Threonine Posttranslational

ARTICLE pubs.acs.org/ac

A Versatile Method for Analysis of Serine/Threonine Posttranslational Modifications by β-Elimination in the Presence of Pyrazolone Analogues Jun-ichi Furukawa, Naoki Fujitani, Kayo Araki, Yasuhiro Takegawa, Kota Kodama, and Yasuro Shinohara* Laboratory of Medical and Functional Glycomics, Graduate School of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan

bS Supporting Information ABSTRACT: Post-translational modifications (PTMs) of serine and threonine occur by diverse mechanisms, including phosphorylation, sulfation, and various types of sugar chain modifications, making characterization of the resulting structures very labor-intensive. Moreover, to fully understand the biological functions of PTMs, both the sites of modification and the modified structures must be analyzed. The present work describes a novel, versatile strategy in which the released O-glycan and the formerly glycosylated/phosphorylated peptide are labeled and thus amenable to further study. In this approach, glycopeptides/phosphopeptides are subjected to β-elimination in the presence of pyrazolone derivatives (BEP), which in the same reaction labels the formerly glycosylated/phosphorylated peptide. The reaction is essentially a β-elimination/Michael addition in which a carboncarbon bond-forming Michael donor rather than a heteroatomic Michael donor is used. The O-glycans released upon BEP are recovered as bis-pyrazolone derivatives, without any detectable side reaction (peeling). Using this technique, the O-glycan profiles of model mucin-type glycoproteins were successfully analyzed. The BEP strategy discriminates between phosphorylated and GlcNAcylated peptides, since cleaved GlcNAc is detectable. In addition, both the released O-glycan and the formerly glycosylated peptide can be selectively labeled by different reagents via a β-elimination reaction performed in the presence of pyrazolone and the thiol Michael donor.

P

osttranslational modifications (PTMs) on serine (Ser) and threonine (Thr) are essential for proteins to perform their intended functions. While analyses of these modifications are challenging, they are indispensable for the detailed elucidation of protein function. Ser and Thr residues can be modified in a number of ways, including phosphorylation, sulfation, and various types of sugar chain modifications,13 such as the O-GalNAc type typical of mucin and the O-GlcNAc, O-Fuc, O-Man, and O-Xyl types. These modifications not only activate proteins but also serve as markers indicating binding to other proteins. Phosphorylation, sulfation, and O-GlcNAcylation involve a single phosphoric, sulfate, and O-GlcNAc residue, respectively, while other modifications subsequently form more complicated oligosaccharide structures through the actions of various glycosyltransferases.1 Protein phosphorylation sites, particularly Ser, Thr, and tyrosine, have been intensively studied by use of phosphopeptide enrichment and separation prior to the analysis, which significantly improves the characterization of phosphorylation sites by mass spectrometry.4 Other methods include affinity-based approaches, such as immobilized metal affinity chromatography (IMAC)5 and metal oxide affinity chromatography (MOAC),6 and the chemical introduction of affinity tags at sites of r 2011 American Chemical Society

phosphorylation. Among the latter, the β-elimination/Michael addition (BEMA) takes advantage of the ease of β-elimination of phosphorylated Ser and Thr residues at basic pH and the ability to subject the resulting dehydroalanine/methyldehydroalanine products to Michael addition with a desired tag (typically thiol Michael donors) for affinity purification or highly sensitive detection.710 For instance, introduced thiol residues, upon reacting with dithiothreitol (DTT) at the formerly phosphorylated site, can functionally serve as the ligand for affinity purification via disulfide exchange with an activated thiol resin.9 However, while BEMA is a well-established and reliable method for the analysis of phosphorylated peptides, it has the ability to act on a variety of O-glycosylated peptides, such that selectivity becomes an issue if several Ser and Thr PTMs are present.11 Characterization of PTMs in which O-glycans are attached to Ser and Thr has remained particularly challenging because no analogous endoglycosidase is known, and alternative chemical protocols are either incomplete, lack specificity, or degrade one or both products (i.e., the glycan and/or the peptide/protein).12 Received: July 30, 2011 Accepted: October 13, 2011 Published: October 13, 2011 9060

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Analytical Chemistry

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Figure 1. Chemical pathways of glycopeptide upon β-elimination under various conditions: (a) released glycan hemiacetal protected from peeling by in situ reduction to the alditol with NaBH4 (Carlson’s method); (b) released glycan degraded by subsequent β-elimination (peeling reaction); and (c) released glycan hemiacetal protected from peeling by in situ derivatization with pyrazolone analogue, in a reaction accompanied by labeling of the formerly glycosylated site by pyrazolone analogue (β-elimination in the presence of pyrazolone, BEP).

