Identification of protein O-GlcNAcylation sites using electron transfer dissociation mass spectrometry on native peptides

Abstract

Protein O-GlcNAcylation occurs in all animals and plants and is implicated in modulation of a wide range of cytosolic and nuclear protein functions, including gene silencing, nutrient and stress sensing, phosphorylation signaling, and diseases such as diabetes and Alzheimer's. The limiting factor impeding rapid progress in deciphering the biological functions of protein O-GlcNAcylation has been the inability to easily identify exact residues of modification. We describe a robust, high-sensitivity strategy able to assign O-GlcNAcylation sites of native modified peptides using electron transfer dissociation mass spectrometry. We have studied the murine postsynaptic density pseudoorganelle and report the assignment of 58 modification sites from a single experiment--significantly increasing the number of sites known in the literature. Components of several repressor complexes, such as NCoR1, polyhomeotic-like protein3, and EMSY, are modified. In addition, 28 O-GlcNAc sites were found on the protein Bassoon, effectively matching the number of phosphorylation sites reported previously on this protein. This finding suggests that on certain proteins, O-GlcNAcylation may be as extensive and important as phosphorylation in regulating protein function. Three of the newly discovered O-GlcNAc sites on Bassoon have previously been reported as phosphorylation sites, highlighting the interplay of the modifications. Surprisingly, several peptides with GlcNAc modifications on asparagines within the N-X-S/T consensus sequence were also observed from membrane protein extracellular domains. This powerful strategy fulfills a long-standing need in the biological community by facilitating modification site identifications that will accelerate understanding of the biological significance of this elusive regulatory posttranslational modification.

Fig. 1.

ETD spectrum of an m/z 843.402 2+ precursor identifies serine 496 as a site of O-GlcNAc modification of actin-binding LIM protein 1. Serine 496 is also known to be a site of phosphorylation.

Fig. 2.

ETD spectrum of an m/z 592.605 3+ precursor identifies a peptide from Disks large-associated protein 1 with 2 O-GlcNAc modifications and a phosphorylation. The sites of O-GlcNAc modification can be identified as threonines 525 and 526. The phosphorylation is on one of the serine residues.

Fig. 3.

ETD spectrum of an m/z 594.946 3+ identifies a peptide from Gamma-aminobutyric acid type B receptor subunit 2 with a single GlcNAc residue attached to asparagine 388.

Fig. 4.

Posttranslational modifications on Bassoon. The position of the 28 O-GlcNAc modification sites on protein Bassoon (3940 aa residues) from this study (Table 1) are indicated in relation to phosphorylation sites and its structural domains (39). Phosphorylated serine/threonine residues found on PSD-associated Bassoon are indicated by long lines (36, 37); short lines indicate additional sites described in preparations of synaptsomes (42, 43, 47). O-GlcNAc sites (at 2029, 2694, and 2703), which have also been reported as phosphorylation sites on Bassoon, are indicated in red. Zn, zinc finger domain; CC, predicted coil–coil domains; CtBP, area of interaction with the CtBP (C-terminal binding protein); CAST, area of interaction with ELKS/CAST/ERC proteins; N, N terminus; C, C terminus.

Fig. 5.

Sequence surrounding sites of O-GlcNAc modification reported in this study. This representation was created using WebLogo (48).