Covalent Modification of Proteins An Overview Wide

Covalent Modification of Proteins An Overview

Wide Range of Application Protein Marking Protein Structure • Fluorescent/radioactive tagging of macromolecules (binding & affinity study) • Enzyme active site query • Crosslinking to matrices/solid support (purification; biochemical function) • Modifications assisting bottomup proteomics • Surface mapping of protein & protein complexes (“footprinting” structural study) • Crosslinking to other proteins & biomolecules (biochemical function; structural study). • Modifications assisting bottomup protein structure

Protein Structure Applications • Crosslinking • Chemical “Footprinting” • Motivation: – As many protein: protein interactions as there are proteins within a cell (von Mering, C. ; Krause, R. ; Snel, B; Cornell, M. ; Oliver, S. G. ; Fields, S. ; Bork, P Nature 2002, 417, 399) – Only ~4000 such crystal structures – Sample ensemble environment: dilute proteins in physiologic sol’n • Advances in MS & source technology elevate covalent modification as a strategy for studying protein structure – Ion trap accumulation of target & subsequent CID, ECD etc. possible – Any protease can be used for digestion

Bottom-Up General Methodology 2. 1. 3. 4. 5. 6. 1. Digest in optimized conditions • Label or cross-link reduction of disulfides, etc. Native/denature; • Cleanup (desalt) Include no-chemistry control • Offline by microbed reverse phase tips • Online by trap column Online HPLC • reverse phase or 2 D chromatography ESI-MS/MS, ion trap or hybrid spectrometer • Low flow rate • MS 2 ion selection: optimize Data analysis a. Protein known? – Use smaller db than full NCBI or Swiss. Prot Data analysis b. Submit to search c. Not what you’re looking for? Manual assignment! – Mascot, SEQUEST, X!Tandem, Protein. Prospector – Define known modification/“Error-tolerant searching” Covalent Chemistry Standard Proteomic Protocols Reconcile Data

Crosslinking

Crosslinking Methodology • Many water and membrane soluble crosslinkers available • NHS-ester reacts at physiologic p. H • Optimize – Concentration or reactant – p. H – Exposure time – Reactions do not require harsh working conditions Bis[sulfosuccinimidyl] glutarate, 7. 7 Å spacer length +2

Crosslinking Methodology Protein 11 Protein Single MS (MALDI-TOF) Protein 2 Cross-link Protein 11 Protein 2 Size Exclusion Chromatography Enzymatic Digestion Nano. HPLC-ESI FTICRMS/MS Crosslinks ID’d Software analysis Intrapeptide cross-linked species Intraprotein cross-linked species Interprotein cross-linked species (Sinz, A. in Mass Spectrometry of Protein Interactions; Downdard, K. M. , Ed; Wiley Inter. Science; John Wiley and Sons, Inc. : Hoboken, NJ, 2007, pp 83 -107)

Crosslinking • Detection by MS – Intra-peptide crosslinks & hydrolyzed linkers standard MS 2 proteomics protocol – Else: manual assignment or by software: • GPMAW (General Protein Mass Analysis for Windows) • Automated Spectrum Assignment Program (ASAP) • MS 2 Assign (http: //roswell. ca. sandia. gov/~mmyoung/ms 2 assign. html, Schilling, B. ; Row, R. H. ; Gibson, B. W. ; Guo, X. ; Young, M. M. J. Am. Soc. Mass Spectrom. 2003, 14, 834) • Insight – What’s near what • low resolution constraints for high resolution – Flexible regions easily ID’d – Whole cell or organelle analysis possible esp. with UVreactive crosslinkers

Crosslinking Pros/Cons Advantages Disadvantages • Wide variety of spacer lengths • -NH 2, -COOH, -SH can be targeted • MS/MS cleavable crosslinkers; photoreactive crosslinkers • Obtaining pure rxn mixtures • Need complete sequence coverage – Missed cleavages – Reduction of charge (amine site links destroy basicity) • Optimization required: – Yield vs. Over-crosslinked • Complexity of analysis