Furthermore, since O-glycosylated proteins exhibit comparable biological functions and are equally diverse,13 their structural characterization has been problematic. In β-elimination reactions that employ a strong base, such as sodium hydroxide, which is typically used for O-glycan release from proteins/peptides, the glycan residues are degraded (peeling). This problem was resolved by in situ reduction of the alditols, a successful strategy that has been frequently applied since it was first reported by Carlson14 in 1968 (Figure 1a,b). However, two major limitations of Carlson’s method are that the proteins and peptides are unstable and reduction causes a loss of the reducing end of the carbohydrate. The latter precludes additional downstream glycomic analysis, such as derivatization suitable for enrichment, chromatographic, and mass spectrometric analyses. While Carlson’s method has been modified, for instance, through the use of milder bases such as ammonia15 and alkyl amines12 instead of a strong base, recent studies clearly demonstrated that considerable peeling is unavoidable if the reduction step is omitted.16 Yu et al.17 reported that even the most successful Carlson degradation results in 5% peeling. The aim of this study was to establish a novel analytical technique applicable to various PTMs of Ser and Thr. Accordingly, we focused on carbohydrate derivatization chemistry with 1-phenyl-3-methyl-5-pyrazolone (PMP), as described by Honda et al.18 Unlike most other glycan derivatization techniques, PMP derivatization proceeds under alkaline conditions. The optimal NaOH concentration is 200400 mM, which is compatible with the conditions of β-elimination employing a strong base. PMP derivatization of the released O-glycans proceeds faster than peeling degradation, thereby allowing the simultaneous release and labeling of O-glycans. In addition, since pyrazolone is a carboncarbon bond-forming Michael donor (referred to as the carbon-Michael donor),19 peptides formerly modified with phosphoric, sulfate, and various O-glycans are concomitantly labeled with the same reagent. We therefore describe herein the first successful strategy to label formerly glycosylated Oglycans and proteins/peptides upon β-elimination. The reaction mechanism is discussed together with its application to phosphopeptides, O-GlcNAcylated peptides, and mucin-type glycoproteins.

’ EXPERIMENTAL SECTION Materials and Reagents. Reagents PMP, 1,3-dimethyl-5pyrazolone (DMP), and 3-methyl-1-p-tolyl-5-pyrazolone (MTP)

were purchased from Tokyo Chemical Industry (Tokyo, Japan). Porcine stomach mucin (PSM), bovine fetuin, and bovine submaxillary mucin (BSM) were purchased from Sigma Aldrich, Inc. (St. Louis, MO). DTT was purchased from Wako Pure Chemicals (Osaka, Japan), and Iatrobeads from Mitsubishi Chemical Medience (Tokyo, Japan). O-Posphorylated peptides H-Phe-Gln-pSerGlu-Glu-Gln-Glu-Gln-Thr-Glu-Asp-Glu-Leu-Gln-Asp-Lys-OH, and H-Val-Asn-Gln-Ile-Gly-pThr-Leu-Ser-Glu-Ser-Ile-Lys-OH were purchased from Anaspec (San Jose, CA), and Waters (Milford, MA), respectively. Other solvents and reagents were of the highest grade commercially available. Preparation of Model Glycopeptides. A model glycopeptide and a peptide with a sequence of PSTPPTPSP((Galβ1 3GalNAc)STPPTPSPSK, in which N-terminal proline and C-terminal lysine are acetylated and amidated, respectively, were synthesized by solid-phase reactions with NovaPEG Rink Amide resin (Novabiochem), with microwave-assisted irradiation (Green Motif II microwave reactor, Tokyo Denshi Co. Ltd., Tokyo, Japan) at 2450 MHz and a temperature of 50 °C.20 Fluorenylmethyloxycarbonyl (Fmoc) amino acids (5 equiv) were assembled onto the resin by treating them with 2-(1H-benzotriazol-1yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU; 5.0 equiv), hydroxybenzotriazole (HOBt; 5.0 equiv), and N,Ndiisopropylethylamine (DIEA; 10.0 equiv) in N,N-dimethylformamide (DMF) (370 μL) for 5 min under microwave irradiation. The Fmoc group was deprotected with 20% piperidine in DMF (1 mL) for 3 min under microwave irradiation. The resulting (glyco)peptidyl-resins were cleaved by treating them with a solution consisting of trifluoroacetic acid (TFA)/ triisopropylsilane/H2O (95/2.5/2.5 v/v/v) for 2 h at room temperature. The resulting solution was evaporated and subsequently precipitated with t-buthylmethyl ether (5 mL) on ice. The precipitations were collected by centrifugation (5000 rpm for 10 min) and then purified by reversed-phase (RP) HPLC on an Inertsil ODS-3 column (10 250 mm) (GL Science Inc.). Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and NMR measurements were used to characterize the glycopeptides of interest. MALDI-TOF MS analysis was performed on an Ultraflex II TOF/TOF mass spectrometer (Bruker Daltonics, Germany); NMR spectra were collected at 27 °C on a Bruker Avance 600 spectrometer at 600.13, corresponding to the proton frequency (Bruker Biospin, Germany). MS data and NMR spectra were analyzed with the FlexAnalysis 3.0 software package (Bruker Daltonics, Germany) and NMRPipe software,21 respectively. 9061

