Purpose: Demonstrate the utility of Electron Transfer Dissociation (ETD) for mapping of neighboring phosphorylated and O-glycosylation sites in peptides.

Methods: Synthetic peptides bearing different posttranslational modifications (PTMs) were analyzed by direct infusion and nESI using a Thermo Scientific LTQ XL mass spectrometer equipped with an ETD option (Thermo Scientific).

Results: Dissociation induced by ETD yields extensive peptide sequence information and in addition preserves labile PTMs. The exact sites of O-linked glycosylation and phosphorylation were unambiguously identified in this work.

Introduction

Modification of Serine/Threonine residues on peptides by phosphorylation or addition of a single O-linked N-acetylglucosamine (O-GlcNAc) plays an important role in cell regulation⁽¹⁾. In many instances, the sites of O-GlcNAcylation or phosphorylation are localized to the same, or neighboring residues on the peptide. Both modifications are extremely dynamic and labile, making them difficult to analyze by traditional mass spectrometry fragmentation techniques such as Collisionally Induced Dissociation (CID). In conventional CID experiments, modifications such as these are often lost prior to fragmentation of the peptide backbone, preventing localization of the site of modification, although the type of modification is often identified. This makes direct identification of sites of O-linked glycosylation almost impossible without employing chemical derivatization techniques.

Figure 1 (left): Schematic of the precisely controlled ion-ion reaction within the LTQ XL segmented linear ion trap for Electron Transfer Dissociation.

A new fragmentation technique, Electron Transfer Dissociation, or ETD, preserves labile PTMs, enabling both PTM identification and site localization.⁽²⁾ Figure 1 (above, left) shows the way the ions are precisely controlled within the LTQ XL linear ion trap mass spectrometer for ETD. In this study, we utilize ETD on a linear ion trap mass spectrometer for the detection and localization of neighboring phosphorylated and O-GlcNacylated sites on peptides.

Methods

Samples
Synthetic peptides bearing 1, 2, 3, 5 or 10 tandem repeats of the RNA Pol II carboxyl terminal domain (CTD), repeat sequence YSPTSPSK (CTD-peptide) or c-Myc proto-oncogene protein sequence KKFELLPTPPLSPSRR (c-Myc peptide) were synthesized by standard Fmoc chemistry. Synthetic peptides bearing O-linked GlcNAc or phosphate were prepared by incorporating Fmocprotected serine/threonine-phospho or O-GlcNAc at the desired position, as described by Greis et al.⁽³⁾ Synthetic peptides corresponding to amino acids 1-21 of human histone H3 with different PTMs were obtained from Upstate (Millipore Corporation, Lake Placid, NY).

Mass Spectrometry

Results

Dissociation induced by ETD yields extensive peptide sequence information and in addition preserves labile PTMs. The exact sites of O-linked glycosylation and phosphorylation were unambiguously identified in this work.

Conclusion

Electron transfer dissociation demonstrated excellent capabilities for mapping neighboring phosphorylated and O-GlcNacylated sites in peptides without chemical derivatization, regardless of charge state.

The exact site of O-glycosylation in the CTD–OGlcNAc peptide was determined to be Serine 5 using ETD on the 2+ species with supplemental activation. High confidence results for ETD data were obtained from the database search by using both BioWorks 3.3.1 (SEQUEST) and Mascot.

Successful ETD fragmentation and sequencing of larger peptides (~25 amino acids) can be performed by selecting optimal charge state species.

Authors
Terry Zhang¹, Tonya P. Second¹, Rosa Viner¹, T. Lakshmanan² and Gerald W. Hart^2
1Thermo Fisher Scientific, ² Johns Hopkins University

References
1 Slawson, C., Hart, G.W. “Dynamic interplay between -GlcNAc and Ophosphate: the sweet side of protein regulation”, Curr.Opin.Struct.Biol, 2003, 13:631-636.

2 Mikesh, L.M., Ueberheide, B., Chi, A., Coon, J.J., Syka, J.E.P, Shabanowitz, J., Hunt, D.F.”The utility of ETD mass spectrometry in proteomic analysis”, Biochim Biophys Acta. 2006; 1764(12):1811-1822.

3 Greis, K. D., Hayes, B.K., Comer, F.I., Kirk, M., Barnes S., Lowary, T.L., Hart, G.W. “Selective detection and site-analysis of O-GlcNAc modified glycopeptides by beta-elimination and tandem electrospray mass spectrometry”, Anal.Biochem. 1996, 234, 38-49.

4 Chou, T-Y., Hart, G.W., Dang, C.V.” c-Myc is glycosylated at Threonine 58, a known phosphorylation site and mutational hot spot in Lymphomas”, J.Biol.Chem. 1995, 270, 18961-18965.

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• BioWorks 3.3.1
• FAIMS Interface
• LTQ XL