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Chemical Labelling Strategies for Mass Spectrometric Peptide Analysis

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Chemical Labelling Strategies for Mass Spectrometric Peptide Analysis Chemical labelling strategies for mass spectrometric peptide analysis Helen Kathryn Robinson Doctor of Philosophy Chemistry University of York September 2015 Abstract The work in this thesis describes the use of self-assembled monolayers (SAMs) to capture peptides on a gold-coated MALDI chip for mass spectrometric analysis. A SAM was formed on the gold-coated MALDI chip, onto which 4-bromophenylalanine was coupled (giving peaks due to 4-bromophenylalanine-SAM species a characteristic isotopic distribution). Simple single peptides and tryptic peptides from single proteins have been analysed using the developed technology. The capture of peptides on SAMs for mass spectrometric analysis has many advantages; all steps (chemistry and analysis) take place in situ on the gold-coated MALDI chip. This means that there are no sample transfer steps, reducing sample loss. Additionally, unreacted reagents and buffers can be removed from the surface of the gold-coated MALDI chip through washing. Buffers which are not compatible with mass spectrometric analysis (e.g. phosphate-based buffers) can be used for peptide capture as the buffer is washed away prior to analysis. This thesis describes an alternative cleaning strategy for the removal of organic material from the surface of the gold-coated MALDI chips. Two plasma instruments were investigated for their abilities to remove organic material from the gold-coated MALDI chips, and both were shown to be a suitable safe alternative cleaning strategy to the caustic, potentially explosive piranha solution currently used. Finally, in-solution dimethyl labelling was investigated, as a labelling strategy which could be adapted for used with the SAM technology. Quantification was performed using standard solutions of light and heavy labelled simple protein digests mixed in a range of ratios. Two different commercially-available software packages were investigated to assess their ability to analyse the generated data in order to determine whether one is more suitable for dimethyl labelling quantification. A set of complex standard samples was dimethyl labelled and analysed, to define parameters for most effectively determining quantification ratios, before application to a set of ‘real’ samples. Muscle protein samples were analysed and a set of potentially differentially abundant proteins identified for further validation. 3 4 Contents Abstract .................................................................................................................................... 3 Contents ................................................................................................................................... 5 List of tables ............................................................................................................................. 9 List of figures .......................................................................................................................... 12 List of equations ..................................................................................................................... 19 Acknowledgements ................................................................................................................ 21 Author’s declaration ............................................................................................................... 23 Chapter 1: Introduction ........................................................................................................... 25 1.1. Protein analysis .......................................................................................................... 27 1.1.1. Gel electrophoresis ............................................................................................ 28 1.1.2. Mass spectrometry ............................................................................................. 30 1.2. Mass spectrometry ..................................................................................................... 31 1.2.1. Ionisation ............................................................................................................ 32 1.2.1.1. Electrospray ionisation (ESI) ...................................................................... 32 1.2.1.2. Matrix-assisted laser desorption/ionisation (MALDI) .................................. 34 1.2.2. Mass analysers .................................................................................................. 38 1.2.2.1. Time-of-flight (ToF)..................................................................................... 38 1.2.2.2. Fourier transform ion cyclotron resonance (FT-ICR) ................................. 41 1.2.3. Tandem mass spectrometry ............................................................................... 45 1.3. Protein labelling strategies ......................................................................................... 47 1.3.1. Stable isotope labelling by amino acids ............................................................. 50 1.3.2. Isotope-coded affinity tag ................................................................................... 51 1.3.3. Tandem mass tags ............................................................................................. 54 1.3.4. Isobaric tag for relative and absolute quantification ........................................... 57 1.3.5. Acid-labile isotope-coded extractants ................................................................ 61 1.3.6. Dimethyl labelling ............................................................................................... 63 1.3.7. N,N-Dimethyl leucine ......................................................................................... 65 1.3.8. 18O oxygen incorporation ................................................................................... 66 1.3.9. Bromine incorporation ........................................................................................ 67 1.3.10. Summary ............................................................................................................ 69 1.4. Self-assembled monolayers ....................................................................................... 71 1.5. Aims of this thesis ...................................................................................................... 76 Chapter 2: Thiol-derived SAM ................................................................................................ 79 5 2.1. Introduction ................................................................................................................. 83 2.1.1. Aims and overview ............................................................................................. 86 2.2. Formation of a thiol-derived SAM............................................................................... 87 2.3. Investigation into the most suitable matrix ................................................................. 90 2.4. Peptide capture on a 4-bromophenylalanine tagged SAM ........................................ 93 2.4.1. Activation of SAM carboxylic acid groups .......................................................... 94 2.4.2. Coupling of 4-bromophenylalanine to activated SAM ........................................ 95 2.4.3. Activation of 4-BrPhe-tagged SAM carboxylic acid groups ............................... 98 2.4.4. Capture of single peptides on activated 4-BrPhe-tagged SAM ....................... 100 2.5. Peptides captured on untagged SAM ...................................................................... 107 2.5.1. Activation of SAM carboxylic acid groups ........................................................ 107 2.5.2. Peptide capture on activated SAM carboxylic acid groups .............................. 108 2.5.3. Use of alternative activating reagents and conditions to investigate peptide capture on untagged SAM ............................................................................................... 114 2.6. Investigation of inefficient activation of carboxylic acid group activation for peptide capture ................................................................................................................................. 116 2.6.1. Natural exchange ............................................................................................. 118 2.6.2. Investigating potential hydrolysis during 4-bromophenylalanine coupling to activated SAM .................................................................................................................. 120 2.6.3. Investigating potential hydrolysis during peptide capture on activated SAM ... 122 2.7. Conclusions and future work .................................................................................... 125 Chapter 3: Disulfide-derived SAM ........................................................................................ 129 3.1. Introduction ............................................................................................................... 133 3.1.1. Aims and overview ........................................................................................... 134 3.2. Formation of a disulfide-derived SAM ...................................................................... 134 3.3. Peptide capture on SAM .......................................................................................... 138 3.3.1. MS/MS analysis of peptides captured on the disulfide-derived SAM .............. 148 3.4. 4-Bromophenylalanine and phenylalanine coupling to
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