Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets

Joshua F. Alfaro, Cheng-Xin Gong, Matthew E. Monroe, Joshua T. Aldrich, Therese R. W. Clauss, Samuel O. Purvine, Zihao Wang, David G. Camp II, Jeffrey Shabanowitz, Pamela Stanley, Gerald W. Hart, Donald F. Hunt, Feng Yang, and Richard D. Smith
PNAS May 8, 2012 109 (19) 7280-7285; https://doi.org/10.1073/pnas.1200425109

  1. Edited by Richard L. Huganir, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved March 20, 2012 (received for review January 11, 2012)

Abstract

O-linked N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification of Ser and Thr residues on cytosolic and nuclear proteins of higher eukaryotes catalyzed by O-GlcNAc transferase (OGT). O-GlcNAc has recently been found on Notch1 extracellular domain catalyzed by EGF domain-specific OGT. Aberrant O-GlcNAc modification of brain proteins has been linked to Alzheimer's disease (AD). However, understanding specific functions of O-GlcNAcylation in AD has been impeded by the difficulty in characterization of O-GlcNAc sites on proteins. In this study, we modified a chemical/enzymatic photochemical cleavage approach for enriching O-GlcNAcylated peptides in samples containing ∼100 μg of tryptic peptides from mouse cerebrocortical brain tissue. A total of 274 O-GlcNAcylated proteins were identified. Of these, 168 were not previously known to be modified by O-GlcNAc. Overall, 458 O-GlcNAc sites in 195 proteins were identified. Many of the modified residues are either known phosphorylation sites or located proximal to known phosphorylation sites. These findings support the proposed regulatory cross-talk between O-GlcNAcylation and phosphorylation. This study produced the most comprehensive O-GlcNAc proteome of mammalian brain tissue with both protein identification and O-GlcNAc site assignment. Interestingly, we observed O-β-GlcNAc on EGF-like repeats in the extracellular domains of five membrane proteins, expanding the evidence for extracellular O-GlcNAcylation by the EGF domain-specific OGT. We also report a GlcNAc-β-1,3-Fuc-α-1-O-Thr modification on the EGF-like repeat of the versican core protein, a proposed substrate of Fringe β-1,3-N-acetylglucosaminyltransferases.

A single O-linked N-acetylglucosamine (O-GlcNAc) attached to Ser and Thr residues of cytosolic and nuclear proteins is a reversible posttranslational modification (PTM) found in some bacteria, some protozoans, filamentous fungi, viruses, and all higher eukaryotes. The enzymes that catalyze the dynamic cycling of O-GlcNAc modification, O-GlcNAc transferase (OGT) and O-GlcNAc hexosaminidase (OGA), are more highly expressed in the pancreas and brain than in other tissues (1, 2). In addition, many proteins involved in neuronal communications, synaptic transmission, and synaptic plasticity are O-GlcNAcylated (3, 4), suggesting a role for this modification in brain function. O-GlcNAc cycling is highly sensitive to nutrients and stress and is regulated by nearly every metabolic pathway. Aberrant O-GlcNAc modification has been linked to Alzheimer’s disease (AD) (5, 6) in which brain glucose metabolism is impaired (7). The reduced O-GlcNAcylation in AD contributes to hyperphosphorylation of tau protein and formation of the neurofibrillary tangles characteristic of AD and related neurodegenerative disorders (8, 9).

Current understanding of the function of O-GlcNAcylation in neurodegeneration has been impeded by difficulties in identifying this modification, even using MS (10, 11). First, the substoichiometric levels of O-GlcNAc modification at given sites on protein substrates necessitate enrichment of O-GlcNAcylated proteins or peptides before sequence analysis by MS (11). Additionally, identifying specific Ser and Thr residues that are O-GlcNAcylated is difficult, because O-GlcNAc is readily lost as an oxonium ion during collision-induced dissociation (CID), a widely used fragmentation mode for peptide sequencing by MS (12). Alternative higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) MS methods have improved detection or have facilitated site-specific identification, but challenges remain (13).

