Anal. Chem. 2009, 81, 9885–9895 Method for the Affinity Purification of Covalently Linked Peptides Following Cyanogen Bromide Cleavage of Proteins Tujin Shi,† Rasanjala Weerasekera,†,‡ Chen Yan,‡ William Reginold,‡ Haydn Ball,§ Thomas Kislinger,| and Gerold Schmitt-Ulms*,†,‡ Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada, Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada, Department of Biochemistry, University of Texas Southwestern Medical School, Dallas, Texas, and Division of Cancer Genomics and Proteomics, Ontario Cancer Institute, Toronto, Ontario, Canada The low resolution structure of a protein can sometimes posttranslational modifications,6 and the mapping of protein-nucleic be inferred from information about existing disulfide acid interactions are increasingly well-developed,7-13 gaining bridges or experimentally introduced chemical crosslinks. insights into the folds of proteins and contact sites between Frequently, this task involves enzymatic digestion of a proteins remains a challenging undertaking. Despite major protein followed by mass spectrometry-based identifica- advances in speed and application range, high-resolution structure tion of covalently linked peptides. To facilitate this task, methods based on X-ray crystallography or nuclear magnetic we developed a method for the enrichment of covalently resonance (NMR) analyses continue to be time-consuming and linked peptides following the chemical cleavage of a are limited by the need to obtain analytes at high levels of purity protein. The method capitalizes on the availability of and quantity. The quest for a robust methodology that fills this homoserine lactone moieties at the C-termini of cyanogen need has arguably become one of the most pressing problems in bromide cleavage products which support selective con- protein science. For proteins which harbor internal disulfide jugation of affinity tags. The availability of two C-termini bridges, a first glimpse into their fold may be obtained by within covalently linked peptides allows for the conjuga- characterizing these linkages. Whenever no disulfide linkages tion of two distinct affinity tags and thereby enables exist in a protein of interest, chemical crosslinking reagents can subsequent removal of unmodified peptides by tandem be employed to experimentally introduce covalent linkages.14 affinity chromatography. Here, we demonstrate the step- Alternatively, cysteine residues can be engineered into a protein wise implementation of this method using a polyhistidine which subsequently can inform one about the structural fold of a tag and a biotin tag for the selective two-step purification protein.15 These methods may become particularly useful for of covalently linked cyanogen bromide fragments from studying the topology and interfaces of macromolecular protein increasingly complex protein samples. The method is complexes that have proven to pose a formidable challenge to independent of the nature of the covalent bond, is adapt- the above-mentioned high-resolution methods. The typical meth- able to fully denaturing conditions, and requires only low odological progression in these studies is the enzymatic digestion picomole quantities of starting material. of proteins followed by a mass spectrometry (MS)-based identi- fication of covalently linked peptides. Multiple reports have The topology of a protein and the interactions it engages in documented the successful application of this approach to explore 16-19 are important determinants of its biology. Proteins do not act in the topology of proteins or to characterize protein-protein isolation but interact with other proteins, nucleic acids, and a range of cellular factors to fulfill their diverse cellular roles.1-4 Whereas (6) Hoffman, M. D.; Sniatynski, M. J.; Kast, J. Anal. Chim. Acta 2008, 627, 50–61 5 . tools for the identification of proteins, the characterization of their (7) Geyer, H.; Geyer, R.; Pingoud, V. Nucleic Acids Res. 2004, 32, e132. (8) Jensen, O. N.; Barofsky, D. F.; Young, M. C.; von Hippel, P. H.; Swenson, * To whom correspondence should be addressed. Address: Centre for S.; Seifried, S. E. Rapid Commun. Mass Spectrom. 1993, 7, 496–501. Research in Neurodegenerative Diseases, University of Toronto, Room 209, Tanz (9) Jensen, O. N.; Kulkarni, S.; Aldrich, J. V.; Barofsky, D. F. Nucleic Acids Neuroscience Building, Toronto, Ontario, M5S 3H2, Canada. Tel: 416-946-0066. Res. 1996, 24, 3866–3872. Fax: 416-978-1878. E-mail: [email protected]. (10) Lenz, C.; Kuhn-Holsken, E.; Urlaub, H. J. Am. Soc. Mass Spectrom. 2007, † Centre for Research in Neurodegenerative Diseases, University of Toronto. 