(LTL) Quantitative Proteomics - ACS Publications - American

Oct 5, 2010 - Disease Research Center, Eastern Virginia Medical School, 700 West Olney Road, Norfolk, Virginia 23508. The accuracy in quantitative ...
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Anal. Chem. 2010, 82, 9201–9210

Investigation of Sialylation Aberration in N-linked Glycopeptides By Lectin and Tandem Labeling (LTL) Quantitative Proteomics Vivekananda Shetty,*,† Zacharie Nickens,† Punit Shah,† Gomathinayagam Sinnathamby,† O. John Semmes,‡ and Ramila Philip*,† Immunotope, Inc., 3805 Old Easton Road, Doylestown, Pennsylvania 18902, and Cancer Biology and Infectious Disease Research Center, Eastern Virginia Medical School, 700 West Olney Road, Norfolk, Virginia 23508 The accuracy in quantitative analysis of N-linked glycopeptides and glycosylation site mapping in cancer is critical to the fundamental question of whether the aberration is due to changes in the total concentration of glycoproteins or variations in the type of glycosylation of proteins. Toward this goal, we developed a lectin-directed tandem labeling (LTL) quantitative proteomics strategy in which we enriched sialylated glycopeptides by SNA, labeled them at the N-terminus by acetic anhydride (1H6/ 2 D6) reagents, enzymatically deglycosylated the differentially labeled peptides in the presence of heavy water (H218O), and performed LC/MS/MS analysis to identify glycopeptides. We successfully used fetuin as a model protein to test the feasibility of this LTL strategy not only to find true positive glycosylation sites but also to obtain accurate quantitative results on the glycosylation changes. Further, we implemented this method to investigate the sialylation changes in prostate cancer serum samples as compared to healthy controls. Herein, we report a total of 45 sialylated glycopeptides and an increase of sialylation in most of the glycoproteins identified in prostate cancer serum samples. Further quantitation of nonglycosylated peptides revealed that sialylation is increased in most of the glycoproteins, whereas the protein concentrations remain unchanged. Thus, LTL quantitative technique is potentially an useful method for obtaining simultaneous unambiguous identification and reliable quantification of N-linked glycopeptides. Altered glycosylation patterns involving a particular type of glycan are considered as biomarkers for early cancer detection1-7

Much of the work has been focused on sialic acid because sialylation plays a crucial role in cell surface interactions,8 protects cells from membrane proteolysis,9 helps in cell adhesion and has been shown to determine the half-life of glycoproteins in blood.10 It has been shown that aberrations in sialylation are associated with a variety of diseases, particularly in cancer, and increased sialylation has been observed on the surface of tumor cells11 during malignant progression.12 In a recent report, Tajiri et al13 showed that the R2,3-linked sialic acid in either free or complex form of PSA potentially could discriminate malignant from benign conditions of the prostate. In view of the significance of sialylation in glycopathology, several methods were developed to examine differential sialylation in various other cancers. The results of many of the sialylation studies involving deglycosylation of proteins and elucidation of the released glycan structures failed to correlate glycan structural features with specific glycosylation sites on proteins due to the complexity of the glycoproteome and occurrence of multiple glycosylation sites within a single protein.10 These observations led to the investigation of glcosylation patterns in intact glycopeptides containing glycan moieties and to maximize glycosylation coverage in terms of the number of glycosylation sites detected and their corresponding glycoforms.14 Lectins have been used in conjunction with reversed-phase chromatography and mass spectrometry to investigate differences in glycosylation of proteins involving a particular type of glycan.10,15 In glycoproteins, sialic acid residues are generally linked via an R-2,3 or an R-2,6 bond to Gal/GalNAc. SNA lectin binds to peptides carrying sialic acid connected to the underlying sugar chains through an R-2-6 linkage. Overexpressed desialylated and partially sialylated glycopeptides have been identified on Kininogen-1 using SNA lectin enrichment of sialylated glycoproteins in pancreatic cancer serum samples.15 These results are consistent

* Address correspondence to either author. Phone: 215 589 6327 (V.S.); 215 489 4955 (R.P.). E-mail: [email protected] (V.S.); [email protected] (R.P.). † Immunotope, Inc. ‡ Eastern Virginia Medical School. (1) Kobata, A.; Amano, J. Immunol Cell Biol. 2005, 83, 429–39. (2) Demetriou, M.; Nabi, I. R.; Coppolino, M.; Dedhar, S.; Dennis, J. W. J. Cell Biol. 1995, 130, 383–92. (3) Orntoft, T. F.; Vestergaard, E. M. Electrophoresis. 1999, 20, 362–71. (4) Patwa, T. H.; Zhao, J.; Anderson, M. A.; Simeone, D. M.; Lubman, D. M. Anal. Chem. 2006, 78, 6411–21. (5) Orntoft, T. F.; Greenwell, P.; Clausen, H.; Watkins, W. M. Gut 1991, 32, 287–93.

