O-GlcNAc Site Mapping by Using a Combination of Chemoenzymatic

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Technical Note

O-GlcNAc Site Mapping by Using a Combination of Chemoenzymatic Labeling, Copper-Free Click Chemistry, Reductive Cleavage, and Electron-Transfer Dissociation Mass Spectrometry Junfeng Ma, Weihan Wang, Zengxia Li, Jeffrey Shabanowitz, Donald F. Hunt, and Gerald W Hart Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05688 • Publication Date (Web): 18 Jan 2019 Downloaded from http://pubs.acs.org on January 19, 2019

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Analytical Chemistry

Anal. Chem. (Technical Note)

O-GlcNAc Site Mapping by Using a Combination of Chemoenzymatic Labeling, Copper-Free Click Chemistry, Reductive Cleavage, and ElectronTransfer Dissociation Mass Spectrometry

Junfeng Ma†¶§*, Wei-Han Wang‡¶Փ, Zengxia Li#¶, Jeffrey Shabanowitz‡, Donald F. Hunt‡, Gerald W. Hart†€*

†Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; ‡Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA; Department of Pathology, Health Sciences Center, University of Virginia, Charlottesville, Virginia 22908, USA; #Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract As a dynamic post-translational modification, O-linked β-N-acetylglucosamine (OGlcNAc) modification (i.e., O-GlcNAcylation) of proteins regulates many biological processes involving cellular metabolism and signaling. However, O-GlcNAc site mapping, a prerequisite for site-specific functional characterization, has been a challenge since its discovery. Herein we present a novel method for O-GlcNAc enrichment and site mapping. In this method, the O-GlcNAc moiety on peptides was labeled with UDP-GalNAz followed by copper-free azide-alkyne cycloaddition with a multifunctional reagent bearing a terminal cyclooctyne, a disulfide bridge, and a biotin handle. The tagged peptides were then released from Neutravidin beads upon reductant treatment, alkylated with (3-acrylamidopropyl)trimethyl ammonium chloride, and subjected to electron-transfer dissociation mass spectrometry analysis. After being validated by using standard synthetic peptide gCTD and model protein α-crystallin, such an approach was applied to the site mapping of overexpressed TGF-β activated kinase 1/MAP3K7 binding protein 2 (TAB2), with four O-GlcNAc sites unambiguously identified. Our method provides a promising tool for the site-specific characterization of O-GlcNAcylation of important proteins.


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INTRODUCTION As a dynamically regulated post-translational modification (PTM), O-linked β-Nacetylglucosamine (O-GlcNAc) modification occurs on serine and threonine residues of proteins.1,2 After over 30 years’ endeavor, O-GlcNAcylation has been found on myriad of proteins localized in cytosol, nucleus, and mitochondria.3-5 OGlcNAc transferase (OGT) adds the O-GlcNAc moiety onto target proteins while O-GlcNAcase removes it. Protein O-GlcNAcylation plays important roles in almost all biochemical processes examined (e.g., DNA transcription, mRNA translation, protein turnover, and regulation of cellular responses to hyperglycemia and starvation/fasting, and stress protection). Besides serving as a nutrient sensor integrating cellular metabolism, protein O-GlcNAcylation is a crucial mediator in multiple signaling pathways. Although much progress has been made to study O-GlcNAc, its site mapping is still a challenging task by using standard mass spectrometric methods.6-8 On one hand, the GlcNAc moiety easily falls off from the peptide backbone in collisionally activated dissociation (CAD) mass spectrometry (MS), which is commonly employed in traditional mass spectrometers, leading to largely failed assignment of modification sites. This approach has been demonstrated successfully by combining with techniques that can convert the glycosidic bond to a CAD-stable covalent bond (e.g., by using β-elimination followed by Michael addition with dithiothreitol (BEMAD), O-GlcNAc modified peptides can be converted to the corresponding DTT-substituted ones).9-12 In contrast, the recently introduced electron-transfer dissociation (ETD) preserves the labile moiety onto peptides, enabling facile and direct assignment of modification sites.13 On the other hand, enrichment is often a perquisite step prior to the mass spectrometric analysis, due to the severe ion suppression by non-O-GlcNAc peptides.6,8 To this end, several enrichment approaches have been developed to facilitate O-GlcNAc detection by mass spectrometry. Lectins (e.g., Ricinus communis agglutinin I (RCA I) and wheat germ agglutinin (WGA)) have been used to the enrichment of O-GlcNAc



