Selective Enrichment of Glycopeptides from Glycoprotein Digests

Oct 31, 2007 - a tryptic digest of ribonuclease B, bovine fetuin, and a complex mixture of glycoproteins, when compared with normal-phase chromatograp...
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Anal. Chem. 2007, 79, 8891-8899

Selective Enrichment of Glycopeptides from Glycoprotein Digests Using Ion-Pairing Normal-Phase Liquid Chromatography Wen Ding,* Jennifer J. Hill, and John Kelly*

Genomics and Proteomics Group, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A 0R6

Detailed structural analysis of glycoproteins requires methods capable of isolating glycopeptides from tryptic digests of purified glycoproteins and complex protein mixtures. Here, we describe the selective and reproducible isolation of glycopeptides from a peptide mixture using ion-pairing normal-phase chromatography (IPNPLC). The addition of inorganic monovalent ions in normal-phase chromatography appears to increase the hydrophobicity difference between peptides and glycopeptides, allowing for more efficient separation. Our data show that IP-NPLC effectively enriches glycopeptides from a tryptic digest of ribonuclease B, bovine fetuin, and a complex mixture of glycoproteins, when compared with normal-phase chromatography alone. The results of the IP-NPLC experiments can be explained using the Wimley-White water/octanol free energy scale to illustrate the hydrophobicity difference of nonglycosylated peptides with and without ion-pairing. We believe that IP-NPLC will be an important tool in glycoprotein characterization and glycoproteomic studies. Protein glycosylation is an abundant and biologically significant post-translational modification, most commonly associated with secreted and membrane proteins.1-3 In fact, many clinical biomarkers and therapeutic targets are glycoproteins.4-9 The full characterization of a single glycoprotein, i.e., glycosylation site identification, glycan structure determination, and analysis of the glycan isoforms at each linkage site, remains very challenging.10-13 * To whom correspondance should be addressed. E-mail: Wen.Ding@ nrc-cnrc.gc.ca; [email protected]. (1) Jaeken, J.; Matthijs, G. Annu. Rev. Genomics Hum. Genet. 2001, 2, 129151. (2) James N.; Arnold, M. R. W.; Robert B.; Sim, Pauline M.; Rudd, Raymond A.; Dwek. Annu. Rev. Immunol. 2007, 25. (3) Rudd, P. M.; Elliott, T.; Cresswell, P.; Wilson, I. A.; Dwek, R. A. Science 2001, 291, 2370-2376. (4) Ferrara, N.; Kerbel, R. S. Nature 2005, 438, 967-974. (5) Burton, D. R.; Dwek, R. A. Science 2006, 313, 627-628. (6) Ferrari, L.; Seregni, E.; Martinetti, A.; Van Graafeiland, B.; Nerini-Molteni, S.; Botti, C.; Artale, S.; Cresta, S.; Bombardieri, E. Int. J. Biol. Markers 1998, 13, 3-9. (7) Urban, D.; Myers, R.; Manne, U.; Weiss, H.; Mohler, J.; Perkins, D.; Markiewicz, M.; Lieberman, R.; Kelloff, G.; Marshall, M.; Grizzle, W. Eur. Urol. 1999, 35, 429-438. (8) Huang, X.; Ushijima, K.; Komai, K.; Takemoto, Y.; Motoshima, S.; Kamura, T.; Kohno, K. Gynecol. Oncol. 2004, 93, 287-291. (9) Myers, R. B.; Grizzle, W. E. Biotechnol. Histochem. 1997, 72, 86-95. 10.1021/ac0707535 CCC: $37.00 Published 2007 Am. Chem. Soc. Published on Web 10/31/2007