(6) Dube, D. H.; Bertozzi, C. R. Nat. Rev. Drug Discov. 2005, 4, 477–88. (7) An, H. J.; Miyamoto, S.; Lancaster, K. S.; Kirmiz, C.; Li, B.; Lam, K. S.; Leiserowitz, G. S.; Lebrilla, C. B. J. Proteome Res. 2006, 5, 1626–35. (8) Paulson, J. C. Trends Biochem. Sci. 1989, 14, 272–6. (9) Gorog, P.; Pearson, J. D. J. Pathol. 1985, 146, 205–12. (10) Qiu, R.; Regnier, F. E. Anal. Chem. 2005, 77, 2802–9. (11) Fischer, E.; Brossmer, R. Glycoconjugate J. 1995, 12, 707–13. (12) Sata, T.; Roth, J.; Zuber, C.; Stamm, B.; Heitz, P. U. Am. J. Pathol. 1991, 139, 1435–48. (13) Tajiri, M.; Ohyama, C.; Wada, Y. Glycobiology. 2008, 18, 2–8. (14) Zhang, Y.; Go, E. P.; Desaire, H. Anal. Chem. 2008, 80, 3144–58. (15) Zhao, J.; Patwa, T. H.; Qiu, W.; Shedden, K.; Hinderer, R.; Misek, D. E.; Anderson, M. A.; Simeone, D. M.; Lubman, D. M. J. Proteome Res. 2007, 6, 1864–74.

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with the data obtained from glycoprotein microarrays where an increased binding of this protein to SNA, MAL, and PNA was detected in cancer samples. Recently Ghesquie‘re et al16 published a new multistep approach for proteome-wide analysis of sialylated N-glycopeptides based on the combined fractional diagonal chromatographic (COFRADIC) technology and 18O/16O labeling during trypsin digestion and indicated that their approach could be used to analyze sera from healthy and diseased states. Kaji et al.17 developed an isotope-coded glycosylation site-specific tagging (IGOT) method for the large-scale identification of N-linked glycoproteins from complex biological samples. In that study, the authors identified hundreds of glycosylation sites in the nematode Caenorhabditis elegans and mouse liver by using lectin, hydrophilic interaction chromatography and 18O/ 16 O labeling quantitative proteomics. A potential pitfall of 18Obased N-linked glycosylation site mapping is the interference of residual active trypsin that incorporates 18O isotope into the C-terminae of the peptides during PNGase F deglycosylation process that has been disregarded until recently. Angel et al.18 addressed the issue of high false positive rate results and recommended procedures to eliminate active trypsin from the solution prior to performing the deglycosylation process. A recent review by Capelo et al.19 further highlighted the significance of 18O-isotopic labeling for mass spectrometrybased proteomics approaches and have discussed methods to overcome the limitation of 18O-labeling efficiency in trypsin digestion. Nilsson et al.20 reported a novel qualitative method in which sialylated glycoproteins were selectively periodateoxidized, captured on hydrazide beads, trypsinized and released by acid hydrolysis of sialic acid glycosidic bonds. In this analysis, mass spectrometric experiments allowed the identification of glycan structures, peptide sequences, and glycan attachment sites without any quantitation strategy. Recently, Atwood et al. using lectin affinity capture method and stable isotope labeling of the glycan attachment sites with H218O, identified 36 glycosylation sites in organelle and cell surface N-linked glycoproteins from a human pathogen Trypanosoma cruzi.21 Majority of the aforementioned methods lack efficient quantitative strategies and have not addressed the issue of quantitative errors due to the contamination from the deamidation product of asparagine, peptides with incomplete trypsin derived 18O labeled carboxylic acids, and back exchange of 18O with 16O under acidic conditions. Unambiguous determination of glycosylation sites and simultaneous quantitation is not possible with the existing methods and there is a need to improve them to obtain accurate quantitative results on the glycosylation changes. One way of probing these glycosylation differences in N-linked (16) Ghesquiere, B.; Buyl, L.; Demol, H.; Van Damme, J.; Staes, A.; Timmerman, E.; Vandekerckhove, J.; Gevaert, K. J. Proteome Res. 2007, 6, 4304–12. (17) Kaji, H.; Saito, H.; Yamauchi, Y.; Shinkawa, T.; Taoka, M.; Hirabayashi, J.; Kasai, K.; Takahashi, N.; Isobe, T. Nat. Biotechnol. 2003, 21, 667–72. (18) Angel, P. M.; Lim, J. M.; Wells, L.; Bergmann, C.; Orlando, R. Rapid Commun. Mass Spectrom. 2007, 21, 674–82. (19) Capelo, J. L.; Carreira, R. J.; Fernandes, L.; Lodeiro, C.; Santos, H. M.; SimalGandara, J. Talanta. 2010, 80, 1476–86. (20) Nilsson, J.; Ruetschi, U.; Halim, A.; Hesse, C.; Carlsohn, E.; Brinkmalm, G.; Larson, G. Nat. Methods. 2009, 6, 809–11. (21) Atwood, J. A., 3rd; Minning, T.; Ludolf, F.; Nuccio, A.; Weatherly, D. B.; Alvarez-Manilla, G.; Tarleton, R.; Orlando, R. J. Proteome Res. 2006, 5, 3376– 84.

