ADPRTs catalyze the transfer of ADP-ribose from β-NAD+
to target proteins (), commonly at arginine residues[1
]. Members of the ADPRT family modify a diverse set of target proteins and share little sequence homology. The mammalian ADP-ribosyltransferase ART-1 modifies defensin-1 which ultimately blocks the antimicrobial and cytotoxic effects of the defensin HNP-1[2
]. Many ADPRTs found in bacteria have been implicated in pathogenicity through the inactivation of host proteins (e.g. Vibrio cholerae
, Bordetella pertussis
and Streptococcus pyogenes
]). S. pyogenes
produces the ADPRT toxin SpyA, which modifies vimentin, actin and tropomyosin, all proteins involved in cytoskeletal structure[3
]. ADP-ribosylation of these proteins prevents polymerization of the cytoskeleton leading to collapse of intermediate filaments. Other ADPRT toxins are known to target proteins involved in signal transduction and regulatory functions[1
]. Although there are numerous molecular assays demonstrating that ADP-ribosylation is an important step in pathogenicity, to date the exact site of ADP-ribosylation and number of modification targets remains unknown for many ADPRTs. A high-throughput mass spectrometry assay designed for the identification of target proteins as well as sites of ADP-ribosylation would provide insight into mechanisms of infection.
ADPRTs catalyze the transfer of ADP-ribose to acceptor proteins from NAD+.
Often LC-MS/MS methods are used to identify sites and protein targets of post-translational modifications (PTMs) in complex biological samples[4
]. Collision induced dissociation (CID) is typically employed as the main fragmentation method during LC-MS/MS analyses because the instrumental duty cycle is high and acquired peptide tandem mass spectra may be easily sequenced. This primarily results from the predictable peptide fragmentation pathways[6
], allowing for the identification of the parent proteins[7
]. However, the presence of a PTM on a peptide can redirect CID fragmentation patterns such that sequence may not be assigned. Previously, Margarit et al. (2006)[8
] demonstrated that CID of an ADP-ribosylated peptide did not result in typical’ peptide fragmentation, interfering with facile sequence interpretation. Additionally, CID analysis of SpyA ADP-ribosylated Vimentin[3
] verified similar fragmentation trends, preventing modified peptide sequence assignment (SM Hengel unpublished data). The inability to directly assign peptide sequences of ADP-ribosylated peptides using CID led us to undertake a detailed mass spectrometry fragmentation pattern analysis of a standard, ADP-ribosylated-Kemptide (LRRASLG). Three common fragmentation methods were investigated: 1)
electron capture dissociation (ECD) and 3)
infrared multiphoton dissociation (IRMPD).
The goals of this study were to determine the most efficient technique for producing reliable and predictable fragment-ion spectra for the identification of ADP-ribosylated peptides and to determine if diagnostic ions exist that might be used in future studies to identify new ADPRT targets. ADP-ribosylated Kemptide was chosen as a standard for examining which of the three methods, CID, ECD and IRMPD, were best suited to 1) assign amino acid sequence and 2) produce ions diagnostic of ADP-ribosylation. Here we discuss the results of this comparison and how these methods may be used alone and in combination to identify ADP-ribosylated proteins in complex mixtures.