18, 869–881. ‡ Department of Laboratory Medicine and Pathobiology, University of Toronto. (11) Pingoud, V.; Geyer, H.; Geyer, R.; Kubareva, E.; Bujnicki, J. M.; Pingoud, § University of Texas Southwestern Medical School. A. Mol. BioSyst. 2005, 1, 135–141. | Ontario Cancer Institute. (12) Urlaub, H.; Hartmuth, K.; Luhrmann, R. Methods 2002, 26, 170–181. (1) Gingras, A. C.; Gstaiger, M.; Raught, B.; Aebersold, R. Nat. Rev. Mol. Cell (13) Urlaub, H.; Kuhn-Holsken, E.; Luhrmann, R. Methods Mol. Biol. 2008, 488, Biol. 2007, 8, 645–654. 221–245. (2) Aloy, P.; Russel, R. B. Trends Biochem. Sci. 2002, 12, 633–638. (14) Sinz, A. Mass Spectrom. Rev. 2006, 25, 663–682. (3) Pandey, A.; Mann, M. Nature 2000, 405, 837–846. (15) Sato, C.; Morohashi, Y.; Tomita, T.; Iwatsubo, T. J. Neurosci. 2006, 26, (4) Sobott, F.; Robinson, C. V. Curr. Opin. Struct. Biol. 2002, 12, 729–734. 12081–12088. (5) Lu, B.; Xu, T.; Park, S. K.; Yates, J. R., 3rd Methods Mol. Biol. 2009, 564, (16) Pearson, K. M.; Pannell, L. K.; Fales, H. M. Rapid Commun. Mass Spectrom. 261–288. 2002, 16, 149–159. 10.1021/ac901373q CCC: $40.75 2009 American Chemical Society Analytical Chemistry, Vol. 81, No. 24, December 15, 2009 9885 Published on Web 11/19/2009 interfaces.20-24 Despite impressive progress in this direction, this or peptides.36,37 Even more promising are approaches which type of investigation has so far been of modest practical benefit selectively remove noninformative peptides from the sample. For to mainstream biochemists because the demand for the purity of this purpose, crosslinkers can be equipped with a functional group samples remains relatively high. A reason for the limitation of such as a biotin moiety which facilitate enrichment following their current protocols is their reliance on the direct detection of conjugation to a peptide.24,38-43 However, the application of more crosslinked peptides by mass spectrometry. This conceptual complex crosslinkers may be counterintuitive if the long-term feature translates into a search not unlike the proverbial search objective is the study of protein complex topologies following in for a “needle in a haystack”, since informative through-space vivo crosslinking of cells and intact tissues. Furthermore, the use covalent linkages, hereafter referred to as intercrosslinks, are of affinity-tagged crosslinking reagents will lead to the concomitant present in such protein samples at substoichiometric levels, often purification of noninformative peptides which are merely deriva- masked by an overabundance of unmodified, derivatized, or tized or contain internal covalent linkages. We, therefore, favor a internally crosslinked peptides. Moreover, upon enzymatic diges- strategy which capitalizes on the fact that intercrosslinked CNBr tion, intercrosslinked peptides give rise to relatively large analytes fragments can be distinguished from all other contaminants by which tend to ionize relatively poorly. Whenever tandem MS the presence of two peptide chains and, therefore, two N- and spectra are obtained, their interpretation is far from trivial due to C-termini. Here, we report on a novel tandem affinity strategy for the population of these spectra with fragments from two different the purification of crosslinked peptides based on these concepts. peptides. In the past 2 years, we developed a protocol which The method employs the presence of homoserine lactone (HSL) addresses this problem by deriving topology and interface moieties at the C-termini of CNBr-cleaved peptides for the information in an indirect manner following consecutive cleavages attachment of polyhistidine and biotin tags. Using model peptides of chemically crosslinked material with cyanogen bromide (CNBr) and proteins, we demonstrate sensitive and selective enrichment and trypsin.25 CNBr cleavage has long been established as a useful of intercrosslinked peptides from samples of increasing complexity. tool for the characterization of endogenous crosslinks present in collagens.26-30 We demonstrated that this approach can be EXPERIMENTAL SECTION adapted for the design of a topology mapping method that does Peptides and Other Reagents. Bovine serum albumin (BSA) not rely on specialized data mining software for the assignment was purchased from Sigma-Aldrich (Oakville, ON, Canada). The of crosslinks. Despite these advances, the application range of N-terminally acetylated model peptide AcCAPQEGILEDMPVD- this method remains limited without a strategy that deals with PDNEAY was synthesized using an automated peptide synthesizer the challenges posed by the overabundance of uncrosslinked (Applied Biosystems, Foster City, CA), and azurin protein was material present in complex protein samples. This problem has generously provided by Dr. Yi Lu (University of Illinois, Urbana, - - - - - - - - also been recognized