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acid (10 mg/mL in 30% acetonitrile) at a 1:1 dilution, and an aliquot (1 μL) was deposited on a stainless steel target plate. MALDI-TOF data were obtained on an Ultraflex II time-of-flight mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a LIFT-TOF/TOF facility controlled by FlexControl 3.0 software according to the general procedure reported previously.23 All spectra were obtained in reflectron mode with an acceleration voltage of 25 kV and a reflector voltage of 26.3 kV in the positive-ion mode. These spectra were the sum of 1000 laser shots. All peaks were selected by FlexAnalysis 3.0 and the sophisticated numerical annotation procedure (SNAP) algorithm, which fits isotopic patterns to the corresponding experimental data. The algorithm provides monoisotopic mass, intensity, area under the envelope of the isotopic cluster, and resolution of the peaks in the cluster. O-linked-type oligosaccharide structures were estimated from inputs of peak masses into the GlycoMod Tool (Swiss Institute of Bioinformatics) and into GlycoSuite (Proteome Systems Inc., Woburn, MA). Figure 2. β-Elimination in the presence of pyrazolone derivatives (BEP). (a) Effect of temperature. The model glycopeptide was subjected to BEP (in 150 mM NaOH/250 mM methanolic PMP) for 16 h at three temperatures: 65 °C (upper panel), 75 °C (middle panel), and 85 °C (lower panel). (b) Comparison of different pyrazolone derivatives. The model glycopeptide was subjected to BEP with three different pyrazolone analogues: DMP (upper panel), PMP (middle panel), and MTP (lower panel). Asterisks denote artifactual peaks resulting from impurities present in the glycopeptide used. The intense signal observed at m/z 974.8 is a peptide with a sequence of AcTPPTPSPSKNH2, which is a byproduct when synthesizing the intended glycopeptide and is present already in the reaction mixture before the BEP reaction. Note also that this peptide cannot be generated from the glycopeptide during BEP because of the N-terminal acetyl modification.

β-Elimination of Glycopeptides, Phosphopeptides, and Mucin-type Glycoprotein in the Presence of Pyrazolone Analogues (BEP Procedure). The model glycopeptide or

glycoprotein (10 μL, 0.140 μg) was treated with 20 μL each of sodium hydroxide (0.30.6 M) and a 0.5 M methanolic solution of pyrazolone, followed by heating at various temperatures (5095 °C) for reaction times of 216 h. The solution was neutralized with 1.0 M hydrochloric acid and the resultant mixture was then subjected to purification on an Iatrobeads silica gel column.22 Briefly, the reaction mixture was diluted with acetonitrile (final concentration 95%) and then applied to ∼25 mg of Iatrobeads silica gel packed in a disposable filter column pre-equilibrated with 1 M acetic acid and acetonitrile. The column was washed first with acetonitrile and then with 96% acetonitrile in water. Glycans labeled with bis-pyrazolone and peptides labeled with pyrazolone were eluted from the silica gel with 50% aqueous acetonitrile. The BEP products of the O-GlcNAcylated and phosphorylated peptides were processed by the same procedures. β-Elimination of Glycopeptide in the Presence of Pyrazolone and a Thiol Michael Donor (BEPT Procedure). A mixture containing the glycopeptide (10 μL, 40 μg) and 10 μL each of 0.8 M sodium hydroxide and 1 M DTT was treated with 20 μL of a 0.5 M methanolic solution of PMP, followed by heating at 75 °C for 16 h. Subsequent neutralization and purification steps were performed by the same procedures as described above. MALDI-TOF Mass Spectrometry. Eluted PMP-labeled O-glycans and peptides were directly mixed with 2,5-dihydroxybenzoic

’ RESULTS β-Elimination in the Model Glycopeptide in the Presence of Pyrazolone Derivatives. Under the typical conditions of

Carlson’s method, glycopeptides are dissolved in 50100 mM NaOH and 0.51 M NaBH4 and then heated at ∼50 °C for ∼16 h.16 Honda et al.18 reported that the derivatization of glucose with PMP proceeds almost quantitatively at 70 °C for 30 min in 0.3 M NaOH and 0.5 M PMP. On the basis of this information, our model glycopeptide (peptide modified with T antigen at a single Ser residue) was incubated in 150 mM NaOH/250 mM PMP at 65, 75, or 85 °C for 16 h. The sample was then neutralized and directly subjected to MALDI-TOF MS analysis. As shown in Figure 2a, almost no products were observed when the β-elimination reactions were performed at 65 °C, whereas at temperatures of 75 and 85 °C, the glycopeptide signal decreased and two new signals clearly appeared, at m/z 736.4 and 2079.5, in the MALDI-TOF mass spectrum. As expected, the signals observed at m/z 736.4 and 2079.5 were confirmed to be glycan-labeled with bis-PMP and peptide-labeled with PMP, respectively. The TOF/TOF spectrum of PMP-labeled peptide is shown in Figure S-1 in Supporting Information. Signals corresponding to peeling degradation products were almost invisible. This result clearly demonstrated that BEP allows simultaneous cleavage and labeling of O-glycan and concomitant labeling of the peptide moiety (Figure 1c). When the same reactions were performed with other pyrazolone analogues, for example, DMP and MTP, both the glycan and the peptide were labeled just as achieved with PMP (Figure 2b). However, the detection sensitivity for the labeled glycan was highly dependent on the reagent used. As shown in Figure 2b (upper panel), the signal intensity of O-glycan labeled with bisDMP was much weaker than that of glycopeptide or peptide. This can be explained by considering that oligosaccharides are known to be generally less sensitive in MALDI-TOF MS analysis than peptides. By contrast, the signal intensities of glycans labeled with bis-PMP and bis-MTP were compatible with those of glycopeptide and peptide (Figure 2b, middle and lower panels), indicating that labeling with PMP and MTP substantially improves the sensitivity of glycan detection by MALDI-TOF MS. Regardless of the reagent, cleaved O-glycan was detected as the bis-pyrazolone derivative while underivatized O-glycan was not detected. Likewise, deglycosylated peptide was strongly detected 9062



