Protein Interactions by Infrared Multiphoton Dissociation-MS. Crosslinker for Identification of. Photoactive Chemical

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1 Photoactive Chemical Crosslinker for Identification of Protein Interactions by Infrared Multiphoton Dissociation-MS Myles W. Gardner, Jennifer S. Brodbelt

2 Chemical Crosslinking of Proteins Protein structural analysis technique Covalently conjugate two specific targets, spatially constrained, with linker molecule Intramolecular crosslinking: tertiary structure Intermolecular crosslinking: quaternary structure Intermolecular Crosslinks Intramolecular Crosslink a 3 S 11.4 Å S 3 a

3 LC MS/MS Protein Crosslinking Protocol Crosslink protein 1. SEC, SDS-PAGE 2. Enzymatic Digest LC-MS/MS Time (min) Identification of Crosslinked Products and Determination of Crosslinked Residues

4 Current Methods for the Identification of Crosslinked Peptides Tri-functional crosslinkers with affinity tag a sulfo-sbed: Biotinylated H S H (CH 2 ) 4 H (CH 2 ) 4 CH H C H S S S 3 a Separate crosslinked peptides from uninformative, unmodified peptides prior to LC-MS/MS Isotope labeled Crosslinkers b BS 3 -d 4 / BS 3 : mass = 4 Da Detect peptide pairs in LC-MS a 3 S D D D D S 3 a a Alley, S.C.; Ishmael, F.T.; Jones, A.D.; Benkovic, S.J. J. Am. Chem. Soc. 2, 122, b Pearson, K.M.; Pannell, L.K.; Fales, H.M. Rapid Commun. Mass Spectrom. 22, 16, 149.

5 Crosslinking Goals Goal: Rapidly distinguish crosslinked peptides from unmodified peptides using quadrupole ion trap-ms IR-active chromophoric crosslinker phospho functional group Infrared Multiphoton Dissociation-MS Selective dissociation of crosslinked peptides Tandem MS/MS information to identify crosslink location

6 1 1 Phospho -groups and IRMPD IRMPD-MS of PMIDA and BIDA PMIDA = ms -H 2 P 3 H P H C 2 laser = 1.6 µm Photoactive Crosslinking Reagent BIDA a = ms Conventional Crosslinking Reagent H H H H Bond Stretch P-H P--C cm µm =P-H P-CH Silverstein, R.M. et al. Spectrometric Identification of rganic Compounds, 4 th ed. Wiley: ew York. 1981: pg a Back, J.W.; Hartog, A.F.; Dekker, H.L.; Muijsers, A..; de Koning, L.J.; de Jong, L.. J. Am. Soc. Mass Spectrom. 21, 12, 222.

7 PMIDA-Peptide Crosslinking H P H H PMIDA H + 2 H 3 C C H EDC 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride CH 3 Cl CH 3 H 3 C H H P H unstable -Acylisourea Active intermediate H CH 3 dry DMS -(phosphonomethyl)- iminodiacetic acid H H H 3 C CH 3 H 3 C CH 3 H P H Protein/Peptide R H 2 H P H H dry DMS 2 2 H H R R Crosslinked Product 9% DMS, 1% 2 mm HEPES ph ~8 (v/v) + 2 H 3 C H PMIDA Crosslinker HS-ester intermediate C H H CH 3 CH 3 Isourea by-product HS -hydroxysuccinimide

8 Experimental: Reaction Procedure Activation Reaction 5. µmol PMIDA, 12.5 µmol EDC, 12.5 µmol HS 3 µl dry DMS 15 min, room temp. Peptide Crosslinking Protein Crosslinking 1:4 molar ratio peptide : PMIDA 1:5 molar ratio Protein:PMIDA 2 mm HEPES, ph 8 2 hours, room temp Desalting by C 18 solid-phase extraction Tryptic digest ESI-MS 18 hours 37 C IRMPD-MS LC-IRMPD-MS

9 ESI-IRMPD-MS Instrumentation ZnSe Window 1.6mm Reflective Mirrors Ring Electrode (5 mm hole) 5 W cw C 2 Laser, 1.6 µm (Synrad) Modified Finnigan LCQ Deca XP Samples: 1-2 µm in 95/4/1 MeH/H 2 /acetic acid (v/v) Flow rate: 3 µl/min, ESI = +4.5 kv IRMPD experiments: 5W for 1 15ms LC-IRMPD-MS C 18 column (2.1 5 mm, 3.5 µm); 98/2/.2 H 2 /AC/formic acid (v/v) to 6/4/.2 data-dependent IRMPD 5 W, 5 ms

10 MS Results: PMIDA + Peptide Crosslinking 1 [P+PMIDAH+2H] 2+ [P+PMIDA+H] 1+ Substance P + PMIDA/EDC/HS H 2 - R P K P Q Q F F G L M [P+PMIDAH+H] 1+ [P+2H] 2+ [P+H] 1+ PMIDA-HS Intra/Inter = +191 Da Dead-end = +29 Da H P H P+PMIDA P H H P+PMIDAH H 2 -R P K P Q Q F F G L M - H 2 H 6.8 Å H R H P K P H H P Q Q F F G L M H 2 H Intramolecular Crosslink Dead-end Modification