Acetylation of Primary Amines

Lysine Acetylation Methodology Acetic anhydride • Acetic anhydride protocol 1. ~ 1 M acetic anhydride 2. RT 1 min-1 hr 3. Acidic p. H, else hydrolysis! • Succinimidyl ester protocol 1. ~0. 1 -10 m. M ester 2. RT 1 hr 3. Neutral p. H • Current Protocols in Protein Science (Coligan, J. E. ; Dunn, B. M. ; Speicher, D. W. ; Wingfield, P. T. ; Ploegh, H. L. ; Talor, G. , Eds; Wiley Inter. Science Current Protocols series; John Wiley and Sons, Inc. : Hoboken, NJ, 2007) Succinimidyl ester

Peptide 487 -500 of human TRF 2 [M+4 H+]4+ 487 IKDRWRTMKRLGMN 500 – 2 possible sites 452. 01 100 % 447. 74 0 + 2 Ac 473. 26 100 Apo: absent DNA + 1 Ac 462. 51 % 479. 30 468. 48 0 + 1 Ac 100 holo + 2 Ac 462. 50 [M+4 H+]4+ 473. 01 452. 01 479. 28 % 458. 26 447. 73 0 445 468. 99 485. 26 477. 26 m/z 450 455 460 465 470 475 480 485

h. TRF 2 Lysine Solvent Accessibility calculated solvent accessibility (Å2) Lysine ID Apo 25 structures Holo 20 structures 446 147 19 118 38 447 134 23 27 5 449 139 20 109 28 459 119 12 107 11 464 123 8 132 15 475 144 10 150 3 488 97 10 11 4 495 111 14 94 10 Avg. All 72 10 61 9 GETAREA – http: //pauli. utmb. edu/cgi-bin/get_a_form. tcl 1. 4 Å probe radius The sidechains of lysine 447 and 488 are not solvent accessible w/ DNA complexation; their backbones are (Sperry, J. B. ; Shi, X. ; Rempel, D. L. ; Nishimura, Y. ; Akashi, S. ; Gross, M. L. Biochemistry 2008, 47, 1797)

Iodination of Tyrosine and Histidine

Iodination of Tyrosine Pierce Iodo-Beads ® • 125 I labeling – Protein marking application • 127 I labeling – X-Ray Crystallography – Tyr & His accessibility • Methods – Lactoperoxidase – N-chloro tosylamide – Iodo-Beads ® Nonpourous polystyrene sphere + Na+I- (Markwell, M. A. K. Anal. Biochem. 1982, 125, 427) I 2

Y 530 in the Active Site of T 7 DNA Polymerase a c b d • Active site: open conformation. Protein (cyan) DNA in CPK • Tyr 530 (brown) • Thioredoxin (purple) • DNA and dd. ATP in active site • Closed conformation (Li, Y. ; Dutta, S. ; Doublie, S. ; Bdour, H. M. ; Taylor, J. S. ; Ellenberger, T. Nat. Struct. Mol. Biol. 2004, 11, 784)

T 7 DNA Polymerase Active Site Tyrosine Iodination Observed Iodinated Peptide T 7 -DNA (open) -dd. NTP Iodinated Residue 64 -YDVPALTK-71 <0. 1 238 -AIEELYVELAAR-249 <0. 1 346 -LQEAGWVPTKYTDKGAPVVDDEVLEGVR-373 <0. 1 409 -YVAEDGKIHGSVNPNGAVTGR-419 <0. 1 523 -TFIYGFLYGAGDEK-536 2. 5 ± 0. 5 2. 4 ± 0. 5 0. 14 ± 0. 03 Y 526, Y 530 Thioredoxin-37 -MIAPILDEIADEYQGK-52 <0. 1 0. 25 0. 03 <0. 1 Y 49 The active site conformation is observed by Iodination MS to close upon dd. NTP substrate binding (Vu, B. Ph. D. Thesis, Washington Unviersity, St. Louis, MO 2006)

Lys Acetylation • Advantages – High yield – Amino group ? group/tag • Disadvantages – Removing basic site Iodination/Acetylation Pros/Cons Tyr Iodination • Advantages – Okay yield – Introduce radioisotope • Disadvantages – Few “interesting” Tyr/protein Targeted Residues • Advantages – Straightforward analysis – High yield • Disadvantages – Requires optimization – Not general – Not necessarily physiologic