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Table 1. Observed Signals of Oligosaccharides Released from (a) Bovine Fetuin, (b) Bovine Submaxillary Mucin, and (c) Porcine Stomach Mucina peak

obsd (m/z)

theor (m/z)

Table 1. Continued peak

deduced composition

(a) O-Glycans of Fetuin 1

574.3

574.23

(HexNAc)1

2

736.4

736.28

(Hex)1 (HexNAc)1

3

1027.5

1027.38

(Hex)1 (HexNAc)1 (NeuAc)1

4

1318.7

1318.47


5

1683.9

1683.60


obsd (m/z)

theor (m/z)

deduced composition

19 20

1508.0 1613.1

1507.57 1612.60

(Hex)2 (HexNAc)4 (Hex)3 (HexNAc)3 (Deoxyhexose)1

21

1654.1

1653.63

(Hex)2 (HexNAc)4 (Deoxyhexose)1

22

1670.1

1669.62

(Hex)3 (HexNAc)4

23

1711.2

1710.65

(Hex)2 (HexNAc)5

24 25 26 27

1759.2 1800.2 1816.3 1857.3

1758.66 1799.69 1815.68 1856.71

(Hex)3 (HexNAc)3 (Deoxyhexose)2 (Hex)2 (HexNAc)4 (Deoxyhexose)2 (Hex)3 (HexNAc)4 (Deoxyhexose)1 (Hex)2 (HexNAc)5 (Deoxyhexose)1c

(b) O-Glycan of Bovine Submaxillary Mucin 574.3 574.23 (HexNAc)1 720.4 720.29 (HexNAc)1 (Deoxyhexose)1

28

1873.3

1872.70

(Hex)3 (HexNAc)5

1 2

29

1962.4

1961.74


30

1978.3

1977.74


3

736.4

736.28

(Hex)1 (HexNAc)1

4

777.5

777.31

(HexNAc)2

31 32

2019.4 2076.4

2018.76 2075.78

(Hex)3 (HexNAc)5 (Deoxyhexose)1 (Hex)3 (HexNAc)6

5

865.5

865.32

(HexNAc)1 (NeuAc)1

6

881.5

881.32

(HexNAc)1 (NeuGc)1

7

882.5

882.34


8

939.6

939.36

(Hex)1 (HexNAc)2

9 10

980.6 1027.6

980.39 1027.38

(HexNAc)3 (Hex)1 (HexNAc)1 (NeuAc)1

33 34 35 36 37

2124.5 2165.5 2181.5 2222.6 2238.6

2123.79 2164.82 2180.81 2221.84 2237.84

(Hex)4 (HexNAc)4 (Deoxyhexose)2 (Hex)3 (HexNAc)5 (Deoxyhexose)2 (Hex)4 (HexNAc)5 (Deoxyhexose)1c (Hex)3 (HexNAc)6 (Deoxyhexose)1 (Hex)4 (HexNAc)6c

11

1043.6

1043.37

(Hex)1 (HexNAc)1 (NeuGc)1

38 39

2270.6 2327.6

2269.85 2326.87

(Hex)4 (HexNAc)4 (Deoxyhexose)3 (Hex)4 (HexNAc)5 (Deoxyhexose)2

12

1068.6

1068.40

(HexNAc)2 (NeuAc)1

40

2384.7

2383.89

(Hex)4 (HexNAc)6 (Deoxyhexose)1c

13

1084.6

1084.40

(HexNAc)2 (NeuGc)1

41

2441.8

2440.92

(Hex)4 (HexNAc)7c

14

1085.6

1085.42


15

1101.6

1101.41

(Hex)2 (HexNAc)2

16

1142.7

1142.44

(Hex)1 (HexNAc)3

17 18

1183.7 1230.7

1183.47 1230.45

(HexNAc)4 (Hex)1 (HexNAc)2 (NeuAc)1

19

1247.7

1247.47


20

1288.8

1288.50


21

1345.8

1345.52

(Hex)1 (HexNAc)4

22

1376.8

1376.51


23

1393.8

1393.53


24

1491.9

1491.58


25

1539.9

1539.59


1

(c) O-Glycans of Porcine Stomach Mucin 736.5 736.28 (Hex)1 (HexNAc)1

2

777.6

777.31

(HexNAc)2

3

882.6

882.34


4

898.6

898.33

(Hex)2 (HexNAc)1

5

939.7

939.36

(Hex)1 (HexNAc)2

6

980.7

980.39

7 8

1044.7 1085.8

1044.39 1085.42


9

1101.8

1101.41

(Hex)2 (HexNAc)2

10

1142.8

1142.44

(Hex)1 (HexNAc)3

11

1183.8

1183.47

(HexNAc)4, internal standard

12 13

1247.9 1288.9

1247.47 1288.50


14

1304.9

1304.49

(Hex)2 (HexNAc)3

15

1345.9

1345.52

(Hex)1 (HexNAc)4

16

1394.0

1393.53


17

1451.0

1450.55


18

1467.0

1466.54

(Hex)3 (HexNAc)3

(HexNAc)3

a

Hex, hexose; HexNAc, N-acetylhexosamine; IS, internal standard (N, N0 ,N00 ,N000 -tetraacetylchitotetraose). c Not annotated in the database of GlycoSuite.