Furthermore, due to the relatively low ionization efficiency of glycopeptides with respect to unmodified peptides as well as the signal dilution that results from the distribution of glycan species at each linkage site, glycopeptide ions are frequently “lost in the noise” in digests of purified glycoproteins. The challenge is even greater when analyzing glycopeptides from digests of complex protein mixtures. For this reason, there is growing interest in methods that can selectively isolate glycopeptides from proteolytic digests of complex protein mixtures for subsequent analysis by reversed-phase liquid chromatography electrospray ionization mass spectrometry (RPLC-ESI-MS).14-18 Several methods have been described for the isolation of glycopeptides from digests of purified glycoproteins and from more complex protein mixtures. Affinity chromatography using lectins, naturally occurring proteins that bind to specific glycans, is used for enriching glycopeptides. However, each lectin species isolates only a subset of glycan species.19-22 Recently, hydrazide chemistry was used to selectively isolate, identify, and quantify N-linked glycopeptides in a very specific and efficient manner.18 This work flow does not provide structural information on the sugar due to the destruction and removal of the glycan moieties.14 Glycopeptides can be isolated from a peptide mixture on the basis of hydrophilicity and electrostatic interactions by using hydrophilic interaction liquid chromatography (HILIC).15,23 However, HILIC could not enrich high-mannose glycopeptides from a tryptic digest of ribonuclease B.15 It should also be possible to (10) Domon, B.; Aebersold, R. Science 2006, 312, 212-217. (11) Dell, A.; Morris, H. R. Science 2001, 291, 2351-2356. (12) Medzihradszky, K. F.; Maltby, D. A.; Hall, S. C.; Settineri, C. A.; Berlingame, A. L. J. Am. Soc. Mass Spectrom. 1994, 5, 350-358. (13) Peterman, S. M.; Mulholland, J. J. J. Am. Soc. Mass Spectrom. 2006, 17, 168-179. (14) Lewandrowski, U.; Moebius, J.; Walter, U.; Sickmann, A. Mol. Cell Proteomics 2006, 5, 226-233. (15) Hagglund, P.; Bunkenborg, J.; Elortza, F.; Jensen, O. N.; Roepstorff, P. J. Proteome Res. 2004, 3, 556-566. (16) Wada, Y.; Tajiri, M.; Yoshida, S. Anal. Chem. 2004, 76, 6560-6565. (17) Tajiri, M.; Yoshida, S.; Wada, Y. Glycobiology 2005, 15, 1332-1340. (18) Zhang, H.; Li, X. J.; Martin, D. B.; Aebersold, R. Nat. Biotechnol. 2003, 21, 660-666. (19) Ghosh, D.; Krokhin, O.; Antonovici, M.; Ens, W.; Standing, K. G.; Beavis, R. C.; Wilkins, J. A. J. Proteome Res. 2004, 3, 841-850. (20) Madera, M.; Mechref, Y.; Novotny, M. V. Anal. Chem. 2005, 77, 40814090. (21) Qiu, R.; Regnier, F. E. Anal. Chem. 2005, 77, 7225-7231. (22) Yang, Z.; Hancock, W. S. J. Chromatogr., A 2004, 1053, 79-88. (23) Thaysen-Andersen, M.; Hojrup, P. Am. Biotechnol. Lab. 2006, 24, 14-17.

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enrich glycopeptides by forming temporary covalent bonds between the hydroxyl groups on R-aminophenylboronic acid media and the glycan moieties containing 1,2 cis-diol groups. However, attempts to efficiently recover high-mannose-type glycopeptides have failed so far.24 Hydrophilic affinity isolation using Sepharose or cellulose media is based on hydrogen bonding between the hydroxyl groups of the media and those of the analyte.16,17 This method was used successfully to isolate various N-linked glycan structures including sialylated, fucosylated, and high-mannose types. In addition, this method preserves the glycan moiety for further structure analysis. However, hydrophilic peptides rich in serine, threonine, aspartic acid, and glutamic acid residues also preferentially bind to the media and are likely to contaminate the enriched glycopeptide sample. Charged peptides are known to form neutral ion pairs with oppositely charged counterions in solution phase.25,26 Wimley et al. demonstrated that peptides in neutral forms have much higher hydrophobicity than those in charged forms.27,28 In this report, we take advantage of ion-pairing (IP) in normal-phase liquid chromatography (NPLC) to increase the hydrophobicity difference between peptides and glycopeptides in order to isolate the latter from tryptic peptide mixtures. We demonstrate here that the neutralization of peptides by ion-pairing is able to effectively reduce, and often eliminate, nonglycosylated peptides in the enriched glycopeptide fraction. EXPERIMENTAL SECTION Materials. Bovine ribonuclease B (RNase B), bovine fetuin, dithiothreitol (DTT), iodoacetamide, wheat germ agglutinin (WGA) agarose, and Sepharose CL-6B were acquired from Sigma (St. Louis, MO) as were all the ion-pairing reagents used in this report. Modified trypsin was purchased from Promega (Madison, WI). The JM01 murine epithelial breast cancer cells were kindly provided by Dr. Anne Lenferink and Dr. Maureen O’ConnorMcCourt (NRC-Biotechnology Research Institute, Montreal, QC, Canada). Tryptic Digestion of the Standard Glycoproteins. A 100µL solution of bovine RNase B or fetuin at 10 µg/µL in 50 mM ammonium bicarbonate was reduced with 100 µL of 2 mM DTT at 37 °C for 1 h and alkylated with 40 µL of 50 mM iodoacetamide at 37 °C for 30 min. The reagents used for reduction and alkylation were removed by centrifugal ultrafiltration (3000 MWCO). The protein solution (1 mL with 50 mM ammonium bicarbonate) was incubated at 37 °C for 16 h after addition of 60 µL of trypsin solution (0.33 µg/µL). Preparation of the JM01 Protein Extracts for IP-NPLCMS Analysis. Approximately 150-µL bead volume of WGA agarose was washed 3× with 300 µL of binding buffer (BB: 50 mM Tris-HCl, pH 6.5, 150 mM NaCl, 0.1 mM MnCl2, 0.5% Triton X-100) in a minispin column, before loading 850 µg of total protein (24) Grace, M, C.; Weibin Chen, P, J.; Lee, Y,-Q, Y,; Gebler, J. C. ASMS 2005, San Antonio, TX, 2005. (25) Muller, P. Pure Appl. Chem. 1994, 66, 1126-1127. (26) Hancock, W. S.; Bishop, C. A.; Prestidge, R. L.; Harding, D. R.; Hearn, M. T. Science 1978, 200, 1168-1170. (27) Wimley, W. C.; Creamer, T. P.; White, S. H. Biochemistry 1996, 35, 51095124. (28) Wimley, W. C.; Gawrisch, K.; Creamer, T. P.; White, S. H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 2985-2990.