peptides.14-18 To improve the enrichment efficiency, long columns packed with WGA resin were used to retard the retention of O-GlcNAc peptides, with the retained fraction usually subjected to several rounds of further enrichment with the same column. Although suffering from relatively low affinity, O-GlcNAc specific antibody enrichment has also been used to pull down O-GlcNAc proteins.19-21 It’s still a challenging task to map the sites on putative target proteins. Recently, it has been demonstrated feasible to perform enrichment at the O-GlcNAc peptide level with several newly developed antibodies.22,23 However, the enrichment efficiency of the proposed method is still to be further validated. Moreover, the integration of chemoenzymatic labeling, streptavidin enrichment, and MS analysis has been exploited as an efficient approach for O-GlcNAc site mapping.24-27 In this approach, using a GalT mutant (GalT1), the O-GlcNAc moiety of peptides is specifically labeled with GalNAc analogs (e.g., azido-substituted form) that enable selective biotinylation (usually by using copper-catalyzed azido-alkyne mediated click chemistry). After captured by streptavidin/neutravidin beads, O-GlcNAc tagged peptides can be released and analyzed by mass spectrometry. As an alternative, cell culture-based metabolic labeling (by feeding cells with peracetylated azido/alkyne analogues) has also been coupled with click chemistry-based enrichment and mass spectrometry for O-GlcNAc site mapping.28-31 Of note, although relatively more chemical reaction steps required, approaches integrating chemoenzymatic or metabolic labeling with click chemistry provide higher affinity and specificity toward O-GlcNAc proteins/peptides. Our previous work for O-GlcNAc site mapping by using chemoenzymatic labeling, copper-catalyzed click chemistry, and UV-cleavage has been a success.25-27 Aiming to further simplify the experimental procedures and improve the enrichment robustness, we present a method for O-GlcNAc site mapping by combining chemoenzymatic labeling, copper-free click chemistry, reductive cleavage, and ETD MS analysis. Specifically, the O-GlcNAc moiety on peptides is labeled with UDP-GalNAz followed by copper-free azide-alkyne cycloaddition with a multifunctional reagent bearing a terminal cyclooctyne, a disulfide bridge, and a


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biotin handle. The tagged peptides are then released from Neutravidin beads upon reductant treatment, alkylated with (3-acrylamidopropyl)-trimethyl ammonium chloride, and subjected to ETD MS analysis. After being tested with a standard synthetic peptide gCTD and model protein α-crystallin, this method was then applied to the O-GlcNAc site mapping of overexpressed TAB2, a crucial scaffold protein involving in multiple cellular signal pathways. Our results demonstrate that such a method will be a useful addition to the repertory toolbox for the comprehensive characterization of O-GlcNAc sites of important proteins. EXPERIMENTAL PROCEDURES Materials. α-crystallin was ordered from Sigma Aldrich (St. Louis, MO). Overexpressed GFP-TAB2 was immunopurified from HEK293A cells by Chromotek GFP-Trap agarose beads (Allele Biotech, CA). The SDS-PAGE gel band corresponding to GFP-TAB2 was cut out and digested.32 gCTD (YSPTgSPS, gS = O-GlcNAcylated Ser) and other chemicals and procedures are described in detail in Supplementary Methods. Chemoenzymatic