Several methods to enrich O-GlcNAcylated proteins or peptides have led to the identification of a limited number of O-GlcNAcylation sites by MS. For example, lectin weak-affinity chromatography (4, 10, 14) enabled identification of up to 142 O-GlcNAcylation sites in 62 proteins from mouse embryonic stem cells (15). Immunoprecipitation, using O-GlcNAc-specific monoclonal antibodies (13, 16), identified 83 O-GlcNAcylated sites from a HEK293T cell extract (13). A recent metabolic labeling study that used alkyne-modified GlcNAc incorporated into OGT substrates and Cu(I)-catalyzed [3 + 2] azide–alkyne cycloaddition (CuAAC) to a chemically cleavable biotin–azide probe, enabled identification of 374 putative O-GlcNAc modified proteins, but yielded no information on sites of O-GlcNAc modification (17). In addition to limited coverage of O-GlcNAcylation sites, a drawback to using these approaches is that they typically require milligram quantities of protein (4, 10, 13, 14, 16) or are limited to cultured cells (17), which makes them generally ill-suited for clinically derived tissue samples often available in small amounts.

The chemical/enzymatic photochemical cleavage (CEPC) method (18, 19) improves upon the highly selective chemical/enzymatic approaches for O-GlcNAcylated proteins/peptides enrichment (20, 21) and increases analytical sensitivity by introducing a photochemical cleavable-biotin probe that allows efficient release of enriched peptides from the avidin affinity column. In this method (Fig. 1A), O-GlcNAcylated peptides are first enzymatically labeled with azidogalactosamine (GalNAz). The free azido group in the GalNAz is then conjugated to the alkyne group in a photocleavable biotin probe (PC-PEG-biotin-alkyne) through CuAAC. The biotinylated peptides are enriched using avidin affinity chromatography, and subsequently released through photochemical cleavage. O-GlcNAc-modified peptides enriched by this method are tagged with a basic aminomethyltriazolacetylgalactosamine (AMT-GalNAc) that facilitates ETD identification and site localization of O-GlcNAc–modified peptides (10, 18, 19, 21). This approach enabled identification of 141 O-GlcNAcylation sites in 64 proteins from <15 μg of spindle and midbody proteins that were enriched from HeLa cells (18, 19). However, to date, this method has not been used for complex tissues or global proteomic analyses. In addition, there remain challenges in reducing contaminants from the CuAAC reaction, which are detrimental to liquid chromatography (LC)-MS measurements.

Fig. 1.
Fig. 1.

Overview of the CEPC method (A) and experimental work flow (B) used in this study for the identification of O-GlcNAc proteins and modification sites. (A) Steps modified in the enrichment strategy are depicted in dashed boxes. PNGase F and calf intestine phosphatase (CIP) are added to the reaction mixture to ensure selective and complete derivatization with GalNAz (18). During the CuAAC reaction, Cu(I) is stabilized with TBTA. (B) The original and modified wash methods for the biotin-avidin enrichment step were compared using peptides from one WT mouse and one 3xTg-AD mouse (female, 1 y old). The number of O-GlcNAc sites and proteins identified from LC-MS/MS analysis of individual samples is depicted at the bottom of the figure.

In the study reported herein, we modified the CEPC protocol and used it to enrich O-GlcNAcylated peptides from tissue samples obtained from the brain cortex of one WT mouse and one 3xTg-AD model (22) mouse. CEPC enrichment of ∼100 μg mouse brain cortex peptides from ∼3 mg WT tissue resulted in the identification of 249 O-GlcNAcylated proteins and 358 O-GlcNAc site assignments from a single LC-MS/MS analysis, using an alternating CID/ETD approach. Enrichment of six 100-μg samples (three each for WT and 3xTg-AD mice) enabled identification of 274 O-GlcNAcylated proteins and 458 O-GlcNAc sites, many of which were previously unknown. Important findings include data that support extensive cross-talk between O-GlcNAcylated and phosphorylated proteins involved in cerebrocortical processes, and unexpected sites of extracellular O-GlcNAc modification. Specifically, we observed O-GlcNAc on a secreted cytokine AIMP1, on EGF-like repeats in the extracellular domain of five other proteins, and on GlcNAc-β-1,3-Fuc-O-EGF of versican that reflects the action of a Fringe β-1,3-GlcNAc transferase.