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from a JM01 cell lysate in BB by gravity. The WGA agarose was washed 3× with 500 µL of BB before eluting with 4 × 125 µL volumes of elution buffer (50 mM Tris-HCl, pH 6.5, 500 mM NaCl, 0.1 mM MnCl2, 0.5% Triton X-100, 0.3 M N-acetylglucosamine). The combined elution fractions were concentrated by acetone precipitation and resuspended in 40 µL of 50 mM Tris, pH 7.4, 150 mM NaCl, and 0.5% SDS. The elution fractions were then reduced, alkylated, and digested with trypsin using a “tube gel” digestion, essentially as described.29-3029-30 Peptides were extracted from the gel in 100 µL of 50% ACN/5%AcOH, dried under vacuum, and resuspended in 30 µL of 2%ACN/1%AcOH for analysis by IP-NPLC. Ion-Pairing Normal-Phase Liquid Chromatography Separation of Glycopeptides. All ion-pairing normal-phase chromatography experiments were performed on a CapLC capillary liquid chromatography system coupled to a Q-TOF-2 hybrid quadrupole/ time-of-flight mass spectrometer (Waters, Milford, MA). The normal-phase column (4 cm × 1 mm) was packed and used at a pressure of less than 40 psi using 15 µL of Sepharose CL-6B gel. The NPLC and IP-NPLC analyses were performed as follows: (1) The column was equilibrated with 75% solvent A, 1-butanol/ ethanol/water (10:3:1, v/v), and 25% solvent B, water + 0.2% formic acid for 15 min at pH 3.7 at a flow rate of 10 µL/min. The flow was split after the column so that ∼300 nL/min was directed to the ESI source to obtain NPLC-ESI-MS or IP-NPLC-ESI-MS chromatograms for each sample. The remainder of the column eluate was directed to a fraction collector or discarded. (2) The sample, typically 10 uL, was then injected onto the column and washed for 10 min using the column equilibration conditions. For NPLC experiments, the sample was simply dissolved in water. For IP-NPLC experiments, ion-pairing reagents were added to the aqueous sample prior to injection. (3) The unbound peptides eluted from the column between 3 and 12 min (referred to below as the “unbound fraction”). (4) Solvent B was increased linearly to 50% in 5 min in order to release the “bound fraction”, rich in glycopeptides. Typically, the retained peptides and glycopeptides eluted between 20 and 30 min. (5) The column was then washed using 100% B for 5 min followed by 15 min of re-equilibration prior to injecting the next sample. In our experience, the Sepharose columns could be used for more than a month of continuous use under these conditions. Nano-Reversed-Phase HPLC Mass Spectrometric (nanoRPLC-MS) Analysis of Peptide Mixtures from Complex Protein Extracts. The tryptic digest of WGA-isolated glycoproteins from the JM01 whole cell lysate (10 µL) was subjected to IP-NPLC for off-line isolation of glycopeptides with 50 mM NaOH added to the sample prior to sample injection. The bound and unbound fractions (∼100 µL for each fraction) were collected and dried to ∼1 µL, which were then resuspended in 10 µL of 1% acetic acid (aq) and analyzed by nanoRPLC-MS using a CapLC capillary liquid chromatography system coupled to a Q-TOF Ultima hybrid quadrupole/time-of-flight mass spectrometer (Waters). The peptides were first loaded onto a 300 µm i.d. × 5 mm C18 PepMap100 trap (LC Packings, San Francisco, CA) and then eluted off to a Picofrit column (New Objective, Woburn, MA) using a linear (29) Lu, X.; Zhu, H. Mol. Cell. Proteomics 2005, 4, 1948-1958. (30) Crabb, J. W.; Carlson, A.; Chen, Y.; Goldflam, S.; Intres, R.; West, K. A.; Hulmes, J. D.; Kapron, J. T.; Luck, L. A.; Horwitz, J.; Bok, D. Protein Sci. 1998, 7, 746-757.

Figure 1. IP-NPLC enrichment of glycopeptides from a tryptic digest of RNase B. (a) Mass spectrum from infusing 0.45 pmol (actual amount infused) of RNase B digest dissolved in 35% solvent B + 65% solvent A; (b) total ion mass spectrum from NPLC-ESI-MS of peptides in the bound fraction from 15 pmol of an RNase B tryptic digest injected in H2O; (c) total ion mass spectrum from IP-NPLC-ESI-MS of peptides in the bound fraction from 15 pmol of tryptic peptides of RNase B injected in H2O + 50 mM NaCl. Note: all abundant ions that are not labeled in Figure 1 are singly charged contaminants. Partial in-source collision-induced dissociation of the high-mannose-type glycopeptides from RNase B occurred leading to the loss of one or two mannose residues from the glycopeptides, as evidenced by the mass spectral peaks at m/z 765.31 (2+) and 684.31 (2+) in Figure 1c.