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glycopeptides is by finding differences indirectly in deglycosylated peptides22 after lectin enrichment of a specific glycan type and differential labeling of glycopeptides. Qiu et al, reported two similar quantitative strategies10,23 but their studies were focused on the comparative analysis of differential branching among complex-type N-linked glycans, and on probing degree of sialylation using serial lectin affinity chromatography (SLAC) strategy in normal human serum. Furthermore, 18O labeling was not performed during the PNGase deglycosylation which may pose problems on glycosylation site determination as well as quantitation of accurate global changes in sialylation especially when dealing with a complex glycoproteome. In view of this, we attempted to develop a method that would allow determination of changes in glycosylation at specific sites in glycoproteins. Herein, we describe a lectin-directed tandem labeling (LTL) quantitative proteomics method to probe sialylation changes based on the ratios of the relative abundance of N-deglycosylated peptides obtained from two different sample sets. We used SNA lectin to capture sialylated glycopeptides and for quantitation we used acetyl (1H3/2D3) labeling at the N-terminus and 18O labeling in PNGase F digestion for glycosylation site mapping. A proof-of-concept study was first carried out on a tryptic digest of bovine fetuin. Using this method, for the first time in prostate cancer (PCa) serum samples, we identified many differentially sialylated N-linked glycopeptides with high confidence. MATERIALS AND METHODS Materials. Bovine fetuin was purchased from QA-Bio (Palm Desert, CA). Serum samples were procured from ten patients diagnosed with primary prostate cancer (Eastern Virginia Medical School) and five age and sex matched healthy control individuals (Research Blood Components, LLC., Brighton, MA). Equal amounts of undiluted serum from each cohort were used to generate composites consisting of cancer and normal serum samples for the study. Serum samples were collected from patients with histological confirmed prostate carcinoma (regardless of stage of the disease), prior to surgery and chemotherapy and were stored at -80 °C until use. HPLC grade acetonitrile (ACN) and water were obtained from Burdick and Jackson (Muskegon, MI). Methanol, calcium chloride (CaCl2), and sodium phosphate (NaH2PO4) were purchased from EMD chemical Inc. (Gibbstown, NJ). Ethanol was purchased from Decon Laboratories, Inc. (King of Prussia, PA). Acetic anhydride (1H6/2D6), H218O water, phenylmethanesulfonyl fluoride (PMSF), ammonium bicarbonate (AB), formic acid (FA), trifluoroacetic acid (TFA), dithiothreitol (DTT), iodoacetamide (IA), manganese chloride (MnCl2), lactose were purchased from Sigma (St. Louis, MO). Sambucus nigra lectin (SNA) agarose was purchased from Vector Laboratories (Burlingame, CA). Spin-x columns were purchased from Corning, Inc. (Lowell, MA). Peptide-N-glycosidase F (PNGase F) was purchased from QA BIO (Palm Desert, CA). Protein A/G beads and C-18 miniprep columns were purchased from Thermo Scientific (Waltham, MA). RapiGest SF, vacuum manifold and C-18 sep-pak cartridges were obtained (22) Liu, Z.; Cao, J.; He, Y.; Qiao, L.; Xu, C.; Lu, H.; Yang, P. J. Proteome Res. 2010, 9, 227–36. (23) Qiu, R.; Regnier, F. E. Anal. Chem. 2005, 77, 7225–31.

from Waters (Milford, MA). Tris, NaCl and NaOH were purchased from Fisher (Pittsburgh, PA). PBS was purchased from Mediatech, Inc. (Manassas, VA). Ultra Centricon 3 kDa filters were purchased from Millipore (Billerica, MA). Trypsin was purchased from Promega (Madison, WI). In-Solution Digestion. A total of 250 µg of fetuin protein solution was divided into two equal aliquots and in-solution digestion was performed using trypsin. Brifely, the protein solution was mixed with 30 µL of 50 mM AB containing 0.1% of Rapigest SF and the protein was reduced with DTT (5 µm/µL in 50 mM AB) by incubating the mixture at 65 °C for 45 min and alkylated with IA (15 µm in 50 mM AB) by incubating the reaction mixture in dark for 30 min. Then the alkylated glycoproteins were digested by Trypsin (5 ng/µL in 50 mM AB) overnight at 37 °C in a water bath. RapiGest was removed according to the vendor’s recommended procedure. Prior to the in-solution digestion, serum samples were precleared with spin-x columns and IgG was removed using protein AG beads and buffer exchanged with 50 mM AB and 0.5% CHAPS detergent using Ultra Centricon 3 kDa filters. The protein concentrations of normal and cancer serum samples were estimated by nanodrop technologies instrument (Wilmington, DE). The trypsin digestion of serum samples was carried out as described above for fetuin. Enrichment of Glycopeptides using SNA Lectin. Tryptic peptides were mixed with 1 mM solution of PMSF prior to the glycoprotein enrichment by SNA lectin. The tryptic peptide sample was diluted to 4:1 with 5× binding buffer (100 mM Tris, 750 mM NaCl, 5 mM CaCl2, and 5 mM MnCl2, pH 6.7). The lectin columns were prepared by adding 200 µL of SNA lectin to the catridges and spun down at 1000g for 1 min to remove storage buffer. The lectin resin in columns was washed with 200 µL (2×) of 1× washing buffer (20 mM Tris, 150 mM NaCl, 1 mM CaCl2, and 1 mM MnCl2). Then the sample was loaded onto the columns, mixed, and incubated at room temperature for 10 min. The columns were centrifuged to collect the flow through and washed with 400 µL of 1× washing buffer (2×). Again, 400 µL of 1× washing buffer was added and incubated for 5 min at RT (2×). The flow through samples were saved for additional labeling experiments. Finally, The glycopeptides were eluted from the lectin columns by adding 200 µL (2×) of elution buffer (20 mM Tris, 150 mM NaCl, 1 mM CaCl2, 1 mM MnCl2, and 200 mM lactose) and incubating for 10 min at RT. The flow through containing the glycopeptides were collected by centrifugation and purified by C-18 chromatography. Acetylation of Peptides. Glycopeptides obtained from fetuin and serum samples and nonglycosylated peptides from serum samples (normal and cancer) were labeled with acetyl (1H3/2D3) groups according to a published protocol.24 Briefly, 2 µL of 1 H6-acetic anhydride or 2D6-acetic anhydride [50% (v/v) in methanol] were added to 100 µL of peptide mixture in 50% methanol/water (v/v). The mixture was allowed to react at 22 °C for 15 min. The reaction was stopped by the addition of 2.2 µL of formic acid and equal aliquots of both samples were mixed. PNGase F Digestion of Glycopeptides. In case of feutin, light, and heavy labeled glycopeptides were mixed in two different

