Analysis of chemically modified proteins by FT-ICR MS
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1 Analysis of chemically modified proteins by FT-ICR MS PETR NOVAK EU FT-ICR MS End User School 1 UEF Chemistry August Joensuu, Finland
2 Structural Mass Spectrometry Disulfide bonds mapping Limited proteolysis H/D exchange Protein covalent labeling Phast photochemical oxidation of proteins Chemical cross-linking ETD/ECD fragmentation Native mass spectrometry and Ion mobility Special Issue on Mass Spectrometry in Structural Biology (2015) Protein Science 24,
3 Protein covalent labeling and chemical cross-linking Available amino acid sidechains for covalent modification Carboxy groups Asp, Glu, C-term, pka (3.8, 4.3, 2.3) ph 7» deprotonation Amino groups Lys, Arg, His, N-term, pka (9.4, 12, 6.8, 7.8) 7 ph» protonation Sulfhydryl groups - Cys. pka 8.9 ph 7» -SH Aromatic groups Trp (indol), Tyr (hydroxyfenyl, pka 9.9) ~ 23% of amino acid can be covalently modified Klapper et. al. Biochem. Biophys. Res. Commun. 1977, 78,
4 Mass Spectrometry: Goal in Protein Structure Characterization Sensitivity Analysis of complex mixtures/high MW protein Rapid data acquisition Protein Bottom-up Top-down Intact mass analysis Sequence analysis Molecular weight determination Fragmentation methods: CID, ETD, and ETD UHPLC-MS/MS Protein Structure analysis Enzymatic digestion Peptides UHPLC 4
5 Protein covalent labeling and the FT-ICR bottom up experiments Suckau et. al. PNAS 1992, 89, 5630 and Glocker et. al. Bioconj. Chem. 1994, 5, 583 Fiedler et. al. Bioconj. Chem. 1998, 9, 236 5
6 Chemical cross-linking and the FT-ICR bottom up experiments Dihazi et. al. Rapid. Commun. Mass Spectrom. 2003, 17,
7 Chemical cross-linking and the FT-ICR Top down experiments MS spectrum Multi-CHEF isolation SORI fragmentation Fragment assignment Kruppa et. al. Rapid. Commun. Mass Spectrom. 2003, 17, 155 7
8 Protein covalent labeling and the FT-ICR Top down experiments ( 1 M~K6~K48~K63) > K33 > K11 > (K27,K29) Novak et. al. J. Mass Spectrom. 2004, 39, 322 8
9 Protein labeling with noble metal Possible amino acids susceptible to tpt modification Met Met tpt adduct His His tpt adduct Lys Transplatinum Lys tpt adduct Ser Ser tpt adduct Platinum isotope pattern 9
10 Protein labeling FT-ICR bottom up approach 10
11 LC/FT-ICR MS and data processing Intens. x Time [min] F_tPt_Tg_ _1610.d: TIC All MS, -Spectral Bkgrnd 11
12 It is a really big mess! Is there any chance to find out modified peptides?... 12
13 LinX algorithm offers seeing the light at the end of the tunnel. 13
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16 16
17 LinX output 17
18 Validation of assigned signals isotopic signature Intens. x Intens. x ' F_tPt_Tg_ _1610.d: MS, min # C₆₃H₁₀₂N₂₀O₂₂S₁PtN₂H₄C₅₉H₁₀₀N₂₂O₁₈, MnH, Time [min] F_tPt_Tg_ _1610.d: TIC All MS, -Spectral 1000Bkgrnd Data-dependent acquisition 500 doesn t work! m/z 18
19 Stable covalent labeling Radical footprinting ( OH, I, CF3) Chemical footprinting and cross-linking Hydroxyl radicals can be generated by various means: Irradiation of water by x-rays or electron beams Fenton reaction Photolysis of hydrogen peroxide FPOP (fast photochemical oxidation of proteins) The relative reactivity of the amino acid side chains Cysteine, Methionine, Tryptophan Tyr > Phe > His > Leu ~ Ile > Arg ~ Lys ~ Val > Ser ~ Thr ~ Pro > Gln ~ Glu > Asp Alanine, Glycine Takamoto K. et al. Annu Rev Biophys Biomol Struct. 2006, 35,
20 Methods and Simulations FPOP a pulsed laser to photolyze hydrogen peroxide generate OH radicals and modify proteins in a flow system Advantageous Covalent modification preserves the primary sequence of modified residues Fast Photochemical Oxidation of Proteins (FPOP) High reactivity of OH the modifications of more than half of amino acid side-chains, providing a higher coverage H 2 O 2 2 OH OH size comparable to a water molecule that able to probe the solvent accessibility of a protein of interest Fagmentation techniques collision induced dissociation (CID) Electron capture dissociation (ECD) OH electron transfer dissociation (ETD) H 2 O 2, hn OH OH OH OH Data analysis OH OH OH 20
21 Experimental part nci Source Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 21
22 Experimental part nci Source Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 22
23 Experimental part nci Source Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 23
24 Collison-induced dissociation (CID) nci Source Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 24
25 Collison-induced dissociation (CID) nci Source Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 25