gradient from 5 to 42% solvent B (acetonitrile, 0.2% formic acid) in 23 min, 42-95% solvent B in 3 min. Solvent A was 0.2% formic acid in water. NanoRPLC-MS/MS analysis was also performed with data-dependent acquisition for the total digest of the WGAisolated proteins from the JM01 whole cell lysate as well as the bound fraction isolated by IP-NPLC. RESULTS AND DISCUSSION IP-NPLC for Isolating Tryptic Glycopeptides. RNase B is a well-characterized glycoprotein containing a single N-linked glycosylation site occupied by five, closely related high-mannosetype glycans.31 Thus, the tryptic digestion of RNase B results in five glycopeptides: NLTK-Glc2Man5-9. The mass spectrum acquired by infusing a tryptic digest of RNase B is presented in Figure 1a. The ions annotated i-ix in Figure 1a correspond to nonglycosylated peptides representing 94% of the amino acid sequence of RNase B. Even though this RNase B digest is a (31) An, H. J.; Peavy, T. R.; Hedrick, J. L.; Lebrilla, C. B. Anal. Chem. 2003, 75, 5628-5637.

relatively simple mixture, the mass spectral peaks of the five N-linked glycopeptides from RNase B are barely detectable (Figure 1a), principally due to the lower ionization efficiency of glycopeptides compared with unmodified peptides. The glycopeptide ions are more prominent when samples are fractionated into bound and unbound fractions by NPLC prior to electrospray ionization (Figure 1b), though the nonglycosylated peptide ions still dominate. However, when 50 mM NaCl is added to the sample prior to injection onto the normal-phase column, the nonglycosylated peptides (i-ix) are almost entirely removed and the spectrum of the bound fraction is dominated by the RNase B glycopeptide ions (Figure 1c). Thus, the presence of sodium chloride in the sample prevents the nonglycosylated peptides, but not the glycopeptides, from binding to the Sepharose column. Lithium chloride and sodium hydroxide were also shown to effectively enrich glycopeptides from RNase B at a concentration of 50 mM (Supporting Information (SI) Figure S-1). These results suggest that NaCl, LiCl, and NaOH all work well as ion-pairing reagents in NPLC. Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

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Figure 2. IP-NPLC enrichment of sialylated glycopeptides from a tryptic digest of fetuin. (a) Mass spectrum from infusing 1 pmol (actual amount infused) of bovine fetuin digest dissolved in 35% solvent B + 65% solvent A; (b) total ion mass spectrum from NPLC-ESI-MS of peptides in the bound fraction from 8.4 pmol of tryptic peptides of fetuin injected in H2O; (c) total ion mass spectrum from IP-NPLC-ESI-MS of peptides in the bound fraction from 8.4 pmol of a tryptic digest of fetuin injected in H2O + 50 mM LiCl. BPI: base peak ion count for the most abundant glycosylated peptides. The ion at m/z 1312.45 (5+) is the monosodium adduct of the pentuply charged glycopeptide, T54-85. The other peaks near the labeled glycopeptide ions are their sodium and lithium adducts. All other abundant ions that are not labeled are singly charged contaminants.

The addition of monovalent salt ions to the sample solution is also effective in isolating more complex glycopeptides from the tryptic digest of bovine fetuin by NPLC (Figure 2). Infusion-MS analysis of the fetuin tryptic digest yielded a spectrum dominated by nonglycosylated peptides, annotated i-xx, representing 63% of this protein’s amino acid sequence (Figure 2a). Here again, no fetuin glycopeptides were observed in the infusion-MS spectrum. Fractionation by NPLC prior to ESI-MS succeeded in removing or reducing peptides i-xi, but the fetuin glycopeptides are still barely detectable (Figure 2b). However, when 50 mM LiCl is added to the sample injection solution, the relative abundance of the N-linked glycopeptides attached to three of the known N-glycosylation sites of fetuin (Asn81, Asn138, Asn158) is increased such that they dominate the upper half of the mass spectrum (Figure 2c).12,13 The fetuin N-linked glycans are complextype glycans and thus are highly sialylated. Therefore, IP-NPLC performs equally well with complex glycans as with the highmannose glycans of RNase B. Due to ion suppression, no O-linked fetuin glycopeptides were observed by direct analysis by IP-NPLC8894 Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

MS. However, the O-linked glycopeptide (T228-288) was detected by reversed-phase LC-MS analysis of the bound fraction (see SI Figure S-3), suggesting that this low-occupancy glycopeptide was retained in the bound fraction. To test the effect of salt concentration on glycopeptide recovery, five different concentrations of LiCl were added. With fetuin, increasing the salt concentration appeared to improve the performance of IP-NPLC to enrich the MS signal of glycopeptides relative to nonglycosylated peptides (Supporting Information Figure S-2). However, with RNase B, increased salt concentrations did not consistently improve performance (data not shown). Thus, we suggest an initial salt concentration of 50 mM for samples that are being analyzed for the first time. The sialic acid-containing glycopeptides from fetuin bind metal ions more strongly than the high-mannose glycopeptides from RNase B. Thus, we observe Li+ and Na+ adduct ions for the fetuin glycopeptides when the bound fraction from the IP-NPLC experiment is eluted directly into the mass spectrometer (see Supporting Information Figure S-2). Interestingly, the sodium adducts are