Labeling,

Copper-Free

Click

Chemistry,

Reductive

Cleavage and Derivatization of O-GlcNAc Peptides. α-crystallin was reduced with DTT, alkylated with iodoacetamide, digested with trypsin, and desalted with a C18 column, as described previously.33 O-GlcNAc peptides from α -crystallin and TAB2 were enriched with a combination of chemoenzymatic labeling, copper-free click chemistry and reductive cleavage approach (Figure 1). Specifically, gCTD, α -crystallin digest, and TAB2 digest in 100 µl 50 mM HEPES (pH 7.9) were chemoenzymatically labeled by incubating overnight with UDP-GalNAz (25 µl; Life Technologies) and GalT1 mutant (15 µl; Life Technologies) in the presence of 22 µl MgCl2 (Life Technologies) at 4 °C, according to the manufacturer instructions and our previous papers.25-27 Calf intestine phosphatase (20 U; New England Biolabs) was then added and incubated for 3 h at room temperature. Excess UDPGalNAz was removed by desalting with a C18 spin column (Nest Group). Copperfree click chemistry was performed in 15 μl PBS and 4 l 1 mM DBCO-S-S-PEG3-



biotin (pre-dissolved in DMSO; Click Chemistry Tools LLC) for 2 h at room temperature. Neutravidin beads were added with gentle shaking for 2 h. After extensive wash, the resin was treated with 20 mM DTT for 30 min at 37 °C, with the released peptides desalted with a C18 spin column. The peptides were immediately derivatized with 3-acrylamidopropyl)trimethyl ammonium chloride (APTA, 500 mM) for 2 h in the dark.34 After desalted with a C18 spin column and dried with a Speed Vac, peptides were analyzed by MALDI-TOF or LC-MS/MS. MALDI-TOF Analysis of Tagged gCTD. A Voyager time-of-flight mass spectrometer (Perseptive Biosystems) with a 337-nm nitrogen laser was used to monitor the O-GlcNAc tagging steps. In brief, a saturated solution of 2,5dihydroxybenzoic acid (DHB) in acetonitrile/water (1:1, v/v) was used as the matrix. A 0.5-µL aliquot of sample from each step in gCTD enrichment procedure was mixed with an equal volume of the matrix solution, which was then loaded on a sample plate. Mass spectra were obtained with the mass spectrometer operated in linear mode. LC-MS/MS Analysis of Enriched O-GlcNAc Peptides. For reversed-phase liquid chromatography tandem MS (LC-MS/MS) analysis, a fraction of the enriched OGlcNAc peptides was pressure loaded onto a precolumn (360 µm o.d. x 75 µm i.d. fused silica capillary) packed with 7 cm of C18 reverse-phase resin (5 µm diameter, 120 Å pore size, Reprosil-Pur, Dr. Maisch GmbH, Germany). The precolumn was desalted by rinsing with 0.1 M acetic acid, followed by connecting to an analytical column (360 µm o.d. x 50 µm i.d.) packed with 12 cm of C18 reverse-phase resin (3 µm diameter, 120 Å pore size, Reprosil-Pur) and equipped with an electrospray emitter tip.35 The peptides were eluted at a flow rate of 60 nL/min using the following gradient: 0-60% B for 60 min, 60-100% B for 5 min, 100% B for 5 min (solvent A: 0.1 M acetic acid in water; solvent B: 70% acetonitrile and 0.1 M acetic acid in water). Full MS (MS1) analyses were acquired in high-resolution Fourier transform mass analyzers (Orbitrap XL or Orbitrap Elite) in LTQ-Orbitrap hybrid instruments (Thermo Fisher Scientific, Bremen, Germany), whereas MS2 spectra