Results and Discussion

Modified CEPC Approach for O-GlcNAc Peptide Enrichment.

To maximize sensitivity and selectivity in the CEPC O-GlcNAc peptide enrichment method (Fig. 1A), we modified the enzymatic reactions (SI Methods and SI Results and Discussion) to ensure high yields in transfer of GalNAz to O-GlcNAcylated peptides by the mutant β-1,4-galactosyltransferase (GalT1Y289L), and effective removal of N-glycans that may contain terminal GlcNAc by peptide:N-glycosidase F (PNGase F). We also incorporated additional aqueous washes and an organic wash [70% (vol/vol) methanol in water] during avidin affinity enrichment (dashed boxes in Fig. 1A; workflow in Fig. 1B). The added washes increased the number of O-GlcNAc protein identifications by ∼16% (Fig. 1B) and reduced the large amount of hydrophobic Tris[(1-benzyl-1H-1,2,3-triazol-4-yl) methyl]amine (TBTA) that remained from the CuAAC reaction (Fig. S1).

Evaluation of MS Fragmentation Methods for Identifying CEPC-Enriched Tagged O-GlcNAcylated Peptides.

In a parallel effort we evaluated the capability of individual MS/MS methods (HCD, CID, and ETD) for identifying tagged O-GlcNAc (AMT-GalNAz-GlcNAc modified) in 100 μg WT mouse cerebrocortical peptides enriched using CEPC. Of the three fragmentation methods, only ETD provided information regarding the location of the modification sites. The basic tag (AMT-GalNAz) increased peptide fragment efficiency of both ETD and HCD compared with that for untagged, native O-GlcNAc peptides (13). Overall, ETD generated the greatest number of O-GlcNAcylated peptides, followed by HCD and CID (Fig. S2). In addition, the AMT-GalNAz-GlcNAc modification produced three major diagnostic oxonium fragment ions for the intact sugars (204.09, 300.13, and 503.21 m/z) compared with one (204.09 for HexNAc) from untagged, native O-GlcNAc peptides. The AMT-GalNAz tag allowed CID to detect at least two of the three major oxonium ions from intact sugars (Fig. S3) despite the one-third low molecular-weight cutoff rule (23). Compared with CID, HCD consistently detected the three major diagnostic ions with higher mass accuracy and generally higher intensity (Fig. S4). The results of this evaluation indicate that combining tandem MS methods to obtain both site-specific (ETD) and diagnostic (HCD or CID) information can increase the confidence of O-GlcNAcylated peptide identification and site localization, consistent with previous studies (10, 13). In addition, alternating CID-ETD analyses have been advocated as a superior method for identification of labile phosphopeptides (24). Our study further demonstrates the need to combine the orthogonal methods of fragmentation for assigning labile PTMs.

Known and Previously Unreported O-GlcNAcylated Proteins and Sites.

In this study, we analyzed cerebrocortical tissue from one 3xTg-AD mouse and one WT mouse (both female, 1 y old). The 3xTg-AD mouse, which overexpresses mutated human amyloid-β precursor protein, tau, and preselinin-1, is a commonly used model of AD (22). Three samples from each mouse were analyzed (Fig. 1B).

We identified 1,575 unique O-GlcNAcylated peptides (Table S1) corresponding to 555 unique peptide sequences (Dataset S1) and 274 O-GlcNAcylated unique proteins (Dataset S2). Overall, 458 unambiguous O-GlcNAc sites (≤5% false localization rate; Dataset S3) were assigned to 195 proteins, tripling the number of O-GlcNAc sites reported in any single study (15, 19). Analysis of the sequence around these O-GlcNAc sites revealed several statistically significant (P ≤ 1E-6) motifs for O-GlcNAcylation (Fig. S5), among which P-X-gT-X-A and P-V-gS are enriched 14- and 23-fold, respectively, compared with a dynamic statistical background of the entire mouse protein database. Some of the motif sequences agree with the previously reported OGT preferred sequence P/V-P/V-V-gS/T-S/T (10, 19). Ontology analysis of the 274 O-GlcNAcylated proteins supports the involvement of O-GlcNAcylation in numerous cellular functions in the brain, such as cytoskeleton organization, neurogenesis, synaptic transmission, learning, and memory (Dataset S4).