Figure 3. Addition of monovalent ions leading to the relocation of a nonglycopeptide from the bound to the unbound fraction. Extracted ion chromatogram of m/z 834.12 (3+) of a hydrophilic nonglycosylated peptide (viii), CKPVNTFVHESLADVQAVCSQK, from the tryptic digest of RNase B. Samples injected with (a) 1, (b) 5, (c) 10, (d) 20, and (e) 50 mM NaCl as an ion-pairing reagent. The insets in (a) and (c) show the combined mass spectra of m/z 834.12 (3+) corresponding to the unbound fractions (2-12 min) and the bound fractions (20-30 min) as shown. The total ion counts (TIC) of m/z 834.12 (3+) in the bound fraction are shown in (a)-(e). TIC in (inset a) and (inset c) shows the abundance of m/z 834.12 (3+) in the unbound fraction for comparison. The peak tailing of the unbound fraction in (b) and (c) and the second peak of the unbound fraction in (d) and (e) were due to the presence of sodium adducts which yielded a spectral peak at m/z 834.12 (3+). The N-terminus of this peptide was cyclized with loss of NH3 to yield m/z 834.12 (3+).30

quite prominent in the bound fraction even when 1 M LiCl is added to sample as the ion-pairing agent. Importantly, these adduct ions disappear when the bound fraction is separated by HPLC prior to ESI-MS, a common analytical work flow. The removal of nonglycosylated peptides from the bound fraction on the NPLC column by the addition of salt is mirrored by an increase in their abundance in the unbound fraction (Figure 3). For example, when the NaCl concentration of the injection solution is 1 mM, the nonglycosylated tryptic peptide from RNase B, CKPVNTFVHESLADVQAVCSQK, elutes late, ∼24 min, as part of the bound fraction (Figure 3a). When the sodium chloride concentration is increased to 10 mM, ∼85% of this peptide elutes early (∼4 min) from the column (Figure 3c). The remaining 15% of the same non-glycopeptide still elutes at ∼24 min, indicative of partial binding to the Sepharose beads at 10 mM NaCl. As the sodium chloride concentration is increased to 50 mM, virtually all of this peptide elutes early from the column as part of the unbound fraction (Figure 3e). Similar retention time shifts are

found for the other nonglycosylated peptides in the RNase B digest (data not shown), suggesting there is a common interaction between the sodium chloride ions and the peptides that effectively increases the observed hydrophobicity of the peptides and prevents them from binding to the column. In addition to improved removal of nonglycosylated peptides from tryptic protein digests, IP-NPLC often increases the actual MS signal intensity of glycopeptides, presumably due to lowered ion suppression. The relative MS signal intensities of fetuin glycopeptides from the bound fraction of IP-NPLC range from 40 to 254% of the MS signal acquired from an unfractionated digest (Supporting Information Figure S-3). In fetuin, four out of five glycopeptides had higher MS signal intensities in the IP-NPLC bound fraction than in the unfractionated tryptic digest. Similar experiments with RNaseB showed a relative glycopeptide signal intensity of 16% after IP-NPLC (data not shown). In all cases, the relative MS signal of glycopeptides to nonglycosylated peptides was improved by IP-NPLC fractionation. Hydrophobicities of Charged and Neutral Nonglycosylated Peptides. In order to better understand the effect of salt on peptide binding in normal-phase chromatography, the hydrophobicities of nonglycosylated tryptic peptides from both RNase B and fetuin were calculated at pH ∼3.7 (the approximate pH of mobile phase for sample loading) using the MPExTotalizer program32 (Figure 4), which is based on the Wimley-White water/octanol free energy scale for the 20 amino acids.27,28,33 According to this scale, a greater energy value (more positive) on the y-axis (the free energy value of transfer ∆Goctw from 1-octanol to water, kcal/mol) of a peptide indicates a higher hydrophobicity of the peptide. The nonglycosylated peptides of the fetuin digest that are removed from the bound fraction in NPLC (i-xi) have charged hydrophobicity values higher (more positive) than -12.2 kcal/mol, while the nonglycosylated peptides that coelute with glycopeptides in the bound fraction (xii-xviii) have hydrophobicity values lower than -12.2 kcal/mol (Figure 4b, white bar, Figure 2b). Similarly, only nonglycosylated peptides with charged hydrophobicity values lower than -12.2 kcal/mol are observed in the bound fraction using IP-NPLC (Figure 4, Figures 1c, 2c). Thus, the NPLC experimental data correlate very well with the calculated hydrophobicities. According to the Wimley-White water/octanol free energy scale, the neutralization of charged moieties on a peptide can significantly increase the overall hydrophobicity of a peptide.27,28,32 The difference in hydrophobicity values for the neutral and charged forms of peptide moieties of interest are shown in SI Tables S-1 and S-2.27,28,32 The hydrophobicity values for neutralized nonglycosylated peptides from RNase B and fetuin are shown in the black bars in Figure 4a and b, respectively. For example, at pH 3.7, the charged form of peptide viii from RNase B, CKPVNTFVHESLADVQAVCSQK, has a hydrophobicity value of -16.9 kcal/mol while its neutral form only has a hydrophobicity value of -3.9 kcal/mol.32 In other words, the neutralization of charged moieties on this peptide causes a significant increase in its hydrophobicity. Similar reductions in hydrophobicity values upon (32) Jaysinghe, S.; Hristova, K.; Wimley, W. C.; Snider, C.; White, S. H. http:// blanco.biomol.uci.edu/mpex, 2006. (33) Hessa, T.; Kim, H.; Bihlmaier, K.; Lundin, C.; Boekel, J.; Andersson, H.; Nilsson, I.; White, S. H.; von Heijne, G. Nature 2005, 433, 377-381.

