XLIM-MS Towards the Development of a Novel approach to Cross-linking Mass Spectrometry Juliette M.B. James University College London Institute of Structural and Molecular Biology Wellcome Trust 4 year Interdisciplinary PhD Programme in Structural, Computational and Chemical Biology For those who changed my world and gave me purpose. For Michael & Asha. 2 Contents Declaration 7 List of Figures 8 List of Tables 21 List of Abbreviations 24 Abstract 26 Impact Statement 27 1 Introduction 29 1.1 History of Mass Spectrometry . 29 1.2 Electrospray Ionisation . 30 1.3 Mass Analysers . 32 1.3.1 Quadrupole . 33 1.3.2 Time of Flight . 35 1.3.3 Orbitrap . 36 1.4 Ion Mobility . 38 1.4.1 Travelling Wave Ion Mobility . 39 1.5 Tandem Mass Spectrometry . 41 1.5.1 Collision Induced Dissociation . 41 1.6 Cross-linking Mass Spectrometry . 43 1.6.1 Experimental Preparation of cross-linked Samples . 44 3 1.6.2 Mass Spectrometry Analysis of Cross-linked Samples . 51 1.6.3 Computational Analysis of Cross-linked Data Sets . 52 1.6.4 xQuest . 54 1.7 Aims and Objectives . 59 2 Materials and Methods 61 2.1 xQuest Installation Requirements . 61 2.2 Preparation of Crosslinked Samples . 61 2.3 LC-MS/MS Analysis . 65 2.4 Raw Data Processing and Cross-link Analysis . 66 2.5 Computational Analysis . 68 3 Analysis of Cross-links identified by xQuest/xProphet in QToF Data 69 3.1 Introduction . 69 3.2 Materials and Methods . 72 3.3 Results and Discussion . 75 3.3.1 Validation of Score Threshold . 75 3.3.2 Effects of Energy Ramps on Cross-link Identification Rates . 80 3.3.3 Cross-link Validation by Solvent Accessible Surface Distance . 83 3.3.4 Effect of Energy Ramps on Fragmentation Patterns . 86 3.4 Conclusion and Further Work . 93 4 Ion Mobility Enhanced Data Dependent Acquisition for the Analysis of Cross-linked Peptides 95 4.1 Introduction . 95 4.2 Materials and Methods . 97 4.2.1 Preparation of uncross-linked BSA . 97 4.2.2 IM-DDA Experimental Design . 97 4.2.3 Extraction of Mobility Time of Linear and Cross-linked BSA . 98 4.3 Results and Discussion . 98 4.3.1 Optimisation of Mobility Parameters for Cross-linked Peptides . 98 4 4.3.2 Mobility of Cross-linked and Linear BSA Peptides . 100 4.3.3 Enhancement of IM-DDA with the Application of Charge Stripping . 102 4.3.4 Comparison of Identified Cross-links across Mobility and Non-Mobility DDA.................................... 103 4.3.5 Effects of SEC on IM-DDA analysis of cross-linked peptides . 105 4.3.6 Effects of Sample Complexity on Cross-link Identification Rates with Mobility and Non-Mobility Methods . 106 4.3.7 Reduction in singly charged precursors . 108 4.4 Conclusion and Further Work . 110 5 High Definition Data Dependent Acquisition for the Analysis of Cross- linked Peptides 112 5.1 Introduction . 112 5.2 Materials and Methods . 116 5.2.1 Preparation and Analysis of Proinsulin C-peptide . 116 5.2.2 Merging of Enhanced High Duty Cycle Data . 117 5.3 Results and Discussion . 119 5.3.1 HD-DDA Analysis of Cross-linked BSA with Proinsulin C-Peptide Wide- band Enhancement . 119 5.3.2 HD-DDA Analysis of Cross-linked BSA with Sample Wideband En- hancement . 123 5.3.3 Role of HD-DDA in Duty Cycle for both Calibrants . 126 5.3.4 Comparison of Spectral Quality Across all Methods . 128 5.4 Conclusion and Further Work . 131 6 Computational Solutions for the Analysis of Cross-linked Peptides 133 6.1 Introduction . 133 6.1.1 Evolution of Crosslinking as a Structural Technique . 133 6.2 Materials and Methods . 136 6.2.1 ValidateXL.py . 136 6.2.2 AnnotateXL . 139 5 6.3 Results and Discussion . 143 6.3.1 Analysis of DDA Datasets with ValidateXL . 143 6.3.2 Effect of Validation and Energy Ramps on Fragmentation Efficiency for Alpha and Beta Peptides . 147 6.3.3 Effects of Validation by ValidateXL on QToF Experiments . 149 6.3.4 Annotate XL: Signal to Noise Improvement for QToF Experiments . 151 6.4 Conclusion and Further Work . 158 7 Conclusion 160 Appendix A: xQuest Ubuntu Installation Protocol 168 Appendix B: xQuest Search Parameters 171 7.1 Kernel Density Estimation . 172 Appendix C: Kernel Density Estimation 173 7.2 Appendix D: BSA Peptides with a Charge State above +3 . 174 Appendix D: BSA Peptides with a Charge State above +3 175 Appendix E: Further Methods for ValidateXL and AnnotateXL 185 6 Declaration I, Juliette James, declare that the work presented in this thesis to be my own. Where infor- mation has been derived from other sources I confirm that it has been explicitly referenced within the text. 7 List of Figures 1.1 Schematic representation of electrospray ionisation. High voltage current is applied to the end of the spray tip producing charged droplets of volatile solvent. The solvent evaporates leaving behind charged particles that are deflected by electromagnetic fields through the mass spectrometer. 