11 Reconstructed Full MS Reconstructed IRMPD-MS, 15ms nly PMIDA-modified peptides undergo IRMPD allowing for confident assignment and differentiation from non-crosslinked peptides Intensity (x 1 6, arb) Screening for Crosslinked Products in a Ten Peptide Mock Mixture 1x Laser off Laser on

12 .6 Relative Decrease in Ion Abundance Upon IR Irradiation Laser ff Laser n, 15 ms Relative Ion Abundance.1. Angiotensin I 3+ Angiotensin II Bradykinin CFIRCPRG-H eurotensin Angiotensin I Substance P Thymopentin Melittin 4+ Substance P -H eurotensin γ-endorphin Melittin Angiotensin II Bradykinin CFIRCPRG-H Angiotensin I Substance P Melittin Substance P Substance P -H eurotensin γ-endorphin PMIDA Relative abundance of PMIDA modified peptides decreases by >85% upon IRMPD whereas unmodified peptides abundance decreases by <5%.

13 1 Irradiation of PMIDA-Modified Peptides: Very Selective Dissociation [Substance P + PMIDA + H] 1+ a 8 +PMIDA b 9 +PMIDA ms H- R P K P Q Q F F G L M P y -H 2 P 8 -H 3 3 y [Substance P+BIDA+H] 1+ 1 [Substance P+H] ms 15ms o dissociation o dissociation

14 1 8 Selective Photodissociation of PMIDA vs. BIDA Modified Substance P Laser off -H 2 -H 6 [Substance P+H] H Laser on 15ms 2 -H -H H -H PMIDA PMIDAH BIDA BIDAH Relative abundance of PMIDA modified peptide decreases by >85% upon IRMPD whereas for BIDA modified peptide, abundance decreases by <1%. -H

15 IRMPD-MS for Elucidation of Crosslink Site [Substance P + PMIDA + 2H] ms H- R P K P Q Q F F G L M x5 b 9 2+ b 1 2+ x5 1 b 3 1+ y 8 1+ [PQ-28] 1+ y 1 1+ y 3 1+ a 6 2+ b 7 2+ b 8 2+ a 5 1+ a 8 -H 2 1+ b FGG b 6 1+ b y 4 PMIDA PMIDA fragment ions and losses of PMIDA IRMPD also allows one to determine sites of modifications, in addition to selectively dissociately only PMIDA modified peptides.

16 LC-IRMPD-MS of Peptide Mock Mixture Full MS Total Ion Chromatogram PMIDA modified peptide A B C D E F G H Time (min) A. [Bradykinin + 2H] 2+ IRMPD-MS, 5W, 5 ms 1 E. [γ-endorphin + 2H] B. [Angiotensin II + 2H] C. [eurotensin + 3H] F. [Substance P + PMIDAH H 2 + MeH+ 2H] G. [Substance P + PMIDA + 2H] D. [Angiotensin I + 3H] H. [Melittin + 4H]

17 Protein Crosslinking: Cytochrome c Deconvoluted MS a B A C Cytochrome c MW=1236 Da Mass ESI-MS PMIDA PMIDAH Protein Databank ore.do?structureid=1hrc a Zhang, Z; Marshall, A.G. J. Am. Soc. Mass Spectrom. 1998, 9,

18 Time (min) LC-IRMPD-MS of Crosslinked Cytochrome c Digest Full MS Total Ion PMIDA modified Chromatogram peptide A B C D E F A. Y74-K IRMPD-MS, 5W, 5 ms 1 D. [E61-K72] B. I9-K E. [I9-K25 + PMIDAH] C. M8-K F. [I9-K25 + Heme + PMIDAH]

19 IRMPD-Mass Spectrum of I9-K25+ PMIDAH [I9 K25 + Heme + PMIDAH + 4H] 4+ Heme I F V Q K C A Q C H T V E K G G K 5 ms b 12 -H 2 2+ y 1 1+ y 2 1+ a 5 -H y 3 or 1+ b 2 y 6 2+ y b 7 2+ y y y y 14 -H 3 3+ b a 1 2+ a 5 -H 3 1+ b a 8 1+ PMIDAH Heme internal fragment ions PMIDAH fragment losses a 16 -H 3 2+ y b LC-IRMPD-MS yields diagnostic tandem MS data to determine crosslinked residues for PMIDA modified peptides.

20 Conclusions Developed 2-step method to crosslink peptides with iminodiacetic acids using EDC/HS coupling PMIDA readily photodissociates when subjected to IR irradiation Rapidly screen for and distinguish PMIDA-modified peptides in complex mixtures using IRMPD or LC-IRMPD-MS IRMPD produces diagnostic fragment ions that allow one to confidently identify the crosslink location

21 Acknowledgements Dr. Jennifer Brodbelt Joe Chipuk Barry Davis Byoung-Joon Ko Carolyn Mazzitelli Sarah Pierce Mike Pikulski Suncerae Smith Jeff Wilson Dr. Matthew Crowe Dr. Courtney Sherman Dr. Eric Anslyn Aaron Wright Himali Hewage Dr. Jae Schwartz Thermo Electron