Crown Ether

Anal. Chem. 2009, 81, 2456–2469

Application of a Combined Weak Cation-Exchange/ Crown Ether Column: First Demonstrations of a Versatile Tool for Proteome Subselection Robin Tuytten,*,† Bart Ruttens,† Katelijne Gheysen,‡ Koen Sandra,† Koen De Cremer,† Dominique Vlieghe,† Natalie Van Landuyt,† Gre´goire Thomas,† Jose´ C. Martins,‡ Pat Sandra,§ Koen Kas,† and Katleen Verleysen† Pronota NV, VIB Bioincubator, Technologiepark 4, B-9052 Zwijnaarde Ghent, Belgium; NMR and Structural Analysis Unit, Department of Organic Chemistry, Krijgslaan 281 S4, Ghent University, B-9000 Ghent, Belgium, and Research Institute for Chromatography (RIC), B-8510 Kortrijk, Belgium The present paper introduces the use of a weak cationexchange/crown ether column in the proteomics field. The 18-crown-6 ether functionality is well-known to selectively complex ammonium and monoalkylammonium ions, which should make this column highly suitable to trap peptides with free r-NH2 or free ε-NH2 groups from lysine side chains. This unique selection mechanism was put to the test in an N-teromics setup which aims for the enrichment of deliberately acetylated protein N-terminal peptides from a serum digest. It was demonstrated that peptides with free r-NH2 groups and peptides with r-amino-acetylated groups can be separated from each other using this weak cation-exchange/crown ether column. The peptides of interest, bearing no free primary amines, were found to be significantly enriched in the column’s flow through. At the same time a favorable coenrichment of N-glycosylated peptides was observed. To obtain more insight in the contributions of the two distinct column functionalities, i.e., the weak cation exchanger and the crown ether, the experimental data were checked against a theoretical prediction of the outcome. Most gel-free proteomics techniques reported over the last years are based on the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of mixtures of peptides derived from the protein pools under study by in vitro proteolysis with well-characterized proteases (bottom-up). They largely subdivide in two major approaches: shotgun and peptide-centric analytical schemes.1,2 Shotgun approaches classically analyze the crude peptide pool as a whole and are therefore (principally) unbiased and little prone to sample preparation artifacts. On the other hand they are likely to suffer from undersampling at the * To whom correspondence should be addressed. Fax: 0032 9 241 11 69. E-mail: [email protected]. † Pronota NV. ‡ Ghent University. § RIC. (1) Lin, D.; Tabb, D. L.; Yates, J. R. Biochim. Biophys. Acta 2003, 1646, 1–10. (2) Gevaert, K.; Van Damme, P.; Ghesquiere, B.; Impens, F.; Martens, L.; Helsens, K.; Vandekerckhove, J. Proteomics 2007, 7, 2698–2718.

