An aptly positioned azido group in the spacer of a protein cross-linker for facile mapping of lysines in close proximity.
Cross-links between amino acid residues in close proximity can provide distance constraints for the validation of models of the 3D structure proteins. The mapping of cross-links by the identification of linked peptides in proteolytic digests is facilitated by cleavable cross-linkers that enable isolation of the cleavage products while preserving information about the linkage. We present an amine-specific cross-linker, bis(succinimidyl)-3-azidomethyl glutarate (BAMG), that fulfils these requirements. Two parallel reaction pathways are induced by tris(carboxyethyl)phosphine (TCEP) in cross-linked peptides from BAMG-treated cytochrome c. One pathway leads to cleavage of the cross-linked species, while in the other the azido group of BAMG is reduced to an amino group without cleavage. Cross-linked peptides and peptides modified by partially hydrolysed BAMG yield distinct sets of TCEP-induced reaction products. These can be isolated by reversed-phase diagonal chromatography and identified by mass spectrometry to reveal the identity of the parent compounds. The ease with which cross-link-derived reaction products can be isolated and identified indicates that the mapping of cross-links in complex biological assemblies and mixtures of protein complexes might become feasible in the near future.
Kasper
Piotr T
PT
Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands.
Back
Jaap Willem
JW
Vitale
Maxime
M
Hartog
Aloysius F
AF
Roseboom
Winfried
W
de Koning
Leo J
LJ
van Maarseveen
Jan H
JH
Muijsers
Anton O
AO
de Koster
Chris G
CG
de Jong
Luitzen
L
D000818:Animals; D001386:Azides / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry*; D045304:Cytochromes c / Q000737:chemistry*; D006736:Horses; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry; D010720:Phosphines / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001386:Azides; D003432:Cross-Linking Reagents; D045304:Cytochromes c; D008239:Lysine; D010455:Peptides; D010720:Phosphines; C080938:tris(2-carboxyethyl)phosphine