ratios (1:1 and 3:1) and the PNGase F digestion was carried out. Along with these fetuin glycopeptides, all the serum glycopeptides were digested with PNGase F to cleave the N-glycans from peptides according to the protocol described elsewhere.25 Briefly, the glycopeptides were dried in vacuum and redissolved in 100 mM NaH2PO4 buffer (pH 7.5). The PNGase F enzyme was dissolved in 50 mM AB. NaH2PO4 buffer solution and AB buffer solution were prepared in H218O before dissolving glycopeptides and enzyme, respectively. The reaction mixture was incubated with 6 µL of PNGase F at 37 °C overnight. The reaction was stopped by adding 0.5% TFA solution. The resulting Ndeglycosylated peptides were purified by C-18 chromatography. Purification of Peptides by C-18 Chromatography. The trypsin digested peptides, SNA enriched glycopeptides and Ndeglycosylated peptides were purified by C-18 reversed-phase (RP) chromatography using either C-18 spin columns or C-18 sep-pak cartridges with the help of a vacuum manifold. Briefly, the C-18 columns were activated with 50% acetonitrile and equilibrated with a buffer containing 2% ACN and 0.1% TFA in water. Then peptide mixture (dissolved in 2% ACN and 0.1% TFA in water) was slowly loaded into the C-18 cartridge. Then the cartridges were washed thoroughly with 0.1% TFA to remove salts and buffers. Finally, the tryptic peptides were eluted with 70% acetonitrile twice (250 µL each), the two eluted fractions were combined and concentrated. The serum glycopeptide mixture was fractionated by C-18 RP column (4.6 mm diameter × 150 mm length) using an offline ultimate 3000 HPLC (Dionex, Sunnyvale, CA). Mobile phase A was 2% acetonitrile (ACN) and 0.1% formic acid (FA) in water, while mobile phase B was 0.1% FA and 90% ACN in water. Peptides were then eluted from the column with an 80 min linear gradient from 5 to 80% buffer B at a flow rate of 200 µL/min. A total of 35 fractions were collected and each fraction was concentrated to 6 µL under vacuum. Mass Spectrometry Analysis. A 3000 nano ultimate HPLC (Dionex, Sunnyvale, CA) was coupled with LTQ mass spectrometer (Thermo Electron, San Jose, CA) equipped with advanced nanospray source to analyze acetyl (1H3/2D3) and 18O labeled peptides. Fetuin N-deglycosylated peptide mixture and serum N-deglycosylated peptide fractions were injected into LC-MS/ MS system to identify deglycosylated peptides. As a part of online sample cleanup step, the peptides were first concentrated in a C-18 RP trap column (Dionex) and then separated by using a 75 µm i.d. × 15 cm C-18 RP analytical column (Dionex, Sunnyvale, CA) equilibrated in 4% ACN/0.1% FA at 250 nL/ min flow rate. Mobile phase A was 2% ACN and 0.1% FA in water, while mobile phase B was 0.1% FA and 90% ACN in water. Peptides were separated with a 4-50% B in 60 min and 50-80% in 90 min and eluted directly into an LTQ MS. The mass range in MS mode was 350-1800 Da and in MS/MS mode it was set as 100-2000 Da. In case of fetuin Ndeglycosylated peptides for quantitation of isotopic peptide ion pairs, triple play zoom scan data-dependent experiments were performed in triplicate to obtain high resolution data by acquiring one MS scan followed by one zoom scan with a mass isolation window of ±5 Da centered around the parent mass. Corresponding MS2 scans were performed with normalized

(24) Lemmel, C.; Weik, S.; Eberle, U.; Dengjel, J.; Kratt, T.; Becker, H. D.; Rammensee, H. G.; Stevanovic, S. Nat. Biotechnol. 2004, 22, 450–4.

(25) Thobhani, S.; Yuen, C. T.; Bailey, M. J.; Jones, C. Glycobiology. 2009, 19, 201–11.