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Figure 4. (a) Hydrophobicity values, free energies of transfer ∆Goctw (kcal/mol) from 1-octanol to water (octw), of nonglycosylated peptides from a tryptic RNase B digest using MPExTotalizer (i-ix). On this scale, a higher (more positive) hydrophobicity value indicates a higher observed hydrophobicity. 0, Charged nonglycosylated peptides (pH3.7); 9, neutral nonglycosylated peptides. (b) Hydrophobicity values of nonglycosylated peptides (i-xx) from a tryptic bovine fetuin digest. 0, Charged nonglycosylated peptides (pH3.7); 9, neutral nonglycosylated peptides. Glu0, Asp0, His+, Lys+, Arg+, N-T NH3+, and C-T COO- are used for calculation of the charged nonglycosylated peptides; Hydrophobicity values of neutral peptides are calculated by assuming the charged groups of peptides are all neutralized by protonation as defined by the MPExTotalizer program.32 For Arg+ and Lys+ to become neutral Arg0 and Lys0 respectively, an empirically estimated value of 2 kcal/mol was further added to the values obtained by using MPExTotalizer since it does not calculate this conversion, due to the difficulties of determining the values for Arg0 and Lys0 (see SI Tables S-1 and S-2 for additional information). The line at -12.2 kcal/mol indicates the estimated hydrophobicity threshold below which (i.e., more negative) a nonglycosylated peptide may be observed in the bound fraction by IP-NPLC.

neutralization were obtained for the other nonglycosylated tryptic peptides of RNase B and fetuin (black bars, Figure 4). Charged peptides are known to form ion pairs with oppositely charged counterions in solution by Coulombic attraction.25 Therefore, when sodium chloride is added to the injection solution, the positively charged moieties of the tryptic peptides associate with negatively charged chloride ions to form neutral ion pairs. Similarly, the negatively charged peptide moieties are neutralized by the positively charged sodium ions. Thus, it is possible for peptide molecules to be converted into their neutral forms by ionpairing and become more hydrophobic as a result. We hypothesize 8896

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that this phenomenon is responsible for the results we observed in our NPLC experiments. Ion-pairing with salt ions may increase the hydrophobicity of the nonglycosylated peptides, thus diminishing their ability to bind to the neutral Sepharose column. On the other hand, glycopeptides can interact via hydrogen bonding with the Sepharose column through the hydroxyl groups present in the saccharide moieties. This form of association is not significantly affected by the presence of salt, and the affinity for the glycopeptides for the Sepharose beads remains high. In fact, addition of monovalent salt ions was illustrated to diminish the hydrophobic/electrostatic interactions between peptides and gel

Figure 5. Effect of different ion-pairing reagents on glycopeptide enrichment. (a) Plot of the ratio of the ion abundance of NLTKGlcNAc2-Man5 at m/z 846.42 (2+) over that of the nonglycosylated peptide (viii), CKPVNTFVHESLADVQAVCSQK (m/z 834.12 (3+)), in the bound fraction at m/z 834.12 (3+) of the RNase B digest versus the concentration of different ion-pairing reagents used (ln scale). (b) Plot of the ratio of ion abundance of NLTK-GlcNAc2-Man5 at m/z 846.42 (2+) over that of a nonglycosylated peptide (ix) rich in hydroxyl groups, QHMDSSTSAASSSNYCNQMMK (m/z 789.91(3+)), in the bound fraction at m/z 789.91 (3+) of the RNase B digest versus the concentration of different ion-pairing reagents used (ln scale). At 50 mM AgNO3, the nonglycosylated peptide (viii) cannot be detected at all, leading to a finite ratio; thus, the last data point was not shown for line 2. To avoid overlapping, only half of the error bars are shown.

