and post-translatinally modified proteins at the low picomole level and below. This work will be developed specifically for sequencing portions of tumor related target proteins, including 0p 18 and Hsp 27. These proteins will be obtained from tumor cells and isolated using 2-D Gel electrophoresis in the 2-D Gel Laboratory at The University of Michigan. These proteins are obtained at very low levels where they are enzymatically cleaved into peptide fractions for sequencing, but they often can not be sequenced by the Edman method due to enter N-terminal blockage, low amounts of sample or the inability to detect post-translational modifications. This proposal will involve development of on-line capillary separation methods interfacedvia miroelectrospry for detection using an ion trap/reflctron time-of-flight mass spectrometer to achieve the ultimate in sensitivity for detection of the vary low levels of peptides available. These methods include the use of capillary electrophoresis, packed and open tubular capillary electrochromatography and isoelectric focusing methods of separation and high sensitivity detection of peptides. These methods will be interfaced to the IT/reTOP MS for detection where the trapping capabilities of the ion trap and various other instrumental modifications will be used to achieve the required detection levels. In addition, the speed of this hybrid device will be shown to be uniquely suited to achieve full resolution in detection on-line for even the fastest separation and even able to perform on-line MS/MS for structural analysis without loss of resolution. Following separation by capillary methods on-line detection and MS/MS in the trap via resonance methods for isolation and excitation will be used to perform structural analysis of these target proteins. In addition, secondary methodology using capillary separation of protein digests followed by off-line MALDI in the trap produce extensive fragmentation via long term decay which can be used for structural analysis at low levels. These ions decay over periods of milliseconds in the trap but are detected as stable fragment ions in the TOF detector. Ultimately these complementary methods will be used to generate structural information on the sequence and modifications of these cellular proteins at the picomole or subpicomole level.
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