We localized known and previously unreported O-GlcNAc sites to specific residues in many of the 106 previously reported O-GlcNAcylated proteins, which include numerous neuronal proteins implicated in AD, such as synapsin I and II, synaptopodin, α-synuclein, several microtubule-associated proteins (Dataset S5), and proteins involved in neurogenesis.

Because neurogenesis is impaired in AD (25), the O-GlcNAcylation status of these identified proteins (Dataset S4) may play a role in AD. Comparison of the O-GlcNAcylated peptides obtained from 3xTg-AD vs. WT cerebrocortical tissue showed that fewer (179 vs. 259) O-GlcNAcylated proteins were identified from the 3xTg-AD mouse (Fig. 1 and Fig. S6). These results are consistent with previous observations of down-regulation of brain protein O-GlcNAcylation in AD due to metabolic impairment (8). In addition, some of the O-GlcNAcylated proteins present only in the 3xTg-AD tissue may represent aberrantly O-GlcNAcylated proteins (Dataset S2). Future quantitative proteomic studies that analyze brain tissue from several WT and 3xTg-AD mice will help to suggest roles for O-GlcNAcylation in AD.

We also identified 168 O-GlcNAcylated proteins previously unknown to be O-GlcNAc-modified, including 114 proteins (Table 1) that have confidently localized O-GlcNAc sites and 54 proteins (Table 2) that have defined tryptic peptide regions for the O-GlcNAc modification, but their modification residues are ambiguous. Many of these O-GlcNAc-proteins are cytoskeleton proteins, signaling proteins, or proteins involved in the regulation of transcription—all classes of proteins known to be O-GlcNAcylated (26, 27). We observed three O-GlcNAcylation sites (T1276, T1796, and T1797) on neurobeachin (Nbea), a protein implicated in membrane protein traffic and autism, and required for the formation and functioning of central synapses (28). This protein is a known phosphoprotein and binds to protein kinase A; however, its O-GlcNAcylation status was previously unknown (29). Literature provides examples of cross-talk between O-GlcNAcylation and phosphorylation (19, 26, 30), as well as examples of O-GlcNAc regulating ubiquitination through E1 ubiquitin-activating enzyme (26, 31). We observed many O-GlcNAcylated proteins previously shown to be involved in the cycling of either phosphorylation or ubiquitination, which provides indirect evidence of cross-talk between these modifications. These proteins were not previously reported to be O-GlcNAc-modified and include 13 kinases (underlined entries in Tables 1 and 2), one putative phosphatase (Dnajc6), three proteins involved in phosphatase regulation (Phactr1, Mprip, and Ppp1r12b), three E3 ubiquitin-protein ligases (SH3RF1, HECTD1, and KCMF1), and one deubiquitinating protein (VCPIP1; Tables 1 and 2).

View this table:
Table 1.

Identified O-GlcNAcylated proteins previously unknown to be O-GlcNAc-modified with confidently localized sites (≤5% false localization rate)

View this table:
Table 2.

Identified O-GlcNAcylated proteins previously unknown to be O-GlcNAc-modified with modification site localized to a specific tryptic peptide region

All previously identified O-GlcNAcylated proteins are also known to be phosphorylated (26), which supports cross-talk between the two modifications. There are 268 (>98%) of the 274 O-GlcNAc-modified proteins identified in this study also known as phosphoproteins (32) (Dataset S2). We found that ∼24% of the identified O-GlcNAc sites have either reciprocal or proximal (±10 aa residues) phosphorylation sites (Dataset S3), which again suggests possible cross-talk between phosphorylation and O-GlcNAcylation in cerebrocortical processes. Some of these potential cross-talk cases that may regulate protein interactions (e.g., EMSY with BRCA2 and HCFC1 with SIN3A) were also observed in HeLa cells (19). Our findings include two mapped O-GlcNAc sites (S539 and T577) on TNIK, a serine/threonine kinase previously unknown to be O-GlcNAcylated. These O-GlcNAc sites (S539 and T577) with proximal known phosphorylation sites (S541, S545, and T552) (33) on TNIK are all located within its predicted (34) binding region to NEDD4-1, an E3 ubiquitin ligase. O-GlcNAcylation of human NEDD4-1 was recently reported (17), but with no site localization.