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were acquired in linear ion traps of LTQ-Orbitrap using collisionally activated dissociation (CAD) and front end ETD.36 MS analyses were completed using a method consisting of one high resolution MS1 scan (resolving power of 60,000 at m/z 400) followed by 10 data dependent low resolution MS2 scans (5 for CAD and 5 for ETD) acquired in the LTQ ion trap. Data dependence parameters were set as follows: repeat count of 2, repeat duration of 15 s, exclusion list duration of 20 s. ETD MS2 parameters were set as follows: 35 ms reaction time, 3 m/z precursor isolation window, charge state rejection “on” for +1 and unassigned charge state precursor ions, 5105 FTMS full automated gain control target, 1104 ITMSn automated gain control target, 2105 reagent ion target with azulene as the electron transfer reagent. Mass Spectrometric Data Analysis. In-house developed software was used to generate peak lists and to remove charge reduction species.35 The Open Mass Spectrometry Search Algorithm (OMSSA, version 2.1.8) was utilized to search cand z-type fragment ions present in ETD MS/MS spectra against a database containing the target protein (either human TAB2 (NCBI accession number Q9NYJ8) or bovine -crystallin A chain (accession number P02470) and B chain (accession number P02510)). Database searches used trypsin as enzyme specificity (allowing 3 missed cleavages) and included the following variable modifications: carbamidomethyl on Cys, oxidation on Met, and tagged HexNAc (+981.4 Da) on Ser and Thr. A precursor mass tolerance of ±0.01 Da was used for MS1 data and a fragment ion mass tolerance of ±0.35 Da was used for MS2 data. All identified peptide sequences were manually validated by inspection of the accurate precursor mass and the corresponding ETD spectra.

RESULTS AND DISCUSSION Development

of

Chemoenzymatic

an

O-GlcNAc

Labeling,

Site-mapping

Copper-Free

Click

Method

Integrating

Chemistry,

Reductive

Cleavage and Derivatization. The whole procedure is shown in Figure 1a.



Chemoenzymatic labeling represents a powerful approach for the activation of ‘chemically inert’ O-GlcNAc moiety.24-27 Indeed, the O-GlcNAc moiety (rather than others) is specifically labeled with GalNAz by GalT1. The tagged GalNAz selectively reacts with alkyne-containing reagents via the copper-catalyzed azidealkyne [3+2] cycloaddition (i.e., Cu+-catalyzed click chemistry).24-27 In comparison to the traditional copper-catalyzed click chemistry, copper-free click chemistry has gained huge popularity in recent years,37,38 mainly due to the simplified reaction system (e.g., no Cu+-producing components needed) and largely quickened reaction dynamics. Moreover, the copper-free click chemistry has recently been shown applicable to the investigation of O-GlcNAc status of proteins in cells fed with peracetylated azidogalactosamine (Ac4GalNAz) for imaging.39,40 To facilitate O-GlcNAc site mapping, a multifunctional reagent (Reagent 1, DBCO-S-S-PEG3Biotin) has been adopted in the current study (Figure 1b). The cyclooctyne moiety of the reagent renders selective reactivity toward GalNAz, while the biotin handle allows capture of tagged O-GlcNAc peptides onto Neutravidin beads. More importantly, the disulfide bond of the reagent enables mild release by reducing agents (e.g., DTT). The released peptides are then derivatized by APTA (Figure 1c), an alkylating reagent which imparts a permanent positive charge, increasing the charge state of peptides (>2) which benefits ETD analysis. Validation of the Method with Standard O-GlcNAc Peptides and Proteins. gCTD, a synthetic O-GlcNAc peptide (YSPTgSPS) was used to test the feasibility of our approach, with each step monitored by MALDI-TOF (Supporting Figure S1ad). The GlcNAc peptide was tagged by GalNAz after GalT1 labeling, with a mass addition of 244.0 Da (Supporting Figure S1b). The further tagging with DBCO-SS-PEG3-biotin and subsequent release with DTT gave a mass addition of 364.3 Da (Supporting Figure S1c). The derivatization by APTA yielded an additional 169.7 Da (Supporting Figure S1d). In total, a mass increase of 981.4 Da was added onto the Ser/Thr residue of modified peptides. The enrichment approach was validated with -crystallin, a protein with extremely low abundance of O-GlcNAc peptides.41 After tryptic digestion, O-GlcNAc peptides were tagged with GalNAz


