Chromogenic cross-linker for the characterization of protein structure by infrared multiphoton dissociation mass spectrometry.
We have developed a new IR chromogenic cross-linker (IRCX) to aid in rapidly distinguishing cross-linked peptides from unmodified species in complex mixtures. By incorporating a phosphate functional group into the cross-linker, one can take advantage of its unique IR absorption properties, affording selective infrared multiphoton dissociation (IRMPD) of the cross-linked peptides. In a mock mixture of unmodified peptides and IRCX-cross-linked peptides (intramolecularly and intermolecularly cross-linked), only the peptides containing the IRCX modification were shown to dissociate upon exposure to 50 ms of 10.6-microm radiation. LC-IRMPD-MS proved to be an effective method to distinguish the cross-linked peptides in a tryptic digest of IRCX-cross-linked ubiquitin. A total of four intermolecular cross-links and two dead-end modifications were identified using IRCX and LC-IRMPD-MS. IRMPD of these cross-linked peptides resulted in secondary dissociation of all primary fragment ions containing the chromophore, producing a series of unmodified b- or y-type ions that allowed the cross-linked peptides to be sequenced without the need for collision-induced dissociation.
Department of Chemistry and Biochemistry, The University of Texas at Austin, 1 University Station A5300, Austin, Texas, USA 78712, USA.
Myles W
MW
Gardner
Lisa A
LA
Vasicek
Shagufta
S
Shabbir
Eric V
EV
Anslyn
Jennifer S
JS
Brodbelt
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003432:Cross-Linking Reagents / Q000138:chemical synthesis / Q000737:chemistry*; D010755:Organophosphates / Q000138:chemical synthesis / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D013388:Succinimides / Q000138:chemical synthesis / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D025801:Ubiquitin / Q000737:chemistry
D003432:Cross-Linking Reagents; D010755:Organophosphates; D010455:Peptides; D013388:Succinimides; D025801:Ubiquitin