matrix in size-exclusion liquid chromatography, though without explanation in terms of an ion-pairing effect.34,35 Effect of Other Ion-Pairing Reagents on IP-NPLC of Glycopeptides. The effect of various ion-pairing reagents on the removal of nonglycosylated peptides was examined. Five different concentrations of each ion pair reagent (1, 5, 10, 20, and 50 mM) were tested for their ability to selectively enrich an RNase B glycopeptide, NLTK-GlcNAc2-Man5, relative to one of two nonglycosylated peptides (peptides viii Figure 5a and ix Figure 5b). On these plots, a higher ratio (ion counts of a glycopeptide over that of a nonglycosylated peptide, e.g., in Figures 1c, 2c) indicates more efficient removal of the nonglycosylated peptide by the specific ion-pairing reagent. Ion-pairing reagents that are commonly used in reversed-phase liquid chromatography, such as weakly hydrophobic triethylamine (TEA) (line 7) and strongly hydrophobic tetrabutylammonium phosphate (TBAP) (line 6) are generally far less effective than LiCl (line 1), NaCl (line 4), KCl (line 3), and AgNO3 (line 2). The inorganic salts, LiCl, NaCl, and KCl, are almost equally effective, while inorganic salts such as Ni(NO3)2 (line 5), CaCl2, and MnCl2 (data not shown for the latter two) are ineffective. We believe that TEA and TBAP, both of which are large in size compared with small inorganic ions, may be ineffective as the result of steric effects that prevent simultaneous (34) Rubin, C. S.; Swergold, G. D. Anal. Biochem. 1983, 131, 295-300. (35) Mant, C. T., Parker, J. M. R., Hodges, R. S. J. Chromatogr. 1987, 397, 99112.

Figure 6. IP-NPLC-MS as a quantitative tool for glycopeptide analysis. (a) 0, Relative abundance of each glycopeptide of RNase B obtained in the bound fraction of IP-NPLC in this report. 9, Relative abundance of each glycopeptide of RNase B as previously reported.31 (b) A plot of the relative ion intensity of the doubly protonated ion at m/z 846.42 corresponding to the RNase B glycopeptide, NLTKGlcNAc2-Man5, in the bound fraction versus the amount of RNase B tryptic digest injected onto the NPLC column. The analysis was performed in triplicate with five sample loadings (3.3, 6.6, 9.9, 19.8, 26.4, and 39.6 pmol, respectively), and the sample injection solution contained 50 mM NaCl. The x-axis is the amount of the RNase B tryptic digest introduced into the mass spectrometer (1/33 of the sample loading after splitting).

Figure 7. Analysis of a complex peptide mixture by IP-NPLC. (a) Base peak ion chromatograms of nano-reversed-phase LC-ESI-MS of tryptic peptides from WGA isolated proteins from a JM01 whole cell lysate. (a) Bound fraction; (b) unbound fraction; (c) total digest. BPI: base peak ion counts. The sample subjected to IP-NPLC for isolation of glycopeptides contained 50 mM NaOH, as an ion-pairing reagent.

ion-pair formation with all of the charged groups of a nonglycosylated peptide.36 In contrast, intermolecular electrostatic interaction involving a single charged site of a peptide may be sufficient for the ion-pairing process to be effective in reversed-phase liquid (36) Katsuta, S.; Ishitani, T.; Suzuki, M.; Ishii, Y.; Kudo, Y.; Takeda, Y. J. Solution Chem. 2004, 33, 437-451.

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Figure 8. Effective enrichment of a sialylated glycopeptide from a complex peptide mixture. The combined MS spectra from a retention time window of 20.7-21.7 min of the nanoRPLC-ESI-MS chromatograms from the (a) bound fraction, (b) unbound fraction, and (c) total digest. The insets show the extracted ion chromatograms (XICs) of 1545.65 (3+) of a glycopeptide from the tryptic digest of WGA isolated glycoproteins from the JM01 cells. (d) MS/MS spectrum of the same glycopeptide in the bound fraction. BPI: base peak ion counts.

chromatography. Unlike monovalent metal ions, the intermolecular electrostatic interaction between divalent metal ions and a single negatively charged site of peptides is unlikely to lead to direct formation of a neutral ion pair. Therefore, Ni2+, Ca2+, and Mn2+ were found to be far less effective than Li+, Na+, and K+ and Ag+ at reducing the abundance of nonglycosylated peptides in the bound fraction using IP-NPLC. Similar plots to Figure 5a can be obtained for other nonglycosylated peptides of RNase B or bovine fetuin digests (data not shown). Silver nitrate, but not LiCl, NaCl, or KCl, was found particularly to be effective in removing RNase B nonglycosylated peptide (ix), a peptide that is rich in serine residues that are capable of forming hydrogen bonds (Figure 5b). Complete removal of this peptide was achieved by addition of 50 mM AgNO3 in the sample injected. This result seems to suggest that Ag+ is capable of preventing 8898

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hydrogen-bonding formation37 or long-range trapping of this particular peptide.38 Another possible explanation may be that Ag+ ion may lead to folding of this peptide, producing a higher effective hydrophobicity favorable for its removal.39 Unfortunately, silver nitrate formed a precipitate when added to the fetuin digests, which excluded its further use for isolating glycopeptides in this report. IP-NPLC for Quantitative Glycoprotein Profiling. To illustrate that IP-NPLC can enrich glycopeptides reproducibly for more quantitative analysis, the relative abundance of each glyco(37) Sachs, J. N.; Nanda, H.; Petrache, H. I.; Woolf, T. B. Biophys. J. 2004, 86, 3772-3782. (38) Xu, X. H.; Yeung, E. S. Science 1998, 281, 1650-1653. (39) Spiga, O.; Scarselli, M.; Bernini, A.; Ciutti, A.; Giovannoni, L.; Laschi, F.; Bracci, L.; Niccolai, N. Biophys. Chem. 2002, 97, 79-86.