as the pyrazolone derivative, indicating that once cleavage occurs, labeling by the pyrazolone derivative of either O-glycan or peptide is quantitative. Time course of the disappearance of glycopeptide and the recovery of PMP-labeled peptide upon reaction in 180 mM NaOH/200 mM pyrazolone at 85 °C were quantified at 0, 2, 4, 7, and 16 h after the reaction by MALDITOF MS by use of a nonglycosylated peptide having the same sequence as an internal standard. As shown in Figure S-2 (upper panel) in Supporting Information, no major new signals were generated during the BEP reaction, suggesting that significant decomposition of peptide is unlikely. The staring material (glycopeptide) steadily decreased with time, which was accompanied by the steady increase of PMP-labeled peptide (Figure S-2, lower panel, left and center, in Supporting Information). On the basis of the results described above, it was concluded that BEP permitted high conversion of model glycopeptide to PMPlabeled peptide. The amount of glycans labeled with bis-PMP also steadily increased with time (Figure S-2, lower panel, right, in Supporting Information). As shown in Figure S-2 (inset) in Supporting Information, signals corresponding to bis-PMPlabeled galactose (m/z 511.2 [M + H]+ and 533.2 [M + Na]+), a peeling product, were hardly visible throughout the BEP reaction. We observed trace levels of peeling products after 16 h incubation, which corresponded to less than 3% of bis-PMPlabeled Galβ13GalNAc (m/z 714.4 [M + H]+, 736.4 [M + Na]+). Recovery of the O-glycan labeled with bis-pyrazolone was accelerated at higher NaOH concentrations and/or higher reaction temperatures, although the peptide moiety degraded accordingly. The simultaneous cleavage and labeling of O-glycan and the concomitant labeling of peptide moiety by BEP was also 9063



Figure 3. MALDI-TOF MS spectra showing the mucin type O-glycan profiles of (a) bovine fetuin, (b) bovine submaxillary mucin, and (c) porcine stomach mucin. Each glycoprotein was subjected to BEP in 180 mM NaOH/200 mM methanolic PMP at 75 °C for 16 h. The putative compositions of the numbered oligosaccharide signals are summarized in Table 1.

observed for a glycopeptide in which the O-glycan was attached to a single Thr residue, indicating that the described approach is applicable to O-GalNAc type modifications occurring on Ser or Thr (data not shown). Mucin-type O-Glycan Analysis. The feasibility of the described method for O-glycosylation analysis of glycoproteins was evaluated by use of various model glycoproteins. First, bovine fetuin, which contains both N- and O-glycans,24 was subjected to BEP. This glycoprotein was chosen to clarify whether the employed BEP conditions result in the selective cleavage of O-glycans, as is the case of β-elimination under Carlson’s conditions, or in the cleavage of not only O-glycans but also small amounts of N-glycans, as is the case of β-elimination under mild alkaline conditions.17 As shown in Figure 3a and Table 1, all O-glycans known to be present on fetuin were detected in the appropriate relative quantities, while no signals corresponding to N-glycans were found, indicating that N-glycans are resistant to BEP. Bovine submaxillary mucin (BSM) belongs to the class of salivary glycoproteins. It has a molecular weight of 4 106 and consists of a long protein chain with numerous disaccharide and oligosaccharide side chains, many of which are known to be sialylated. As shown in Figure 3b and Table 1, the observed O-glycan profile was similar to that previously reported.25 Thus, as in the case of fetuin, the results obtained with BSM indicated that the BEP method exclusively cleaves O-glycans whereas sialic acid residues are resistant and thus stable. The observed O-glycan profile of porcine stomach mucin (PSM) was also similar to previously reported profiles (Figure 3c and Table 1).26 The detection limit of the described technique was investigated by analyzing 10 μg, 1 μg, and 100 ng of PSM. As shown in Figure S-3 in Supporting Information, 100 ng of PSM yielded quantitatively comparable O-glycan profiling results. As an example of O-glycosylation site analysis of glycoprotein by BEP, fetuin was subjected to S-alkylation by iodoacetoamide and was subjected to BEP, followed by digestion with pronase. As a control, S-alkylated fetuin was directly subjected to pronase digestion. As shown in Figure S-4a in Supporting Information, signal observed at m/z 1334.65, which was assigned to be 269AP(sialyl T)

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Figure 4. MALDI-TOF spectra showing the application of BEP to (a) O-GlcNAcylated and (b) phosphorylated peptides. MALDI-TOF spectra acquired before and after BEP reaction (upper and lower panels, respectively) were compared. The model GlcNAcylated peptide was subjected to BEP in 180 mM NaOH/200 mM methanolic PMP at 75 °C for 16 h. The phosphorylated peptide was subjected to BEP in 180 mM NaOH/200 mM methanolic PMP at 85 °C for 16 h. Unannotated signals observed in panel a are artifactual peaks resulting from impurities present in the glycopeptide used.

