Analysis of chemically modified proteins by FT-ICR MS

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Analysis of chemically modified proteins by FT-ICR MS PETR NOVAK EU FT-ICR MS End User School 1 UEF Chemistry August 19-24 2018 Joensuu, Finland

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, 1173-1332 2

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, 1018. 3

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

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

Chemical cross-linking and the FT-ICR bottom up experiments Dihazi et. al. Rapid. Commun. Mass Spectrom. 2003, 17, 2005 6

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

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

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

Protein labeling FT-ICR bottom up approach 10

LC/FT-ICR MS and data processing Intens. x10 10 8 6 4 2 0 10 15 20 25 30 35 40 Time [min] F_tPt_Tg_04102016_1610.d: TIC All MS, -Spectral Bkgrnd 11

It is a really big mess! Is there any chance to find out modified peptides?... 12

LinX algorithm offers seeing the light at the end of the tunnel. http://peterslab.org/downloads.php 13

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15

16

LinX output 17

Validation of assigned signals isotopic signature Intens. x10 10 8 Intens. x10 6 6 5 '631.90332 F_tPt_Tg_04102016_1610.d: MS, 10.9-13.0min #111-151 6 4 4 2 2 0 2000 5 631.90349 5 632.50460 C₆₃H₁₀₂N₂₀O₂₂S₁PtN₂H₄C₅₉H₁₀₀N₂₂O₁₈, MnH, 631.90341 1500 0 10 15 20 25 30 35 40 Time [min] F_tPt_Tg_04102016_1610.d: TIC All MS, -Spectral 1000Bkgrnd 5 631.70301 Data-dependent acquisition 500 doesn t work! 5 632.70493 5 632.90527 5 633.10562 5 633.30600 0 630.5 631.0 631.5 632.0 632.5 633.0 633.5 m/z 18

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, 251-276 19

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

Experimental part nci Source Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 21

Experimental part nci Source Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 22

Experimental part nci Source Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 23

Collison-induced dissociation (CID) nci Source Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 24

Collison-induced dissociation (CID) nci Source Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 25

Collison-induced dissociation (CID) nci Source Ar Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 26

Electron-transfer dissociation (ETD) nci Source CH 4 -- - - Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 27

Electron-transfer dissociation (ETD) nci Source CH 4 -- - - Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 28

Electron-transfer dissociation (ETD) nci Source CH 4 -- - - Detector ESI -- -- - cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 29

Electron-transfer dissociation (ETD) nci Source CH 4 -- - - Detector ESI -- -- - cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 30

Electron-capture dissociation (ECD) nci Source Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 31

Electron-capture dissociation (ECD) nci Source Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 32

Electron-capture dissociation (ECD) nci Source Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 33

Electron-capture dissociation (ECD) nci Source Detector ESI electron - - - - - beam cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 34

Electron-capture dissociation (ECD) nci Source Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 35

MultiCASI nci Source Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 36

MultiCASI nci Source Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 1 oxidation 37

MultiCASI nci Source Ar Detector ESI cathode Dual Octapole Quadrupole Ion selector collision cell ICR Cell 15T Magnet 1 oxidation 38

Data Analysis: ms2links Evaluation Young MM Proc Natl Acad Sci U S A. 2000; 97:5802-6. Schilling B J Am Soc Mass Spectrom. 2003;14:834-50. 39

Data Analysis: ms2links Evaluation 40

Data Analysis: ms2links Evaluation Select Quality Factor threshold Select max charge state 41

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

Data Analysis: ms2links Evaluation mgf format Text file select mgf format Save as: mgf file 26

Data Analysis: ms2links Evaluation mgf format Text file Mass of ions Intensity of ions select mgf format Save as: mgf file 26

Data Analysis: ms2links Evaluation 45

Data Analysis: ms2links Evaluation Select internal ions for CID Select b ions for CID Select y ions for CID 46

Data Analysis: ms2links Evaluation Select internal ions for CID Select b ions for CID Select y ions for CID 47

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

Data Analysis: Result ms2links Evaluation 49

Data Analysis: Result ms2links Evaluation 50

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

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

ETD of single oxidized ubiquitin 53

ECD of single oxidized ubiquitin 54

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, 531-544 (1987). 55

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, 531-544 (1987). 56

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.731077 CZECH SCIENCE FOUNDATION (GRANT NUMBERS 16-24309S) THE MINISTRY OF EDUCATION OF THE CZECH REPUBLIC (PROJECT LH15010; PROGRAMS NPU II - LQ1604 AND LM2015043 CIISB FOR CMS BIOCEV - LTC17065) AND THE EUROPEAN REGIONAL DEVELOPMENT FUNDS (BIOCEV - CZ.1.05/1.1.00/02.0109)

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