We identified an O-GlcNAc site (T375) that is located within the experimentally determined TNIK binding region of NEDD4-1 (35). A known (S381) and a predicted (S385) (34) proximal phosphorylation site on NEDD4-1 also occur within this binding domain. NEDD4-1, TNIK, and Rap2A are known to form a complex that regulates NEDD4-1–mediated Rap2A ubiquitination (35). Together, our findings suggest that cross-talk between phosphorylation and O-GlcNAcylation may be involved in the NEDD4-1/TNIK/Rap2A (35) signaling pathway that regulates neurite growth. We also suggest that this cross-talk may extend to ubiquitination, given that TNIK is required in this complex to enable ubiquitination of Rap2A (35) and that its interaction with NEDD4-1 may be regulated by O-GlcNAcylation/phosphorylation.

O-GlcNAcylation on a Secreted Protein and on the Extracellular Domains of Membrane Proteins.

Fig. 2 shows subcellular localization of the 274 identified O-GlcNAcylated proteins. Previous studies have demonstrated that O-GlcNAc modifies numerous nuclear and cytoplasmic proteins (26). Unexpectedly, we also identified O-GlcNAc modification on the extracellular EGF domain of five membrane proteins (Table 3), and on one secreted cytokine. Fig. S3 shows modification and site assignments derived from MS/MS spectra for the tryptic peptide CACLAGYTGQR from the EGF domain of Pamr1. To our knowledge, there are only two previous reports of extracellular O-GlcNAcylation, i.e., the extracellular domain of NOTCH and Dumpy in Drosophila (36, 37).

Fig. 2.
Fig. 2.

Cellular component gene ontology annotation of identified O-GlcNAcylated proteins.

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Table 3.

O-GlcNAc modification sites on a secreted protein and on the extracellular domains of membrane proteins

The O-GlcNAc transferase that attaches O-β-GlcNAc to NOTCH is a distinct enzyme that is genetically unrelated to OGT (36). It resides in the endoplasmic reticulum (ER) within the secretory pathway and is termed EGF domain-specific O-GlcNAc transferase (EOGT) (36, 37). EOGT is conserved from Drosophila to mammals (37, 38). There is no evidence that intracellular forms of OGT or O-GlcNAcase occur in the extracellular or luminal spaces. Four O-GlcNAcylated peptides from these proteins were located within an EGF-like domain, sharing a similar motif sequence CXXGXS/TGXXC to the reported extracellular O-GlcNAcylation on NOTCH and Dumpy in Drosophila and Notch1 in mammals (36⇓–38). We also identified an O-GlcNAc site on the EGF-like domain of NOTCH2 protein of the Notch signaling pathway, which we infer is on the T residue in the YSCVCSPGFTGQR sequence, consistent with the CXXGXS/TGXXC motif (Table 3). Our findings suggest that this EOGT (36) has additional substrates. In fact, we determined that 91 mouse proteins, 104 human proteins, and 18 Drosophila proteins contain the CXXGXS/TGXXC motif (Datasets S6, S7, and S8). These proteins are involved in the Notch signaling pathway, extracellular matrix (ECM)-receptor interactions, and other signaling pathways. Among the proteins in both the human and mouse proteome that contain this motif, ∼30% are localized within the ECM. The importance of this modification within the ECM has been demonstrated in Drosophila, where loss of EOGT causes defects in the apical ECM (37). The proteins containing the CXXGXS/TGXXC motif appear conserved across species, with 83 proteins conserved between human and mouse, and 15 of 18 Drosophila proteins having orthologs in both human and mouse.