SAVPD275 (theoretical m/z 1334.54), disappeared upon BEP treatment, while a new intense signal appeared at m/z 812.47, which was assigned to be 269AP(PMP)SAVPD275 (theoretical m/z 812.39). Compared to the TOF/TOF spectrum of glycopeptide (m/z 1334.7) (Figure S-4b, Supporting Information), where cleavages of glycan moieties are dominant and structural information of peptide moieties are scarce, that of PMP-labeled peptide (m/z 812.4) provided many fragments due to the cleavage of peptide bonds (Figure S-4c, Supporting Information), thus advantageous to identify the peptide identification and the O-glycan bonding site. These results demonstrate the feasibility of BEP for the analysis of O-glycosylation profile as well as the identification of O-glycosylation sites. Research focusing on large-scale O-glycosylation site analysis by BEP is currently under investigation in our laboratory and will be present elsewhere. Application of BEP to O-GlcNAcylated and Phosphorylated Peptides. The described procedure was further applied to O-GlcNAcylated and phosphorylated peptides, both of which are known to be labile to β-elimination, as O-GlcNAc and phosphoric acid are cleaved upon β-elimination, respectively.11 When the model GlcNAcylated and phosphorylated peptides were subjected to BEP, GlcNAc and phosphoric acid, respectively, were cleaved (Figure 4). Moreover, in both cases, peptides formerly GlcNAcylated or phosphorylated were labeled by PMP. To the best of our knowledge, the Michael donors used in BEMA thus far have been limited to heteroatomic nucleophiles involving thiols and amines. Therefore, BEP may be the first example of a BEMA that employs a carbon Michael donor. When BEP was applied to the O-GlcNAcylated peptide, the cleaved GlcNAc was detected as a bis-PMP derivative (Figure 4a, lower panel). Thus, compared to BEMA, in which the detection/ quantitation of GlcNAc or phosphoric acid is not possible, BEP offers an important advantage. 9064



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Figure 6. Chemical pathways of a glycopeptide upon β-elimination reaction in the presence of both pyrazolone and thiol Michael donor (BEPT).

Figure 5. Distinct modification of the released O-glycan and of the formerly glycosylated peptide by β-elimination reaction in the presence of both pyrazolone and thiol Michael donor (BEPT). The model glycopeptide was subjected to (a) BEP with PMP, (b) BEMA with DTT, and (c) BEPT with both PMP and DTT Asterisks denote artifactual peaks resulting from impurities present in the glycopeptide used.

β-Elimination Reaction in the Presence of Both Pyrazolone and a Thiol Michael Donor. As described above, pyrazo-

lones, acting as carbon Michael donors, label O-glycans as well as the peptide of a glycopeptide, in both cases via a β-elimination reaction. Thus, our interest was further directed to the β-elimination of glycopeptide in the presence of both a carbon Michael donor and a thiol Michael donor. It was reasoned that although O-glycan labeling by a thiol Michael donor is unlikely, this might not be the case for peptides. The model glycopeptide was therefore subjected to the βelimination reaction in the presence of pyrazolone and the model thiol, Michael donor DTT. MALDI-TOF MS was then used to compare the reaction products with those obtained by BEP, employing PMP as the pyrazolone. As shown in Figure 5 (upper panels), BEP carried out with PMP produced O-glycan and deglycosylated peptide, both of which were PMP-labeled. BEMA carried out with DTT produced DTT-labeled formerly glycosylated peptide (Figure 5, middle panel) while the released O-glycan was degraded through a peeling reaction (data not shown). However, BEPT produced O-glycan labeled with bisPMP and peptide labeled with DTT (Figure 5. lower panel). There were no signals corresponding to glycans labeled with PMP and DTT, nor was the peptide labeled with PMP, indicating that BEPT allowed the highly selective labeling of released O-glycan and formerly glycosylated peptide by PMP and DTT, respectively (Figure 6). This is the first report of successful selective labeling of released O-glycan and peptide by different reagents upon β-elimination.

