Title

An Esi-Ms/Ims/Ms Study of Protein interactions with Complexes of Palladium(Ii) Cations in the Presence of Detergents

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

Date of Award

Spring 2012

Abstract

Presently many labeling and tagging techniques exist that retain both spectral and electrochemical properties for the study of proteins; however, current challenges with these tags are the ability to locate, identify, and quantify them. Conversely, tagging proteins with cis-[Pd(en)(H 22 O) 2 ] 2+ in tandem with an ESI-Q/TOF mass spectrometer are novel and indispensable for the study of proteins because it allows for the detection of spectroscopically silent tags, as well as the identification and quantification of multiple tags. Thus, this study explores the sensitivity and key use of mass spectrometry in examining, detecting, and quantifying various bovine proteins, namely ubiquitin, β-casein, and serum albumin (BSA) labeled with a specific palladium(II) complex tag. Moreover, the study of ligation states will be explored. Collision-induced dissociation (CID) was done at varying collision energies and demonstrated the possibility of en detachment from the Pd(II) tags, as well as the greater bond strength seen between the palladium(II) tag with the protein versus the en ligand on the Pd(II) complex. Proteolytic enzymes are common agents for site-specific protein cleavage. However, because they become denatured and inactivated by detergents, these enzymes are inefficient for proteomic analysis of hydrophobic proteins which require detergents as solubilizing agents. In this study, protein cleavage via a palladium(II) complex in the presence of detergents is accomplished. The artificial protease cis-[Pd(en)(H 22 O) 2 ] 2+ is used to effect cleavage of the three aforementioned bovine proteins in separate experiments. Cleavage took place in aqueous solutions containing up to 1.0 % wt/vol of either 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or Zwittergent 3-14 (Zwitt 3-14) detergent at 2.0 < pH < 2.9 and 55 � C for 3-72 h with a 2:1 molar excess ratio of Pd(II) complexes to protein concentrations. Digests were separated by HPLC and analyzed by tandem mass spectrometry, specifically ESI-Q/TOF. Identification of original parent proteins is done by utilizing a proteomic database software known as PEAKS, which matches identified peptide fragments from the digest against proteomic databases. Peptides were identified by de novo sequencing, compared and confirmed by PEAK's peptide fragment identification, and matched against the bovine genome.

Advisor

Laurence Angel

Subject Categories

Chemistry | Physical Sciences and Mathematics

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