We mapped an O-GlcNAc site (T91) on AIMP1 that is known to regulate angiogenesis, inflammation, and wound healing (39, 40), and report O-GlcNAc modification on a secreted cytokine. A recent study (41) showed AIMP1 plays a glucagon-like role in glucose homeostasis and its secretion is induced by TNFα or heat shock (40, 42). Motif analysis indicates two proximal phosphorylation sites (S99 and S107) of T91 are potential targets of the intracellular kinase MAPK. In addition, because AIMP1 is present in multiple subcellular locations besides the extracellular space (34) and lacks an EGF repeat, the AIMP1 form we detected from mouse cerebrocortical tissue is likely cytosolic, and the T91 is the substrate of OGT instead of EOGT. The O-GlcNAc site (T91), together with its reported five proximal phosphorylation sites (S99, S101, T105, S107, and S138), are all located within the HSP90B1 interaction region of AIMP1. The potential cross-talk between these sites may be relevant to AIMP1 and HSP90B1 interactions that regulate KDELR1-mediated retention of HSP90B1/gp96 in the endoplasmic reticulum (34).

Interestingly, we also identified a GlcNAc-β-1,3-Fuc-α-1-O-Thr site (T3103) in the EGF-like domain 2 (EGF2) of versican core protein (Fig. S3) within the tryptic peptide sequence NGAT#CVDGFNTFR (# indicates the modification). The addition of O-fucose to EGF repeats is catalyzed by Pofut1 (43), and elongation of the monosaccharide is initiated by Fringe, an O-fucose–specific β-1,3-N-acetylglucosaminyltransferase (44, 45) to form the disaccharide modification we detected. GlcNAc-β-1,3-Fuc may be an intermediate species before subsequent elongation by galactosyltransferase and sialyltransferase to form a tetrasaccharide (46). The Thr modified by O-fucose is in a predicted consensus site (C2XXXXS/TC3) between C2 and C3 of EGF repeats for O-fucosylation (47, 48) and within EGF2 (C2RNGATC3) of versican.

Summary.

To our knowledge, the present study has produced the most comprehensive O-GlcNAc proteome to date in terms of both protein identifications (274) and O-GlcNAc site assignments (458) for mouse brain tissue, and used much smaller samples (∼100 μg tissue peptide per enrichment) than in previous studies (10, 15, 20). Our studies suggest roles for extensive cross-talk between O-GlcNAc and other posttranslational modifications in not only the regulation of normal neuronal functions, but also in the etiology of neurodegeneration. We demonstrate the suitability of the CEPC method for global proteomic analysis of O-GlcNAcylated peptides, and the potential for rapidly expanding the O-GlcNAc proteome in brain and other tissues. This approach will enable high-resolution spatial mapping of O-GlcNAcylation patterns in brain samples from laser-capture microdissection to gain insights into roles for O-GlcNAcylation in neurodegenerative disease.

Methods

Sample Preparation.

Mouse cerebrocortical tissue was homogenized in a solution containing 6 M guanidine HCl, 10 mM DTT, 50 mM ammonium bicarbonate [NH4HCO3 (pH 8.1)] with 1% (vol/vol) phosphatase inhibitor mixture 2% (vol/vol) (Sigma), and 100 nM PUGNAc. Details of protein digestion are described in SI Methods.

O-GlcNAc Enrichment.

Enrichment was performed as previously described (18) except where specified otherwise in SI Methods.

LC-MS/MS Analysis.

Samples were analyzed using a LTQ Orbitrap Velos MS (Thermo Scientific) coupled to an automated dual-column metal-free nanoLC platform (49). Details of the separation and mass spectrometer parameters are described in SI Methods.

Data Analysis.