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collision energy of 33 on the top three most intense peaks. Whereas, serum N-deglycosylated peptides were analyzed by normal data dependent mode method in which the instrument was set to acquire fragment ion (MS/MS) spectra on the 4 most abundant precursor ions from each MS scan with a repeat count set of 1 and duration of 30 s. Dynamic exclusion was enabled for 180 s and the collision energy was 33. In a separate experiment, serum nonglycosylated peptide mixture was analyzed by LC-MS/MS experiments (in triplicate) with a long HPLC linear gradient (4-50% B in 120 min and 50-80% in 180 min) following the experimental parameters described above for N-deglycosylated peptide analysis. Protein Identification. The raw data were converted into DTA files using Bioworks 3.1 software (Thermo electron, San Jose, CA). The DTA generation parameters were scan limits, all scans; molecular weight range, 375-1500 Da; threshold, absolute (100); precursor tolerance, 1.5 Da; group scan, 0; minimum group count, 1; minimum ion count, 10; charge state, auto; MSn level, MS2; activation type, CID. All the “dta” files were merged and converted into a single text file using an in-house software program. The proteins were identified by searching this text file of tandem mass spectrometry data in IPI human database with mascot 2.0 software (MatrixScience, London, U.K). The database search parameters were mass type-monoisotopic precursor and fragment; enzyme-trypsin; threshold-100; peptide tolerance-1.5 Da; and fragment ion tolerance-1.0. Fixed modifications: N-terminus-acetyl (1H3/2D3), C-carbamidomethylation; variable modifications: K-acetyl (1H3/2D3), N-deamidation (3 Da), M-oxidation. Proteo IQ software (Bioinquire, Athens, GA) was used to filter the search results and to group proteins and isoforms that have similar peptides. The search results, as well as the raw tandem mass spectrometry data were further verified manually and all glycopeptide sequences were identified with high confidence based on the presence of NXT/S motif and a 3 Da mass shift in b or y ions due to the modification at the N-terminus or K, respectively, by heavy acetyl (2D3) group. Quantitative Analysis. In case of fetuin glycopeptides, the mixed theoretical ratios of peptides were compared with that of observed ratios in high resolution zoom scan mass spectra. The isotopic peak envelops of fetuin glycopeptides were confirmed by MS-Isotope software (http://prospector.ucsf.edu). In order to calculate the relative standard deviation of LTL method, isotope ratios were calculated for light and heavy forms of a fetuin deglycosylated peptide LCPDCPLLAPLNDSR identified in triplicate experiments. The ratios were calculated manually by Xcalibur software (Thermo Electron, San Jose, CA) using peak areas derived from the XIC’s of the heavy isotope divided by the peak area of the light isotope. Similarly, acetyl (1H3/2D3) and 18O labeled glycopeptides in the serum analysis were quantified manually by using the Xcalibur software program. In case of multipeptide protein identification, quantification was performed for at least one glycopeptide of each modification (acetyl (1H3/2D3)+18O/acetyl (1H3/2D3)+18O+M-oxdation/acetyl (1H3/ 2 D3)+18O+esterification). The ratios of the light and heavy isotope-labeled peptides were determined by calculating the peak areas found in the corresponding extracted ion chromatograms (XIC’s) of deglycosylated peptides obtained with 1 Da mass window. These peptide ratios were also confirmed by the 9204

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relative intensities of multiply charged peptide peaks in the MS spectrum. Mean ratios were also calculated for the peptides identified with multiple modifications as light and/or heavy forms. Final protein ratios were calculated by averaging the peptide ratios of all modifications. Since there is only a 3 Da mass difference between differentially labeled peptides and overlapping of light and heavy isotopic envelops could introduce small errors in the quantitaion, we considered sialylation is increased in a peptide only if heavy/light ratio of that peptide is g2 fold. RESULTS AND DISCUSSION To demonstrate LTL proteomics strategy and to test the reproducibility of this multistep quantification method, we analyzed an N-glycosylated model glycoprotein, bovine fetuin. Two equal aliquots of fetuin samples were digested separately by trypsin and glycopeptides were enriched by using SNA lectin. Bound glycopeptides were then eluted, and labeled with light (1H6) and heavy (2D6) forms of the acetic anhydride reagent as described in the material and methods section. Light and heavy labeled glycopeptides were mixed in two different ratios (1:1 and 3:1) as outlined in Figure 1 and digested by PNGase F in the presence of H218O. The resulted N-deglycosylated peptides were analyzed by nanoLC-ESI-MS/MS in triple play mode. Fetuin protein was identified by searching tandem mass spectrometry data in bovine database using mascot search engine. The identified fetuin glycopeptides are shown in Table 1. We identified 3 N-linked glycopeptides of fetuin and similar numbers of N-linked glycopeptides were identified in previous studies that have been reported in the literature.22,26-28 The sequences of all these peptides were characterized based on their tandem mass spectrometry data and a representative tandem mass spectrum (MS/MS) in Figure 2a shows all the signature ions (b and y ions) confirming the sequence of light isotope of CH3COLCPDCPLLAPLNDSR glycopeptide. And in the MS/MS spectrum of Figure 2b, a shift of 3 Da (N f D modification) in b8, b9, and b14 ions (shown in circles) confirms the identity of heavy isotope of CD3CO-LCPDCPLLAPLNDSR glycopeptide sequence. It should be noted that asparagine in both peptides is modified to aspartic acid and the total mass is increased by 3 Da. The quantitative information was obtained from triple play LC-MS/MS experiment in which zoom scan event was used to record the high resolution MS spectra of glycopeptide precursor ions in profile mode. The mass spectra corresponding to zoom scan high resolution data of identified peptides with lowest molecular weight [LCPDCPLLAPLNDSR] and highest molecular weight [RPTGEVYDIEIDTLETTCHVLDPTPLANCSVR] are shown in Figures 3 and 4, respectively, in order to accurately delineate the changes in N-glycosylation levels. As there can be a 3 Da/6 Da shift in mass due to light and heavy acetyl groups at the N-terminus/N-terminus + K and because of the formation of multiply charged peaks and relatively a low zoom scan resolution, overlapping of isotopic peak envelope is evident (26) Thaysen-Andersen, M.; Mysling, S.; Hojrup, P. Anal. Chem. 2009, 81, 3933– 43. (27) Ritchie, M. A.; Gill, A. C.; Deery, M. J.; Lilley, K. J. Am. Soc. Mass Spectrom. 2002, 13, 1065–77. (28) Ding, W.; Nothaft, H.; Szymanski, C. M.; Kelly, J. Mol. Cell Proteomics 2009, 8, 2170–85.