peptide, NLTK-Glc2Man5-9, enriched from an RNase B tryptic digest is presented in Figure 6a. The relative abundance of these five glycopeptides obtained using IP-NPLC-ESI-MS (black bars) is the same as that obtained by direct LC-MS analysis of the tryptic digest (data not shown) and is essentially identical to that reported recently (white bars).31 To further illustrate the quantitative nature of IP-NPLC-MS, we plotted the relative ion abundance of the RNase B glycopeptide, NLTK-GlcMan5, that resulted from loading five different amounts (between 3.3 and 39.6 pmol) of RNase B onto the normal-phase column (Figure 6b). The linearity of the plot (r2 ) 0.98) and the good reproducibility of the replicates (RSD e16%) indicates that IP-NPLC is capable of recovering this glycopeptide quantitatively over the concentration range examined. Isolation of Glycopeptides from Complex Peptide Mixtures Using IP-NPLC. Glycoproteins from JM01 murine epithelial breast cancer cells were enriched on a lectin affinity column and subjected to tryptic digestion and analysis by IP-NPLC/RPLCMS. The base peak chromatograms (BPCs) of the bound and flowthrough IP-NPLC fractions, as well as the total digest, are presented in Figure 7. The BPCs of the flow-through fraction and total digest (Figure 7b and c, respectively) are very similar while that of the bound fraction (Figure 7a) is far less complex because of effective removal of nonglycosylated peptides from the bound fraction (SI Figure S-5). The bulk of this digest consists of nonglycosylated peptides, which should not be bound by the Sepharose column. In fact, the signal intensity ratio between the bound and flow-through fractions for the majority of the nonglycosylated peptides is ∼1:99. IP-NPLC enriched many glycopeptides in a very efficient manner, and an example is presented in Figure 8. A relatively large ion (m/z 1545.65 (3+)) was observed at ∼21 min in the RPLC-MS run of the bound fraction (Figure 8a). Though this ion was quite prominent in the bound fraction (inset in Figure 8a), it was not observed in the analyses of the unbound fraction or the total digest (insets of Figure 8b and c, respectively). The MS/MS spectrum of this ion clearly indicates that it is a sialylated glycopeptide (Figure 8d), though the determination of its amino acid sequence would require MS3 analysis. This impressive degree of enrichment was observed for other glycopeptides as well, though not all performed to the same level. These preliminary data, combined with the results obtained from standard proteins, suggest that IP-NPLC will be an effective tool for enriching glycopeptides from the digests of complex protein extracts. IP-NPLC may be used in partnership with, or as a viable alternative to, lectin affinity enrichment for glycoproteomic analysis. Future studies will further develop IP-NPLC in order to fully explore its potential in this regard.

CONCLUSIONS We report here the development of a new NPLC method for enriching glycopeptides from protein digests using Sepharose media. Significant improvement in the selectivity of the enrichment process is achieved by the addition of monovalent ions to the sample solution prior to injection. We believe that this effect is due to neutralization of charged peptides via ion-pairing. This significantly reduces the ability of nonglycosylated peptides to bind to the Sepharose media. On the other hand, glycan chains are rich in hydroxyl groups that are not affected by ion-pairing, ensuring that the glycopeptides can still interact with the Sepharose beads. We have attempted to provide a more quantitative explanation of this ion-pairing phenomenon using the Wimley-White water/octanol partitioning free energy hydrophobicity scale. Furthermore, we have determined that only the salts of singly charged metal ions such as Li, Na, K, and Ag will produce this effect, adding further credence to the hypothesis that this is a “charge neutralization” phenomenon. IP-NPLC worked very well for the efficient enrichment of glycopeptides from the digests of purified glycoproteins (RNase B and fetuin). We have also demonstrated the potential of this technique for enriching glycopeptides from complex protein extracts. In general, we found that 50 mM NaCl worked well in IP-NPLC experiments. Changes in the ion-pairing reagent species or concentration may allow for further optimization of the relative signal intensity between glycopeptides and nonglycosylated peptides in a sample-dependent manner. We believe that IP-NPLC has the potential to make a significant contribution to glycoprotein characterization and glycoproteomic studies. ACKNOWLEDGMENT The authors thank Dr. Anne Lenferink and Dr. Maureen O’Connor-McCourt of the Biotechnology Research Institute in Montreal, QC, for providing the JM01 cells and Dr. Stephen H. White, University of California at Irvine, Department of Physiology & Biophysics, for providing critical expertise in MPExTotalizer. This work was supported by NRC’s Genomics and Health Initiative. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review April 16, 2007. Accepted September 5, 2007. AC0707535

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