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followed by enrichment and derivatization, with the final product analyzed by both CAD and ETD MS/MS using a LTQ-Orbitrap mass spectrometer. Shown in Figure 2a is the CAD spectrum recorded on [M+4H]+4 ions at m/z 531.5 from EEKPAVgTAAPK of -crystallin. Note that the signals labeled as Fragment 1 and Fragment 2 are cleaved fragments at the glycosidic linkage in CAD, serving as diagnostic oxonium ions. Figure 2b shows the ETD spectrum recorded on [M+4H]+4 ions for the same peptide, with predicted fragment ions of type c and z shown above and below the insert while those ions observed underlined. As expected, the tag was well retained on the peptide fragments, allowing direct assignment of O-GlcNAc site based on ETD fragment ions. The tagged form of another O-GlcNAc peptide (AIPVgSREEKPSSAPSS) of -crystallin was also observed (data not shown). O-GlcNAcylation of Endogenous TAB2. TAB2, the binding protein 2 of TGF-αactivated kinase 1 (TAK1), is an upstream adaptor protein in the IL-1 signaling pathway and others in the regulation of multiple cellular processes (including inflammation).42-44 Although previous work found one O-GlcNAc site (i.e., T456) of TAB2,17,45 totally four O-GlcNAc sites (i.e., S166, S350, S354, T456) were unambiguously identified from GFP tagged TAB2 after in-gel digestion, enrichment, and ETD MS/MS analysis in this study (Table 1). Next we validated the OGlcNAcylation of TAB2 both in vitro and in vivo (Supporting Figure S2). By exploiting the in vitro OGT labeling assay, the unglycosylated recombinant TAB2 protein (purified from E. Coli) was found glycosylated by OGT in the cell-free system (Supporting Figure S2a), showing that TAB2 is a direct substrate for protein O-GlcNAcylation. Also we confirmed the physiological interaction between TAB2 and OGT in cells by using co-immunoprecipitation (Supporting Figure S2b, S2c). In addition, treating cells with TMG, a specific inhibitor to O-GlcNAcase, increased the overall O-GlcNAcylation of TAB2. While treating cells with AC4-S-GlcNAc, an inhibitor to OGT, decreased the O-GlcNAcylation, demonstrating a dynamical response of O-GlcNAcylation of TAB2 toward the pharmaceutical inhibition of OGlcNAc cycling (Supporting Figure S3). These results indicate that O-



GlcNAcylation of TAB2, which is tightly regulated, might be an important player controlling the activity of IL-1 signaling pathway and others. Ongoing site-specific mutation analysis will facilitate elucidation of functional roles of TAB2 OGlcNAcylation. CONCLUSION In summary, here we present a refined method for O-GlcNAc site mapping by combining chemoenzymatic labeling, copper-free click chemistry, and ETD MS analysis. Different from previous work,25-27,45 this procedure employs a novel reductant-cleavable biotin tag that allows for reliable and efficient release of the enriched O-GlcNAc peptides from the solid affinity support. The released peptides can be derivatized by -SH reactive reagents (e.g., APTA herein), allowing for the addition of positive charges and thus better fragment efficiency when subjected to ETD. Besides being used for the comprehensive site-mapping of individual proteins, this method is directly applicable for complex samples, with which a cancer O-GlcNAc proteomics project is undergoing. Last but not least, peptides enriched with this method can also be subjected to BEMAD for CAD/HCD-based O-GlcNAc site mapping if an ETD-based mass spectrometer is not available (as exemplified in: Zeiden Q, Ma J, Hart GW. Manuscript in preparation). It should be noted that, performing O-GlcNAc enrichment using chemoenzymatic labeling and click chemistry usually requires strong understanding of each reaction step and thus chemical expertise of investigators. However, the method herein with improved simplicity and robustness shall be facilely adopted by more biomedical labs for their research on the site-specific functional elucidation of biological functions of O-GlcNAc protein(s). Taken together, we believe this method will provide a useful tool to the repertoire for efficient site-specific characterization of important O-GlcNAcylated proteins individually and globally.