’ DISCUSSION A novel and versatile method for the analysis of serine/ threonine posttranslational modifications by β-elimination in the presence of pyrazolone analogues (BEP) is described. During the preparation of our paper, the same strategy to analyze

O-glycans after combined O-glycan release by β-elimination and PMP labeling was reported by Zauner et al.,27 who used dimethylamine as the releasing agent and PMP in the reaction with the reducing end of the released glycans. However, BEP is unique in that it allows concomitant labeling of both the released O-glycan and the deglycosylated peptide. Under the optimized conditions described above, the peeling reaction was found to be almost negligible, as demonstrated by our analyses of the model glycopeptide and glycoproteins. Although the peptide was found to be more labile than the glycan moiety under BEP, as in Carlson’s method, BEP nonetheless allows simultaneous analysis of both products, without significant degradation of either one when the reaction is performed under the optimized conditions. We conducted a number of trials aimed at avoiding reduction of the reducing end of the carbohydrate, as it was highlighted recently that stark discrepancies are observed when reduction is omitted during the β-elimination reaction.16,17 BEP avoids the peeling reaction, perhaps due to the fact that the reaction of the pyrazolone derivative is much faster than β-elimination. While the PMP labeling of glucose is reportedly complete in 30 min under optimized conditions, β-elimination typically requires ∼16 h. It is also worth mentioning that pyrazolones exhibit ketoenol tautomerism, and thus they behave as weak acids due to their weakly acidic enol groups.28 The NaOH concentration in the optimized reaction conditions is as high as ∼180 mM, whereas the presence of the pyrazolone derivative at ∼200 mM lowers the pH of the reaction solvent to below 9.0, such that the conditions for β-elimination are milder. Of particular interest was the inability of the BEP procedure to cleave N-glycans, which is extremely advantageous considering that O-glycans and N-glycans may have the same molecular weight and are sometimes difficult to differentiate; in addition, analytical procedures for N-glycans are already wellestablished.23,29 As noted above, BEP results in the labeling of not only the released O-glycan but also the deglycosylated peptide. In addition, deglycosylated and dephosphorylated peptides are labeled by the same pyrazolone derivatives. The reaction can be viewed as a kind of BEMA, in which a carbon Michael donor, instead of heteroatomic Michael donors involving thiols and amines, is employed. This successful demonstration of the feasibility of carbon Michael donors in BEMA will broaden the applications of this approach by increasing the available options for introducing various functionalities into the formerly glycosylated/phosphorylated peptide. For instance, incorporation of signature tag will lead to improved ionization efficiency and facilitate sequencing. 9065


Analytical Chemistry The ability to label both O-glycan and peptide moieties upon β-elimination will be particularly useful in the discrimination between phosphorylation and O-GlcNAcylation. Ser/Thr phosphorylation and O-GlcNAc modifications are mutually exclusive at a given residue. Nonetheless, several site-mapping studies have established the same site on a protein as being phosphorylated and O-GlcNAcylated. It was therefore suggested that there is a reciprocal, competitive yinyang relationship between these two dynamic modifications.30 The selectivity of β-elimination toward O-GlcNAcylated and phosphorylated peptides has yet to be addressed properly, such that it is difficult to discriminate between O-GlcNAcylation and phosphorylation by only the βelimination method. Since BEP allows the detection of both the peptide and the GlcNAc derived from parent glycopeptide/ glycoprotein, the reaction can be applied to directly discriminate between GlcNacylated and phosphorylated peptide/protein by detecting the GlcNAc residue. In the case of samples in which both phosphorylated and O-GlcNAc peptides are present, the two species could be relatively quantified by introducing a strategy to quantify the GlcNAc and the peptide, for instance by introducing a fluorescent tag. We further described the concept of BEPT, in which β-elimination is performed in the presence of PMP (carbonbond-forming Michael donor) and DTT (thiol Michael donor). The O-glycans and formerly glycosylated peptides were found to be exclusively labeled by PMP and DTT, respectively. The exclusive labeling of peptide by DTT may be explained by the higher reactivity of thiol than pyrazolone as Michael donor. You et al.31 proposed a reaction mechanism for the production of glycan labeled by bis-pyrazolones, in which the first pyrazolone is added to the reducing end via an aldol condensation reaction and the second pyrazolone via a Michael addition. Therefore, we expected to detect heterobis-type labeling (incorporation of pyrazolone via aldol condensation, followed by Michael addition of DTT). However, no such product was observed and glycan was solely recovered as a bis-PMP derivative. This suggests that the stabilization caused by bis-PMP formation at the reducing end of a glycan (e.g., intermolecular hydrogen bond formation)28 may compensate for the lower reactivity of the Michael donor compared to that of DTT. Otherwise, the reaction mechanism of the second attachment of pyrazolone may need to be reconsidered. From a practical viewpoint, the ability to selectively label glycans and peptides by different reagents will facilitate systematic glycomic and proteomic analyses. For instance, peptide and glycan moieties may be labeled by reagents to facilitate sequencing and selective enrichment, respectively. We examined BEP for phosphorylated and O-glycosylated (i.e., O-GalNAc and O-GlcNAc type) peptides and determined that BEP is generally applicable to all Ser/Thr modifications so far tested. Therefore, it is likely that BEP and BEPT can also be applied to the other types of Ser/Thr modifications, although this must be experimentally confirmed, as is currently being done in our laboratory.

’ ASSOCIATED CONTENT

bS

Supporting Information. Four figures showing MALDITOF/TOF spectrum of PMP-labeled model peptide, time course of disappearance of glycopeptide and recovery of PMP-labeled peptide, detection limit of the described technique, and O-glycosylation site analysis of glycoprotein by BEP. This material is available free of charge via the Internet at http://pubs.acs.org.

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’ AUTHOR INFORMATION Corresponding Author

*Phone: +81 11 706 9091. Fax: +81 11 706 9087. E-mail: [email protected].

