Determination of Protein Contacts by Chemical Cross‐Linking With EDC and Mass Spectrometry

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

This unit describes a method for analyzing protein complexes by chemically cross?linking closely positioned amino acids. The cross?linked products are isolated by SDS?PAGE and the bands of interest are excised. Proteins in the excised gel piece are digested with trypsin and the resulting peptides recovered. Chemically bonded peptides are distinct from the multitude of unmodified single peptides by the presence of two carboxy?terminal ends. The protocol also describes incorporation of ¹⁸ O at the carboxy?terminal ends and purification and preparation of the peptide mixture for mass spectroscopy.

Keywords: protein contacts; cross?links; isotopic exchange; mass spectrometry

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Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1:

Materials

Purified protein(s) to be cross‐linked, of a known concentration (6 to 8 nmol minimum)
0.5 M sodium N‐morpholinoethanesulfonate (MES buffer), pH 6.5 (see recipe )
0.2 M [1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide hydrochloride] (EDC; Pierce) in 50 mM MES; prepare immediately before use
0.02 M sulfo‐N‐hydroxysuccinimide (Sulfo‐NHS;Pierce) in 50 mM MES; prepare immediately before use.
0.5 M hydroxylamine hydrochloride
25 mM Tris⋅Cl, pH 8.0 ( appendix 2E ) in acetonitrile:water (1:1)
100% acetonitrile
Trypsin working solution (see recipe )
25 mM Tris⋅Cl, pH 8.0 in water
25 mM Tris⋅Cl, pH 8.0 in 20% (v/v) acetonitrile
80% H₂ ¹⁶ O/20% (v/v) acetonitrile
80% H₂ ¹⁸ O/20% (v/v) acetonitrile
Modified trypsin, sequencing grade (Promega)
95% isotopically enriched H₂ ¹⁸ O (Sigma‐Aldrich)
2.5% (v/v) trifluoroacetic acid in water
0.1% (v/v) trifluoroacetic acid in 50% (v/v) acetonitrile
0.1% (v/v) trifluoroacetic acid in water
0.1% formic acid in 50% (v/v) acetonitrile or methanol

Dialysis membrane or micro dialysis cups of appropriate MWCO (Pierce) or gel filtration spin column (MacroSpin columns; http://nestgrp.com, no. SMM 025.25; also see unit 8.3 )
Scalpel
Speedvac evaporator
Clear 0.5‐ or 1.5‐ml microcentrifuge tubes
30°C incubator
Reversed‐phase C18 ZipTip pipet tips (Millipore) or OMIX Tip C18 (Varian)

Additional reagents and equipment for dialysis ( appendix 3B ) or buffer exchange using a gel filtration column (unit 8.3 ), mass spectrometry (Chapter 16), SDS‐PAGE gel electrophoresis (unit 10.1 ), and gel staining and destaining (unit 10.5 )

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Figures

Figure 19.16.1 The mechanism of condensation (cross‐linking) reaction between a protein side chain carboxyl (I) and amine (II) group. (A ) Reaction with EDC alone. (B ) NHS forms a more stable active ester (V) and increases the yield of the isopeptide (cross‐link) bond (IV).

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Figure 19.16.2 Portion of a MALDI‐TOF spectrum displaying the isotopic distribution of a cross‐linked H₂ ¹⁶ O/H₂ ¹⁸ O‐double‐labeled peptide.

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Videos

Literature Cited

	Back, J.W., Notenboom, V., de Koning, L.J., Muijsers, A.O., Sixma, T.K., de Koster, C.G., and de Jong, L. 2002. Identification of cross‐linked peptides for protein interaction studies using mass spectrometry and 18O labeling. Anal. Chem. 74:4417‐4422.
	DeTar, D.F. and Silverstein, R. 1966. Reactions of carbodiimides. II. The reactions of dicyclohexylcarbodiimide with carboxylic acids in the presence of amines and phenols. J. Am. Chem. Soc. 88:1013‐1019.
	Schnolzer, M., Jedrzejewski, P., and Lehmann, W.D. 1996. Protease‐catalyzed incorporation of 18O into peptide fragments and its application for protein sequencing by electrospray and matrix assisted laser desorption/ionization mass spectrometry. Electrophoresis 17:945‐953.
	Sehgal, D. and Vijay, I.K. 1994. A method for the high‐efficiency of water‐soluble carbodiimide‐mediated amidation. Anal. Biochem. 218:87‐91.
	Sinz, A. 2003. Chemical cross‐linking and mass spectrometry for mapping three‐dimensional structures of proteins and protein complexes. J. Mass Spectrom. 38:1225‐37.
	Staros, J.V., Wright, R.W., and Swingle, D.M. 1986. Enhancement by N‐hydroxysulfosuccinimide of water‐soluble carbodiimide‐mediated coupling reactions. Anal. Biochem. 156:220‐222.
	Trakselis, M.A., Alley, S.C., and Ishmael, F.T. 2005. Identification and mapping of protein‐protein interactions by a combination of cross‐linking, cleavage, and proteomics. Bioconjugate Chem. 16:741‐751.
	Yao, X., Afonso, C., and Fenselau, C. 2003. Dissection of proteolytic 18O labeling: endoprotease catalyzed 16O‐to‐18O exchange of truncated peptide substrates. J. Proteome Res. 2:147‐152.
Key References
	Sinz, 2003. See above.
	An extensive review of cross‐linkers, types of cross‐links and methods of MS analysis.
	Trakselis et al., 2005. See above.
	Recent advances and current experimental techniques for identifying protein contacts and networks by mass spectrometry.
Internet Resources
	http://www.gpmaw.com
	GPMAW (General Protein/Mass Analysis for Windows). Software with many useful features for handling and analyzing protein and peptide sequences.
	http://www.piercenet.com
	The company offers a wide choice of cross‐linking reagents with varying spacer lengths and reactive towards different amino acid side chains.