26 Collison-induced dissociation (CID) nci Source Ar Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 26
27 Electron-transfer dissociation (ETD) nci Source CH Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 27
28 Electron-transfer dissociation (ETD) nci Source CH Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 28
29 Electron-transfer dissociation (ETD) nci Source CH Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 29
30 Electron-transfer dissociation (ETD) nci Source CH Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 30
31 Electron-capture dissociation (ECD) nci Source Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 31
32 Electron-capture dissociation (ECD) nci Source Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 32
33 Electron-capture dissociation (ECD) nci Source Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 33
34 Electron-capture dissociation (ECD) nci Source Detector ESI electron beam cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 34
35 Electron-capture dissociation (ECD) nci Source Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 35
36 MultiCASI nci Source Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 36
37 MultiCASI nci Source Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 1 oxidation 37
38 MultiCASI nci Source Ar Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 1 oxidation 38
39 Data Analysis: ms2links Evaluation Young MM Proc Natl Acad Sci U S A. 2000; 97: Schilling B J Am Soc Mass Spectrom. 2003;14:
40 Data Analysis: ms2links Evaluation 40
41 Data Analysis: ms2links Evaluation Select Quality Factor threshold Select max charge state 41
42 Data Analysis: ms2links Evaluation Data file format: For an MS/MS Ions Search, the data file contain one or more MS/MS peak lists. In the Mascot generic format, (MGF), each MS/MS dataset pairs of mass and intensity values select mgf format Save as: mgf file 26
43 Data Analysis: ms2links Evaluation mgf format Text file select mgf format Save as: mgf file 26
44 Data Analysis: ms2links Evaluation mgf format Text file Mass of ions Intensity of ions select mgf format Save as: mgf file 26
45 Data Analysis: ms2links Evaluation 45
46 Data Analysis: ms2links Evaluation Select internal ions for CID Select b ions for CID Select y ions for CID 46
47 Data Analysis: ms2links Evaluation Select internal ions for CID Select b ions for CID Select y ions for CID 47
48 Data Analysis: ms2links Evaluation Select internal ions for CID Select b ions for CID Select y ions for CID Inactivate for unmodified Choose number of modification 48
49 Data Analysis: Result ms2links Evaluation 49
50 Data Analysis: Result ms2links Evaluation 50
51 Data Analysis: Result ms2links Evaluation Experimental mass of ions Theoretical mass of ions Ions sequence Intensity of ions Read the intensities of signals for the modified (I ox ) and the unmodified species (I) for each. Detected of unmodified and modified ions 51
52 Extent of Modification CID of single oxidized ubiquitin Plot of the yield of oxidized residues in ubiquitin Extent of Modification Read the intensities of signals for the modified (I ox ) and the unmodified species (I) for each The extents of modification: were calculated by using the following equation: Extent of Modification = σ I ox σ I ox I 52
53 ETD of single oxidized ubiquitin 53
54 ECD of single oxidized ubiquitin 54
55 Crystal structure of oxidized ubiquitin_cid and ETD a b Colored residue side-chain residues that are modified (violet) Modified structure from ref. Kumar V. et al. J. Mol. Biol. 194, (1987). 55
56 Crystal structure of oxidized ubiquitin_ecd a b Colored residue side-chain residues that are modified (violet) Modified structure from ref. Kumar V. et al. J. Mol. Biol. 194, (1987). 56
57 ACKNOWLEDGEMENT GHAZALEH YASSAGHI, LUKAS SLAVATA, ZDENEK KUKACKA, PETR POMPACH, PETR MAN, DANIEL KAVAN, MICHAL ROSULEK, RUZENA LISKOVA DANIELE FABRIS, WILL MCINTIRE, MIKE MILLER, MATEO SCALABRIN GARY KRUPPA, JOE SCHOENIGER, MALIN YOUNG H2020 EUROPEAN NETWORK OF FOURIER-TRANSFORM ION-CYCLOTRON- RESONANCE MASS SPECTROMETRY CENTERS - PROJECT AGREEMENT NO CZECH SCIENCE FOUNDATION (GRANT NUMBERS S) THE MINISTRY OF EDUCATION OF THE CZECH REPUBLIC (PROJECT LH15010; PROGRAMS NPU II - LQ1604 AND LM CIISB FOR CMS BIOCEV - LTC17065) AND THE EUROPEAN REGIONAL DEVELOPMENT FUNDS (BIOCEV - CZ.1.05/1.1.00/ )
Other Methods for Generating Ions 1. MALDI matrix assisted laser desorption ionization MS 2. Spray ionization techniques 3. Fast atom bombardment 4.
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