30 1.2 Definition of resolution for mass spectrometry as defined by.71 ........ 33 1.3 Principles of separation and stable ion trajectory through a Quadrupole mass analyser. a) Schematic showing ion trajectory through a quadrupole. b) Re- lationship between RF (V ) and DC (U) voltage and stable trajectory. Arrow indicates the scan function or DC/RF. Filled triangles indicate stable trajec- tories for ions of three different masses where m3<m2<m1. Length of line in shaded areas indicates spectral peak area that will be generated. 34 1.4 Path of a packet of ions through a ToF analyser. Ion beam is shown as a dashed tan line, high energy ion (yellow), low energy ion (brown). Pusher lens is shown in green and pulses a packet of ions from the ion beam into the analyser. In between pusher pulses the ion beam continues to the TIC monitor (in blue) where total ion count is recorded. 35 1.5 Schematic of Thermo Velos Orbitrap mass analyser. Reproduced with permis- sion from Thermo Fisher Scientific [106]. Locations of ESI source, linear ion trap, C-trap, reagent source and the orbitrap mass analyser are marked. The ion beam through the C-Trap to the orbitrap analyser is shown in red. Ions rotate around the central spindle in the orbitrap analyser generating an image current. 37 1.6 Principles of Ion Mobility separation by Travelling Wave Ion Mobility. 40 8 1.7 Representation of tandem mass spectrometry. Precursor ion masses are recorded and isolated generating MS spectra. Fragmentation occurs as represented by arrow. Fragment ions are generated from the isolated precursor and recorded by a mass analyser generating MS/MS spectra. This may be done in space or in time. 41 1.8 Principles and nomenclature of peptide fragmentation. A) Fragmentation at different bonds in the peptide backbone yields: A or X ions (orange), B or Y ions (green) and C or Z ions (blue). ABC ions are generated by fragmenta- tion at the N’ terminal side of the peptide. XYZ ions are generated through fragmentation at C’ terminal side of the peptide. B) B ion formation proceeds through a cyclic oxazolone structure as shown. This prevents the observation of a B1 ion as two carbonyl groups are required. 42 1.9 A cross-linking mass spectrometry workflow. Nomenclature as discussed in Leitner et al. [62]. Following exposure to the cross-linker the protein of inter- est is digested to produce a number of cross-linked products. Of these only the inter and intralinks are structurally informative. The cross-linked prod- ucts are analysed by LC-MS/MS to sequence the peptides. Cross-links can then be mapped onto 3 dimensional structures or models to aid in structural determination and refinement. 44 1.10 Biotinylated Azo-Leiker 1 (bAL1) cross-linker. Biotin group shown in ma- genta. Image produced using ChemDraw Professional version 16.0 . 45 1.11 PIR cross-linker. Biotin tag shown in red, CID cleavable D-P shown in blue with dashed line representing scissile bond and leaving group shown in green. Image produced using ChemDraw Professional version 16.0 . 46 1.12 Schematic representation of cross-linker DSSO and CDI cleavable cross-linkers. Image produced using ChemDraw Professional version 16.0 . 47 1.13 Schematic representation of cross-linker BS3 and BSG cross-linkers that can be deuterated to create Heavy and Light Pairs. Image produced using ChemDraw Professional version 16.0 . 48 9 1.14 Reaction scheme for conjugation of NHS ester with a primary amine. Opti- mal pH for reaction is shown. Image produced using ChemDraw Professional version 16.0 . 49 1.15 Sulfosuccinimdyl 4,4’-azipentanoate (sulfo-SDA) cross-linker. Leaving group following cross-linking shown in green. Leaving group following UV exposure shown in red. Image produced using ChemDraw Professional version 16.0 . 49 1.16 Example of collision energy ramping in a Synapt G2-Si. Low mass ramp shown in blue, high mass ramp shown in green. An ion of a particular m/z is exposed to the range of energies between the two ramps over the course of a scan. 1200 m/z is indicated on the image. Under these conditions an ion of this m/z will experience energies from 42 eV to 59 eV. 51 1.17 Molecular structure of DSS cross-linker. Cross-linker may be isotopically la- belled. X represents Hydrogen (d0) or Deuterium (d12). Image produced using ChemDraw Professional version 16.0 . 54 1.18 Calculation of inner product vector for XCorr score.