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The E. coli effector EspJ blocks Src kinase activity via amidation and ADP-ribosylation

ABSTRACT: In vitro ADP-ribosylated Src-K295M was separated by SDS-PAGE and the Coomassie stained protein band excised and digested with either trypsin, thermolysin or elastase. All digests were analysed by nanoLC-MS/MS using Higher-energy C-trap Dissociation (HCD) as well as Electron-Transfer Dissociation (ETD) fragmentation.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Andreas Schlosser

LAB HEAD: Andreas Schlosser

PROVIDER: PXD001461 | Pride | 2014-12-19

REPOSITORIES: Pride

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			Action	DRS
	2014-04-08_velos_7346.raw	Raw
	2014-04-08_velos_7347.raw	Raw
	2014-04-08_velos_7348.raw	Raw
	2014-04-08_velos_7349.raw	Raw
	2014-04-08_velos_7350.raw	Raw

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Sequential digestion with Trypsin and Elastase in cross-linking/mass spectrometry

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Attenuation of Global Transcript Changes Induced by Elastase with Symplostatin 5 Cotreatment

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Project description:ADP-ribosylation is a reversible post-translational modification of proteins that has been linked to many biological processes. The identification of ADP-ribosylated proteins and of their acceptor amino acids remains a significant challenge. The attachment sites of the modification are difficult to study by mass spectrometry (MS) because of its labile nature and its complex fragmentation pattern in MS/MS experiments. In this study we performed a detailed analysis of higher-energy collisional dissociation (HCD) spectra acquired from ADP-ribosylated peptides which were modified on arginine, serine, glutamic acid, aspartic acid, tyrosine or lysine. In addition to the fragmentation of the peptide backbone, various cleavages of bonds within the ADP-ribose, and between the modification and the amino acid residue, have to be considered. We focused on gas-phase fragmentations that are specific either to ADP-ribosylated arginine or to ADP-ribosylated serine and other O-linked ADP-ribosylations. The O-glycosidic linkage between ADP-ribose and serine, glutamic acid or aspartic acid is the major cleavage site, making localization of these modification sites difficult. In contrast, the bond between ADP-ribose and arginine is relatively stable. The main cleavage site is the inner bond of the guanidine which results in the formation of ADP-ribosylated carbodiimide and of ornithine in place of modified arginine. Taking this specific cleavage into account, a considerably larger number of peptides containing ADP-ribosylated arginine were identified in database searches, and their modification sites were assigned with increased confidence.

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