Oxidation by Hydroxyl Radical

Hydroxyl Radicals Insert Oxygens Unoxidized +16 Da +32 Da +48 Da +64 Da +16 Da +14 Da Typical modification pattern of hydroxyl radical chemistry. Scheme 1: General aliphatic oxidation (Xu, G. ; Chance, M Chem. Rev. 2007, 107, 3514)

Hydroxyl Radicals Target Solvent Accessible Sidechains of 8 Residues Hydroxyl reactivity with 20 amino acids. http: //allen. rad. nd. edu/browse_compil. html

H 2 O Synchrotron X-rays (e. g. Brookhaven) 100 e. V [ H 2 O·+ + edry-] HO· + 2. 7 eaq- + 0. 61 H· + 0. 03 HO 2· + 0. 61 H 2 O 2 + 0. 43 H 2 + 2. 7 H+ (Maleknia, S. D. ; Ralston, C. Y. ; Brenowitz, M. D. ; Downard, K. M. ; Chance, M. R. Anal. Chem. 2001, 289, 103) High Voltage Electrical Discharge 8 k. V ESI needle voltage, O 2 nebulizer gas Protein(aq) Oxidized Protein(g) (Maleknia, S. D. ; Chance, M. R. ; Downard, K. M. Rapid Commun. Mass Spectrom. 1999, 13, 2352) H 2 O g-radiation 137 Cs [ H 2 O·+ + edry-] HO· + 2. 7 eaq- + 0. 61 H· + 0. 03 HO 2· + 0. 61 H 2 O 2 + 0. 43 H 2 + 2. 7 H+ (Venkatesh, S. ; Sharp, J. S. ; Tomer, K. B. Rapid Commun. Mass Spectrom. 2007, 21, 3927) Fenton chemistry H 2 O 2 Fe(II)/EDTA 2 HO· (Tullius, T. D. ; Dombroski, B. A. Proc. Natl. Acad. Sci. U. S. A. 1986, 83, 5469) Photolysis H 2 O 2 248 nm 2 HO· (Hambly, D. M. ; Gross, M. L. J. Am. Soc. Mass. Spectrom. 2005, 16, 2057)

X-Ray Dose Response Curve • Monitor ratio • QC check: – • If 1 st order, protein not unfolding during labeling Relative reactivity/residue – Quantitative measure of solvent accessibility Dose response for an exemplary peptide from a reacted protein (Kiselar, J. G. ; Maleknia, S. D. ; Sullivan, M. ; Downard, K. M. ; Chance, M. R. Int. J. Radiat. Biol. 2002, 78, 101)

Fast Photochemical Oxidation of Proteins (FPOP) Radical Scavenger + H 2 O 2 at start 248 nm 1) H 2 O 2 2 ·OH 2) 2) ·OH + Residue HO-Residue + ·H Kr. F excimer laser, 248 nm

X-Ray H 2 O Method • Advantages – No additives – Dose/response residue rxn rate • Disadvantages – Synchrotron XRay source Oxidation Pros/Cons FPOP H 2 O 2 Method • Advantages – Laser only investment – High Yield & tunable • Disadvantages – Handling peroxide – No direct dose/response OH Labeling • Advantages – Non specific labeling: M, C, F, Y, W, H, P, L/I – Water-sized probe – Physiologic context – No optimization • Disadvantages – Post labeling oxidation (esp. Met) – X-Ray/FPOP: more than chemicals investment

Summary: Covalent Modification Pros/Cons Advantages Protein complexes Membrane proteins Fast Sensitive: ~fmoles -NH 2, -COOH, -SH, tyrosyl, … can be targeted • Multiple functionalities added • Covalent modification • • • – MS 2 analysis – Multiple digest enzymes – Top-down too! Disadvantages • Labeling not ubiquitous: – Lower resolution probe • Often need pure rxn mixtures • Optimization of rxn: – Prevent over-labeling – Avoid sampling non-native structure • Complexity of analysis • Downstream modifications

Thank You! • John R. Engen • Robert L. Hettich • HD exchange and covalent labeling interest group • Michael R. Gross • Washington University Center for Biomedical and Bioorganic Mass Spectrometry