Figure 1. LTL quantitative proteomics strategy used for the identification and quantification of sialylated N-linked glycopeptides in fetuin, normal serum, and PCa serum. Table 1. Mascot Search Results of N-Linked Glycopeptides Identified in LTL Quantitative Proteomics Analysis of Fetuin

in these spectra as reported by other studies.22 Furthermore, the observed ratios of light and heavy peptide LCPDCPLLAPLDDSR are in good agreement with their mixed theoretical ratios, 1:1 and 3:1 as shown in Figure 3a and 3b, respectively. We also observed similar pattern for RPTGEVYDIEIDTLETTCHVLDPTPLADCSVR peptide as corroborated by light and heavy peptide ratios in Figure 4a and 4b. The accuracy of our LTL labeling method is relevant to the high resolution capability of the mass spectrometer and concentration of glycopeptides. Accordingly, minor differences in the relative ratios of fetuin glycopeptides could be attributed to the low resolution LTQ ion trap instrument although we conducted the LC-MS/MS experiments in zoom scan mode. Therefore, we have carried out mass spectrometry experiments in triplicate and reproduced the zoom scan high resolution data for isotopic peaks of light and heavy fetuin glycopeptides (Figure S1 and S2 in Supporting Information). The measurement error associated with this method was determined based on the identification and quantification of a fetuin N-deglycosylated

Figure 2. Tandem mass spectra of light (A) and heavy (B) acetylated and 18O labeled LCPDCPLLAPLDDSR glycopeptides obtained by LTL quantitative proteomics method. Analytical Chemistry, Vol. 82, No. 22, November 15, 2010

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Figure 3. Zoom scan mass spectra showing the ratio of isotopic envelops of light and heavy acetylated and deglycosylated LCPDCPLLAPLD _ DSR peptide mixtures: (A) 1:1 and (B) 3:1.

Figure 4. Zoom scan mass spectra showing the ratio of isotopic envelops of light and heavy acetylated and deglycosylated RPTGEVYDIEIDTLETTCHVLDPTPLAD _ CSVR peptide mixtures: (A) 1:1 and (B) 3:1.

peptide LCPDCPLLAPLNDSR in triplicate experiments and relative standard deviation of the method was 9%. By and large, these results indicate that there is no significant variability and manual errors occurred in the sample preparation during the steps in LTL workflow, despite the fact that we carried out lectin enrichment before N-terminal labeling of glycopeptides as opposed to previously reported reverse order method.10 Thus, the quantitative data obtained with fetuin glycopeptides strongly suggest that this method could be applied to investigate the global glycosylation differences in cancer samples based on the relative abundance of N-deglycosylated peptides. 9206