We used two database search engines, SEQUEST and Protein Prospector, to obtain more comprehensive peptide identifications. Data were searched against a decoy protein database. Fully tryptic peptide identifications were filtered in a way that no reversed hits were left, with an estimated zero false discovery rate (Datasets S9 and S10) (50). All identifications were within ±5 ppm measured mass accuracy and required observation of oxonium ion fragments (204.0872, 300.1308, and 503.2101 m/z for HCD; 300.1308 and 503.2101 m/z for CID) in their corresponding HCD or CID scans with a mass tolerance of 0.0025 Da for HCD and 0.3 Da for CID, which were extracted using our updated MASIC software (51). Ascore (52) and SLIP score (built into the Protein Prospector search engine) (53) were used to estimate the confidence of O-GlcNAc modification site assignment for SEQUEST and Protein Prospector search results, respectively. See SI Methods and SI Results and Discussion for more details. All of the peptides identified from NEDD4-1, TNIK, AIMP1, and EGF-like repeats in the extracellular domain of six proteins were manually confirmed by authors J.F.A., F.Y., and J.S. The six N-linked GlcNAc peptides (Dataset S11) were also manually confirmed.

Bioinformatics.

Gene ontology annotation, cellular component, and biological process was performed using the Software Tool for Researching Annotations of Proteins, or STRAP (54). The pathway analysis was performed using DAVID Bioinformatics Resources 6.7 as previously described (55). Briefly, all identified O-GlcNAc modified proteins were queried against the mouse proteome as a background. The statistical enrichment was calculated for KEGG pathways identified from a protein list obtained during this study, and pathways with P ≤ 0.05 were reported as significant. Prealigned sequence was generated with six residues on either side of all of the unambiguous O-GlcNAc sites. The sequence was subject to motif analysis online by motif-x (http://motif-x.med.harvard.edu/) (56), and the motifs were built through comparison with a dynamic statistical background of mouse protein database. The occurrences threshold was set at 20, and the significance (P value) was 1E-6.

Acknowledgments

We thank Dr. Joshua Adkins at Pacific Northwest National Laboratory (PNNL) for helpful suggestions regarding the manuscript, Robert Chalkley (University of California, San Francisco) for help with using Protein Prospector, and Ronald J. Moore for discussions regarding MS analysis. This work was funded by PNNL Laboratory Directed Research Development funding (to F.Y.); three National Institutes of Health (NIH) grants (to R.D.S.), National Center for Research Resources Grant 5P41RR018522-10, National Institute of General Medical Sciences Grants 8 P41 GM103493-10 and AG027429; NIH Grant GM 037537 (to D.F.H); NIH Grants N01-HV-00240, R01 CA42486, and P01HL107153 (to G.W.H); AG027429 and TW008123 (to C.-X.G.); and NIH National Cancer Institute Grant R01 36434 (to P.S.). Samples were analyzed using capabilities developed under the support of the NIH National Center for Research Resources Grant RR018522 and the US Department of Energy Biological and Environmental Research (DOE/BER). Work was performed in the Environmental Molecular Science Laboratory, a DOE/BER national scientific user facility at PNNL in Richland, WA. PNNL is operated for the DOE by Battelle under Contract DE-AC05-76RLO-1830.

Footnotes

  • ↵1To whom correspondence may be addressed. E-mail: feng.yang{at}pnnl.gov or rds{at}pnnl.gov.

  • Author contributions: J.F.A. and F.Y. designed research; J.F.A., T.R.W.C., and F.Y. performed research; J.F.A., M.E.M., J.T.A., S.O.P., Z.W., F.Y., and R.D.S. contributed new reagents/analytic tools; J.F.A., J.S., P.S., G.W.H., D.F.H., and F.Y. analyzed data; and J.F.A., C.-X.G., D.G.C., P.S., G.W.H., D.F.H., F.Y., and R.D.S. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1200425109/-/DCSupplemental.

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MS identifies many brain O-GlcNAcylated proteins
Joshua F. Alfaro, Cheng-Xin Gong, Matthew E. Monroe, Joshua T. Aldrich, Therese R. W. Clauss, Samuel O. Purvine, Zihao Wang, David G. Camp, Jeffrey Shabanowitz, Pamela Stanley, Gerald W. Hart, Donald F. Hunt, Feng Yang, Richard D. Smith
Proceedings of the National Academy of Sciences May 2012, 109 (19) 7280-7285; DOI: 10.1073/pnas.1200425109
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