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Title:Automated data acquisition and analysis for high-resolution Fourier transform mass spectrometry of proteins and peptides
Author(s):Robinson, Dana E.
Director of Research:Gennis, Robert B.
Doctoral Committee Chair(s):Gennis, Robert B.
Doctoral Committee Member(s):Scheeline, Alexander; Sweedler, Jonathan V.; Yau, Peter M.
Department / Program:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):mass spectrometry
cytochrome c oxidase
oxygen reductase
microbial respiration
proton pumping
Abstract:This thesis details the progress made in automating the data acquisition and data processing of MS and MS/MS data from Fourier transform ion cyclotron resonance mass spectrometers. These instruments have made great strides in the past few years, moving from labor-intensive, largely manual systems to the current flagship of the Kelleher group – a 12 tesla hybrid LTQ FT capable of analyzing hundreds of proteins per week with minimal user intervention. In early automation work centered around an 8.5 tesla hybrid quadrupole FT-ICR mass spectrometer, three strategies for automated operation of the instrument were developed. In one method, a charge state deconvolution algorithm was used to identify species of interest for isolation using a SWIFT waveform, which were then fragmented in the ICR cell using IRMPD. In another method, broad 20-60 m/z wide sections of the mass spectrum of a complex mixture were fragmented in parallel and this multiplexed fragment data was analyzed using an iterative algorithm. In the third method, samples were analyzed using a THRASH-based "quad march" method where data were acquired using consecutive, wide (20-60 m/z) isolation windows, analyzed using the THRASH algorithm, and then selected species were selected for MS/MS fragmentation. Results from the application of this platform to a survey of the Methanosarcina acetivorans proteome are presented. Later work focused on a commercial 12 tesla hybrid linear ion trap FT-ICR mass spectrometer. This automation scheme used hybrid online/offline data acquisition to take advantages of the features of both online LC-MS (efficient separation and rapid data collection) and offline direct infusion MS/MS (project-wide target selection, better MS/MS data). The workflow for the online portion of this scheme is a modified form of the THRASH-based "quad march" data acquisition scheme from the earlier 8.5 T automation work. The centerpiece of this - iii - platform is a database known as the Automation Warehouse, which acts as a repository for the intact mass data observed in a proteome project and stores the overall state of the project. Custom software binds together the raw data, the data stored in the warehouse and ProSight. Results from the application of this platform to a survey of proteins from HeLa cell nuclei are presented. In an attempt to begin applying the above platforms to membrane proteins, MS analysis of a putative cross-link in the active site of the C-type heme-copper oxygen reductase from Vibrio cholera was performed. Though the sequences in the region differed, other members of the HCO superfamily contained a similar cross-link (confirmed by mass spectrometry and crystal structures) that is important in the catalytic cycle of the enzyme. Computer modeling of the C-type oxidase suggested that the cross-link would be present, though the key amino acid would be located on a different helix. MS/MS analysis of a tryptic digest confirmed the presence of the cross-link and the evolutionary migration of the key amino acid.
Issue Date:2011-05-25
Rights Information:Copyright 2011 Dana E. Robinson
Date Available in IDEALS:2011-05-25
Date Deposited:2011-05

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