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Next, we applied the method outlined in Figure 1, to investigate the differences in the amount of sialylated glycopeptides in normal and prostate cancer (PCa) serum samples. In this analysis, equal protein concentrations of normal and cancer serum samples (5.6 mg each) were used for in-solution digestion by trypsin. N-linked glycopeptides were enriched using SNA lectin as described for fetuin and N-glycans were removed by PNGase F digestion in the presence of H218O. The resulted N-deglycosylated peptides were further purified, fractionated and individual fractions were analyzed by data-dependent nanoLC-ESI-MS/MS experiments. The quantitative analysis of these N-deglycosylated peptides were carried out as described in the materials and methods section. The standard deviation in serum glycopeptides analysis was assumed to be 9% based on the standard deviation obtained from fetuin glycopeptide data in triplicate analysis. Table 2 summarizes the results of the identification and quantification of N-deglycosylated peptides and corresponding proteins. As evident from Table 2, sialylation is increased in majority of the proteins that are identified by multiple N-deglycosylated peptides in PCa serum sample. Only one glycoprotein is identified in which sialylation is decreased and there is no change observed in the concentration of other proteins. It should be noted that all the peptides possess NXT(S) consensus sequence and N f D modification that resulted in the PNGase F mediated removal of glycans as confirmed by tandem mass spectrometry data. For example, Figure 5 shows the tandem mass spectra of CD3COAGLQAFFQVQECD _ K peptide (ceruloplasmin) in which sialylation increased 6.8 fold and CH3CO-FDVSAMEKDASNLVKOCCH3 peptide (isoform B of transforming growth factor beta2) in which a 2 fold decrease in sialylation is observed. Quantitative ratios are very different and varied significantly between different peptide sequences of each protein suggesting the prominent differences at the site of sialylation (Table 2). In case of ceruloplasmin, except for ELHHLQEQNVSNAFLDK peptide all other peptide ratios were increased more than 2 fold (Table 2). These results are in consistent with reported findings that sialylation is independently regulated at various sites in glycoproteins.10 Indeed, our results correlate with previous findings of sialylation changes on N-linked glycopeptide glycans in other cancer serum samples. For example, immunodepletion of the abundant serum proteins in combination with WGA lectin detection enabled the observation of increased sialylation in several N-linked glycopepetides in pancreatic cancer.15 In this study, sialylated LNAENNATFYFK glycopeptide (isoform HMW of Kininogen-1) was found to be overexpressed in pancreatic cancer and we also observed a dramatic increase (5 fold) in sialylation of this peptide in PCa serum samples. Similarly, sialylation in haptoglobin (NLFLNHSENATAK) was observed to be increased in pancreatic cancer serum.15 This is consistent with our results in which we observed an increase in sialylation of two glycopeptides NLFLNHSENATAK (3.1 fold) and LHPNYSQVDIGLIK (3.8 fold) from haptoglobin. In contrast, no change in the sialylation was noticed in a recent study on glycosylation status of haptoglobin when serum samples from prostate cancer were compared to benign prostate disease or healthy subjects.29 In addition, the amount of sialylation in GLNVTLSSTGR peptide of (29) Fujimura, T.; Shinohara, Y.; Tissot, B.; Pang, P. C.; Kurogochi, M.; Saito, S.; Arai, Y.; Sadilek, M.; Murayama, K.; Dell, A.; Nishimura, S.; Hakomori, S. I. Int. J. Cancer 2008, 122, 39–49.

Table 2. List of Identified Glycosylation Sites and the Results of Quantitation of N-Desialylated Glycopeptides Obtained from Normal and Prostate Cancer Sera by Using Ltl Quantitative Proteomics Method

a The RSD of this method is 9% as determined by the reproducibility studies with fetuin. † The ratio change in these proteins is due to increase in sialylation and not due to the increase in protein concentration. * New sialylated glycopeptides identified in the current analysis.

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Figure 5. Tandem mass spectra of doubly charged ions of CD3CO-AGLQAFFQVQECD _ K and CH3CO-FDVSAMEKD _ ASNLVK peptides as identified by LTL quantitative proteomics in serum analysis. The insets in this figure show the XIC’s and quantitative ratios of light and heavy peptides as calculated manually by using Xcalibur software.

C4a protein, a putative serum biomarker to predict prostate cancer biochemical recurrence in radical retropubic prostectomy patients,30 did not change in PCa in our experiments. Our data is in agreement with previously reported studies in emphasizing that the sialylation status of the glycoproteome in cancer is highly dependent on the type of samples compared and stage of the disease and independent of the concentration of the glycoproteins. Majority of the N-deglycosylated peptides identified in this study were identified previously by various methods as sialylated glycopeptides and most of the glycosylation sites characterized in our study were also previously characterized in either normal or different cancer serum samples,10,15,21,22 demonstrating the strength of our LTL quantitative method to investigate glycosylation sites and glycosylation aberrations in cancer. Furthermore, we have also identified low abundant serum proteins including Thrombospondin131 (3.5 pg/mL), and PON 132 (25 µg/mL) in our analysis indicating the high sensitivity of the LTL strategy. Although there is high glycosylation heterogeneity in serum glycoprotein mixture, we were able to identify a small fraction of low abundant sialylated glycoproteins using the LTL method. Importantly, we identified two previously uncharacterized glycopeptides in our analysis (showed as asterisks in table 2), which further signifies the sensitivity of the method. In order to determine whether the change in serum Ndeglycosylated peptides is due to the alterations in the level of glycans at identified site or the change is at the concentration of the parent glycoprotein levels, we performed quantitative analysis of the nonglycosylated peptides obtained from the flow through (30) Rosenzweig, C. N.; Zhang, Z.; Sun, X.; Sokoll, L. J.; Osborne, K.; Partin, A. W.; Chan, D. W. J Urol. 2009, 181, 1407–14. (31) Manero, M. G.; Olartecoechea, B.; Royo, P.; Alcazar, J. L. J. Ovarian Res. 2009, 2, 18. (32) Blatter Garin, M. C.; Abbott, C.; Messmer, S.; Mackness, M.; Durrington, P.; Pometta, D.; James, R. W. Biochem. J. 1994, 304Pt 2, 549–54.