’ ACKNOWLEDGMENT This work was supported by the Special Coordination Funds for Promoting Science and Technology of Japan’s Ministry of Education, Culture, Sports, Science and Technology. ’ REFERENCES (1) Jensen, O. N. Nat. Rev. Mol. Cell. Biol. 2006, 7, 391–403. (2) Medzihradszky, K. F.; Darula, Z.; Perlson, E.; Fainzilber, M.; Chalkley, R. J.; Ball, H.; Greenbaum, D.; Bogyo, M.; Tyson, D. R.; Bradshaw, R. A.; Burlingame, A. L. Mol. Cell. Proteomics 2004, 3 429–440. (3) Endo, T. Biochim. Biophys. Acta 1999, 1473 (1), 237–246. (4) Grimsrud, P. A.; Swaney, D. L.; Wenger, C. D.; Beauchene, N. A.; Coon, J. J. ACS Chem. Biol. 2010, 5, 105–119. (5) Andersson, L.; Porath, J. Anal. Biochem. 1986, 154, 250–254. (6) Sugiyama, N.; Masuda, T.; Shinoda, K.; Nakamura, A.; Tomita, M.; Ishihama, Y. Mol. Cell. Proteomics 2007, 6, 1103–1109. (7) Meyer, H. E.; Hoffmann-Posorske, E.; Korte, H.; Heilmeyer, L. M., Jr. FEBS Lett. 1986, 204, 61–66. (8) Oda, Y.; Nagasu, T.; Chait, B. T. Nat. Biotechnol. 2001, 19 379–382. (9) McLachlin, D. T.; Chait, B. T. Anal. Chem. 2003, 75, 6826–6836. (10) Arrigoni, G.; Resj€o, S.; Levander, F.; Nilsson, R.; Degerman, E.; Quadroni, M.; Pinna, L. A.; James, P. Proteomics 2006, 6, 757–766. (11) Poot, A. J.; Ruijter, E.; Nuijens, T.; Dirksen, E. H.; Heck, A. J.; Slijper, M.; Rijkers, D. T.; Liskamp, R. M. Proteomics 2006, 6, 6394–6399. (12) Maniatis, S.; Zhou, H.; Reinhold, V. Anal. Chem. 2010, 82, 2421–2415. (13) Brockhausen, I. EMBO Rep. 2006, 7, 599–604. (14) Carlson, D. M. J. Biol. Chem. 1968, 243, 616–626. (15) Huang, Y.; Mechref, Y.; Novotny, M. V. Anal. Chem. 2001, 73, 6063–6069. (16) Wada, Y.; Dell, A.; Haslam, S. M.; Tissot, B.; Canis, K.; Azadi, P.; B€ackstr€om, M.; Costello, C. E.; Hansson, G. C.; Hiki, Y.; Ishihara, M.; Ito, H.; Kakehi, K.; Karlsson, N.; Hayes, C. E.; Kato, K.; Kawasaki, N.; Khoo, K. H.; Kobayashi, K.; Kolarich, D.; Kondo, A.; Lebrilla, C.; Nakano, M.; Narimatsu, H.; Novak, J.; Novotny, M. V.; Ohno, E.; Packer, N. H.; Palaima, E.; Renfrow, M. B.; Tajiri, M.; Thomsson, K. A.; Yagi, H.; Yu, S. Y.; Taniguchi, N. Mol. Cell. Proteomics 2010, 9, 719–727. (17) Yu, G.; Zhang, Y.; Zhang, Z.; Song, L.; Wang, P.; Chai, W. Anal. Chem. 2010, 82, 9534–9542. (18) Honda, S.; Akao, E.; Suzuki, S.; Okuda, M.; Kakehi, K.; Nakamura, J. Anal. Biochem. 1989, 180, 351–357. (19) Li, X. L.; Wang, Y. M.; Tian, B.; Matsuura, T.; Meng, J. B. J. Heterocycl. Chem. 1998, 35, 129–134. (20) Matsushita, T.; Hinou, H.; Kurogochi, M.; Shimizu, H.; Nishimura, S.-I. Org. Lett. 2005, 7, 877–880. (21) Delaglio, F.; Grzesiek, S.; Vuister, G.; Zhu, G.; Pfeifer, J.; Bax, A. J. Biomol. NMR 1995, 6, 277–293. (22) Kita, Y.; Miura, Y.; Furukawa, J.; Nakano, M.; Shinohara, Y.; Ohno, M.; Takimoto, A.; Nishimura, S. Mol. Cell. Proteomics 2007, 6, 1437–1445. (23) Furukawa, J.; Shinohara, Y.; Kuramoto, H.; Miura, Y.; Shimaoka, H.; Kurogochi, M.; Nakano, M.; Nishimura, S. Anal. Chem. 2008, 80 1094–1101. (24) Merry, A. H.; Neville, D. C.; Royle, L.; Matthews, B.; Harvey, D. J.; Dwek, R. A.; Rudd, P. M. Anal. Biochem. 2002, 304, 91–99. (25) Karlsson, N. G.; Packer, N. H. Anal. Biochem. 2002, 305 173–185. 9066



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