ASSOCIATED CONTENT Supporting Information Additional Information as noted in text.


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AUTHOR INFORMATION ¶ These authors contributed equally. § Current address: Department of Oncology, Georgetown University Medical Center, Washington DC 20057 Փ Current address: Janssen Research & Development, LLC, 200 Great Valley Parkway, Malvern, PA 19355 € Current address: Department of Biochemistry and Molecular Biology, Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602 Corresponding Authors *Address: Dept. Biochemistry and Molecular Biology, Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, Tel. 706-5835550. Email: [email protected] *Address: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington DC 20057, Tel. 202-6873802. E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS The authors would like to thank the Hart lab for their great help. Stimulating discussions from sister labs of the NHLBI-Johns Hopkins Cardiac Proteomics Center and NHLBI-Program of Excellence in Glycosciences Center at Johns Hopkins are also appreciated. Research reported in this publication was supported by NIH N01-HV-00240, P01HL107153, R01DK61671, R01GM116891 (to GWH), NIH GM037537 (to DFH), and National Natural Science Foundation of China (NCSF) 81772962 (to ZL). Dr. Hart receives a share of royalty received by the



university on sales of the CTD 110.6 antibody, which are managed by the Johns Hopkins University.

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Figure 1. (a) Schematic for the enrichment of O-GlcNAc peptides with the chemoenzymatic labeling, copper-free click chemistry, and reductant-cleavable DBCO-S-S-PEG3-Biotin. (b-c) The structure of Reagent 1 (i.e., DBCO-S-S-PEG3Biotin) and Reagent 2 (i.e., APTA).

Figure 2. (a) CAD and (b) ETD MS/MS spectra of the [MH4]+4 ions (m/z 531.5) of O-GlcNAc peptide EEKPAVgTAAPK (gT = O-GlcNAcylated Thr) enriched from crystallin. In the CAD spectrum (a), two +2 charged signature fragment ions at m/z 390.4 and 492.0 result from cleavage at the two sugar ketal linkages and confirm the presence of the tagged O-GlcNAc moiety. In the ETD spectrum (b), The predicted monoisotopic singly and selected average mass doubly charged c- and z-type fragment ion masses, as well as the average mass of the precursor and charge-reduced precursor ions are listed above and below the peptide sequence, respectively. Observed c- and z-type fragment ions are underlined within the peptide sequence and allowed for the O-GlcNAc site localization at Thr. Unreacted precursor and charge-reduced precursor ions are labeled as MH4, and the ions resulting from neutral losses are labeled as . The peak labeled with * correspond to the singly charged signature fragment ion, C30H39O3N7S+, dissociated from the modified tag upon ETD. Other ions with superscript * correspond to species due to loss of the signature fragment from other assigned ions.

Table 1. O-GlcNAcylated peptides and sites of endogenous TAB2. (gS, OGlcNAcylated Ser; gT, O-GlcNAcylated Thr)



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Figure 1.

(a)

(b)

(c)


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Figure 2. (a)

(b)



Table 1. O-GlcNAcylated peptides and sites of endogenous TAB2. (gS, O-GlcNAcylated Ser; gT, O-GlcNAcylated Thr) Peptide sequence

AA startstop GTSgSLSQQTPR 163173 TSgSTSSSVNSQTLNR 348362 TSgSTSSgSVNSQTLNR 348362 VVVgTQPNTK

453461

Major OMSSA Experimental Manual charge search m/z error verification state (ppm) +3 Yes 1 13 c- and zions in ETD +3 Yes 2 13 c- and zions in ETD +4 No 2 7 c- and zions, weak ETD +3 Yes 1 12 c- and zions in ETD


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Abstract Graphic (For Table of Contents Only)


O-GlcNAc Site Mapping by Using a Combination of Chemoenzymatic

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