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Electron transfer dissociation (ETD): the mass spectrometric breakthrough essential for O-GlcNAc protein site assignments-a study of the O-GlcNAcylated protein host cell factor C1.

ABSTRACT: The development of electron-based, unimolecular dissociation MS, i.e. electron capture and electron transfer dissociation (ECD and ETD, respectively), has greatly increased the speed and reliability of labile PTM site assignment. The field of intracellular O-GlcNAc (O-linked N-acetylglucosamine) signaling has especially advanced with the advent of ETD MS. Only within the last five years have proteomic-scale experiments utilizing ETD allowed the assignment of hundreds of O-GlcNAc sites within cells and subcellular structures. Our ability to identify and unambiguously assign the site of O-GlcNAc modifications using ETD is rapidly increasing our understanding of this regulatory glycosylation and its potential interaction with other PTMs. Here, we discuss the advantages of using ETD, complimented with collisional-activation MS, in a study of the extensively O-GlcNAcylated protein Host Cell Factor C1 (HCF-1). HCF-1 is a transcriptional coregulator that forms a stable complex with O-GlcNAc transferase and controls cell cycle progression. ETD, along with higher energy collisional dissociation (HCD) MS, was employed to assign the PTMs of the HCF-1 protein isolated from HEK293T cells. These include 19 sites of O-GlcNAcylation, two sites of phosphorylation, and two sites bearing dimethylarginine, and showcase the residue-specific, PTM complexity of this regulator of cell proliferation.

SUBMITTER: Myers SA

PROVIDER: S-EPMC3988289 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Electron transfer dissociation (ETD): the mass spectrometric breakthrough essential for O-GlcNAc protein site assignments-a study of the O-GlcNAcylated protein host cell factor C1.

Myers Samuel A SA Daou Salima S Affar El Bachir el B Burlingame Al A

Proteomics 20130301 6

The development of electron-based, unimolecular dissociation MS, i.e. electron capture and electron transfer dissociation (ECD and ETD, respectively), has greatly increased the speed and reliability of labile PTM site assignment. The field of intracellular O-GlcNAc (O-linked N-acetylglucosamine) signaling has especially advanced with the advent of ETD MS. Only within the last five years have proteomic-scale experiments utilizing ETD allowed the assignment of hundreds of O-GlcNAc sites within cel ...[more]

PMID: 23335398

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Project description:In the biotechnology industry, the generation of incorrectly folded recombinant proteins, either from an E.coli expression system or from an overexpressed CHO cell line (disulfide scrambling), is often a great concern as such incorrectly folded forms may not be completely removed in the final product. Thus, significant efforts have been devoted to map disulfide bonds to ensure drug quality. Similar to ECD, disulfide bond cleavages are preferred over peptide backbone fragmentation in ETD. Thus, an online LC-MS strategy combining collision-induced dissociation (CID-MS(2)), electron-transfer dissociation (ETD-MS(2)), and CID of an isolated product ion derived from ETD (MS(3)) has been used to characterize disulfide-linked peptides. Disulfide-linked peptide ions were identified by CID and ETD fragmentation, and the disulfide-dissociated (or partially dissociated) peptide ions were characterized in the subsequent MS(3) step. The online LC-MS approach is successfully demonstrated in the characterization of disulfide linkages of recombinant human growth hormone (Nutropin), a therapeutic monoclonal antibody, and tissue plasminogen activator (Activase). The characterization of disulfide-dissociated or partially dissociated peptide ions in the MS(3) step is important to assign the disulfide linkages, particularly, for intertwined disulfide bridges and the unexpected disulfide scrambling of tissue plasminogen activator. The disulfide-dissociated peptide ions are shown to be obtained either directly from the ETD fragmentation of the precursors (disulfide-linked peptide ions) or indirectly from the charge-reduced species in the ETD fragmentation of the precursors. The simultaneous observation of disulfide-linked and disulfide-dissociated peptide ions with high abundance not only provided facile interpretation with high confidence but also simplified the conventional approach for determination of disulfide linkages, which often requires two separate experiments (with and without chemical reduction). The online LC-MS with ETD methodology represents a powerful approach to aid in the characterization of the correct folding of therapeutic proteins.

Dataset Information

Electron transfer dissociation (ETD): the mass spectrometric breakthrough essential for O-GlcNAc protein site assignments-a study of the O-GlcNAcylated protein host cell factor C1.

Publications

Electron transfer dissociation (ETD): the mass spectrometric breakthrough essential for O-GlcNAc protein site assignments-a study of the O-GlcNAcylated protein host cell factor C1.

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