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Peptide Fragmentation Studies on Doubly Charged Proline and Pipecolic Acid Containing Pentapeptides and Methods Development for an HPLC/MS Instrument for Proteomics Experiments
Smith, Hannah Emily
Smith, Hannah Emily
Abstract
This thesis presents fragmentation studies as well as proteomics studies performed using mass spectrometry. Peptide fragmentation was performed on both singly and doubly charged peptides containing either a proline or pipecolic acid residue, as well as either an arginine or lysine residue using collision-induced dissociation. Results showed that altering the position of pipecolic acid or proline in relation to arginine or lysine can affect the formation of doubly charged product ions, which may be a result in change of the proton affinity of the basic residue. Proteomics experiments were performed on a new HPLC/LTQ instrument using a standardized sample of six digested proteins in order to optimize a method by which future proteomics studies can be performed. The protein sample underwent separation via the HPLC with a 90 or 63.3-minute gradient, then data dependent scanning in the LTQ mass spectrometer. These data dependent scans were processed using the SEQUEST searching algorithm in order to identify all possible proteins. Results showed that a 63.5-minute linear gradient of acetonitrile/H2O was sufficient to successfully identify all proteins in the standard sample using SEQUEST, at a flow rate of 0.2 mL/min in the HPLC with initial injection size of 10 µl into a ACE superC18 reverse phase column, using CID only in the mass spectrometer to induce fragmentation.
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2016-05-01
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Chemistry