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samples of the lectin enrichment step. The nonglycosylated peptides were further purified, labeled, and analyzed by -LC-MS/ MS experiments (in triplicate) with a long gradient (180 min) and quantitative analysis was performed as described in the Materials and Methods section. We identified approximately 50 serum proteins out of which 15 glycoproteins were identified in the N-deglycosylated peptide analysis using the LTL method. Quantitative ratios were obtained for 10 out of the 15 proteins as they were identified in two or three replicates with high confidence (Table 3). For 7 of the 10 proteins (indicated with asterisks), the protein concentration remain unchanged between normal and cancer with overall heavy to light ratios ranged from 0.6 to 1.4. However, for the remaining three proteins (serotransferrin, beta2-glycoprotein and complement C4-A), we observed no change in sialylation (Table 2), and protein concentration levels (Table 3). Consequently, based on the N-deglycopeptide and nonglycosylated peptide analysis, it is evident that sialylation and not the protein levels is clearly increased in prostate cancer for these 7 up-regulated glycoproteins identified by the LTL method (see footnote † in Table 2). Interestingly, similar results were reported in a pancreatic cancer study wherein only sialylation of Haptoglobin and Hemopexin was increased without an increase in protein concentration as confirmed by lectin blot analysis.15 In summary, our quantitative data clearly demonstrates that in prostate cancer, alterations in sialylation with regard to the seven glycoproteins is independent of the concentration changes in the glycoprotein. Advantages and Limitations of LTL Strategy. There are several advantages associated with our LTL quantitative method apart from simple and efficient acetylation laleling.33 By carrying out tandem labeling (N-acetylation and 18O after lectin enrich(33) Noga, M. J.; Lewandowski, J. J.; Suder, P.; Silberring, J. Proteomics 2005, 5, 4367–75.

Table 3. List of Glycoproteins Identified and Their Quantitative Ratios Obtained from the Analysis of Nonglycosylated Peptides of Normal and Prostate Cancer Sera

ment (i) the intervention of peptides containing E, D, and C-terminus carboxylic acid esters and left over acetylation labeling reagents with lectins can be avoided while maintaining the natural peptide mixture composition during lectin enrichment process, (ii) one can eliminate additional step of treating with hydroxylamine to cleave esters, (iii) since the residual active trypsin cannot be carried over after the lectin enrichment, it will not interfere during PNGase F digestion and cannot exchange 16O isotope of C-terminus carboxylic acid with 18O isotope which could lead to the ambiguity in glycosylation site determination, (iv) there will not be any contamination from in vivo or in vitro asparagine (N) deamidation products since we introduce 18O isotope and rely on a 3 Da mass shift at the N-linked glycosylation site in both normal and cancer samples and (v) one can avoid quantitative errors due to partial reversible PNGase reaction34 that incorporates two 18O into the beta-carboxyl groups of the Asp residue and back exchange of 18O with 16O (vi) SNA lectin used in the current study is specific to R2,6-linked sialic acids and not useful to identify and probe differences in R2,3-linked sialic acids, however, this issue may be resolved by using Maackia amurensis (MAM) lectin (vii) this method can be applied to other glycan types such as fucose while the current study is focused on sialic acid glycan. This LTL method, however, can only be applied to (34) Xiong, L.; Regnier, F. E. J. Chromatogr., B 2002, 782, 405–18.

probe the differences in glycosylation in N-linked glycopeptides and cannot differentiate those glycopeptides that differ in the number of glycans. Lectin enrichment of glycopeptides, iTRAQ labeling combined with 18O labeling will be a method of choice for an alternative multiplex analysis strategy. CONCLUSIONS We have developed a method for comprehensive comparative analysis of sialylated glycopeptides and glycosylation sites in a model glycoprotein, fetuin, and normal and prostate cancer serum samples. SNA lectin affinity extraction was used for effective enrichment of N-linked sialylated glycopeptides. These peptides were differentially labeled at the N-terminus by acetyl reagents and glycosylation sites were labeled further by 18O isotope during PNGase F digestion. The PNGase F modified peptide mixtures were separated and sequenced by nanoLC-ESI-MS/MS experiments. A 3 Da mass shift due to the conversion of N f D and the presence of NXT(S) consensus sequence in glycopeptides were used as criteria for the identification of N-glycosylation sites and simultaneous quantification was achieved by calculating ratios of the relative abundance of heavy and light labeled N-deglycosylated peptide precursor ions. The applicability of this method was demonstrated by comparing normal and prostate cancer serum samples. In this analysis, we identified 45 sialylated glycopeptides and characterized 46 glycosylation sites in 30 glycoproteins. Sialylation was altered in 29 glycoAnalytical Chemistry, Vol. 82, No. 22, November 15, 2010

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peptides derived from 13 proteins in PCa serum sample. The sialylation increase and not the total protein concentration of seven glycoproteins was confirmed by the quantitative analysis of nonglycosylated peptides. Development of this LTL quantitative proteomics method opens the possibility to investigate further the glycosylation differences in cancer and benign diseases of prostate (BPH) and other cancers. In view of the fact that a disease might not only be linked with a change in protein concentration but also in the structure of a given posttranslational modification, such as glycosylation, our current results on the sialylation changes in glycoproteome provides valuable information for the development of glycopeptides as biomarkers of prostate cancer.

NOTE ADDED AFTER ASAP PUBLICATION This paper was published on October 5, 2010 with the name of the fourth author transposed. The corrected version was reposted on October 18, 2010.

ACKNOWLEDGMENT We are thankful to Bioinquire for providing us a copy of Proteo IQ software.

Received for review June 4, 2010. Accepted September 8, 2010.

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SUPPORTING INFORMATION AVAILABLE The zoom scan high resolution data for isotopic peaks of light and heavy fetuin glycopeptides obtained from triplicate analysis are provided in Figure S1 and S2. This material is available free of charge via the Internet at http://pubs.acs.org.

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