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level of the mass spectrometer3 when applied to highly complex mixtures such as blood serum,4 making, e.g., intersample comparisons for biomarker discovery purposes difficult. To overcome this issue iterative analyses can be performed.3 Other research efforts focus on increasing the discriminative power of shotgun schemes by maximizing the peptide separation space at the chromatographic level with the development of highly efficient uni- or multidimensional chromatographies,5,6 as well as at detection/identification level with the introduction of mass spectrometers with unsurpassed resolution, accuracy, duty cycle, and fragmentation merits.7,8 Nevertheless, it is increasingly apparent that these high-level shotgun schemes come at the expense of throughput and cost, while the outcome not necessarily meets up with the expectations.2 Opposite the shot gun schemes, peptidecentric approaches aim for a drastic simplification of the initially created peptide pool by a targeted enrichment of a subset of (modified) peptides that is representative for (a relevant subgroup of) the constituent proteins: a so-called “signature”.9 The choice of which peptide subset to enrich is often driven by a biological premise, e.g., the enrichment of phosphopeptides to study the kinome,10 and/or the availability of a common denominating (chemical) property that can be used as the selection handle.11,12 More importantly, a sample complexity reduction can bring the analytical challenge within the scope of more modest LC-MS/ MS setups, without compromising the protein information sought after. Unlike shotgun schemes, however, peptide-centric approaches often involve lengthy preparation procedures, (negatively) affecting peptide recoveries and technical variability, and/ (3) Liu, H. B.; Sadygov, R. G.; Yates, J. R. Anal. Chem. 2004, 76, 4193–4201. (4) Jacobs, J. M.; Adkins, J. N.; Qian, W. J.; Liu, T.; Shen, Y. F.; Camp, D. G.; Smith, R. D. J. Proteome Res. 2005, 4, 1073–1085. (5) Sandra, K.; Moshir, M.; D’hondt, F.; Verleysen, K.; Kas, K.; Sandra, P. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2008, 866, 48–63. (6) Fournier, M. L.; Gilmore, J. M.; Martin-Brown, S. A.; Washburn, M. P. Chem. Rev. 2007, 107, 3654–3686. (7) Domon, B.; Aebersold, R. Science 2006, 312, 212–17. (8) Chakraborty, A. B.; Berger, S. J.; Gebler, J. C. Rapid Commun. Mass Spectrom. 2007, 21, 730–744. (9) Ji, J. Y.; Chakraborty, A.; Geng, M.; Zhang, X.; Amini, A.; Bina, M.; Regnier, F. J. Chromatogr., B 2000, 745, 197–210. (10) Jalal, S.; Kindrachuk, J.; Napper, S. Curr. Anal. Chem. 2007, 3, 1–15. (11) Mirzaei, H.; Regnier, F. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2005, 817, 23–34. (12) Leitner, A.; Lindner, W. Proteomics 2006, 6, 5418–5434. 10.1021/ac801975b CCC: $40.75  2009 American Chemical Society Published on Web 03/10/2009

or multiple chemical derivatizations often introducing undesired/ unanticipated peptide artifacts. The latter can lead to new and undesired complexity increments and chemical noise.13 However, together with the increased interest in the blood proteome as the preferable source for biomarker discovery, the necessity for (targeted) sample complexity reductions becomes more and more clear.14-16 Blood is a dynamically changing body fluid with an assumed tens of thousands of protein species present, spanning a concentration dynamic range over 1010 and which can be subjected to a multiplicity of posttranslational modifications.17 Therefore, the analytical challenge to describe its proteome by identifying (and ultimately quantifying) all of the digestionderived peptides is well beyond the currentsunbiasedsshotgun schemes. Within this context, mapping the blood proteome with a peptide-centric approach that focuses on the N-termini of the proteins is interesting because methods that selectively isolate the proteins’ N-terminal peptides, i.e., N-teromics, will principally produce one peptide per protein and thus provide ultimate complexity reduction. Yet as the blood proteome is very prone to protein processing and/or proteolytic (i.e., enzymatic) degradation, multiple other protein fragments will also be present. These fragments differ in their N-terminal amino acid sequence,18 and by subselecting the proteins’ N-termini this degradome information is also conserved, increasing the chances of finding a relevant protein biomarker (event). To this extent, several analytical schemes to (co)enrich N-terminal peptides have been reported.19-26 Direct (unmodified) N-terminal peptide enrichment methods, i.e., positive selection, selectively block the lysine residues after which the free Nterminal amino groups are derivatized with a tag designed to enrich/retrieve the N-terminal peptides from the peptide pool created during the subsequent digestion.24,26-30 Indirect Nterminal peptide enrichment methods, i.e., negative selection, do (13) Abello, N.; Kerstjens, H. A. M.; Postma, D. S.; Bischoff, R. J. Proteome Res. 2007, 6, 4770–4776. (14) Jiang, X. G.; Ye, M. L.; Zou, H. F. Proteomics 2008, 8, 686–705. (15) Zhou, Y.; Aebersold, R.; Zhang, H. Anal. Chem. 2007, 79, 5826–5837. (16) Zhang, H.; Li, X. J.; Martin, D. B.; Aebersold, R. Nat. Biotechnol. 2003, 21, 660–666. (17) Anderson, N. L.; Anderson, N. G. Mol. Cell. Proteomics 2002, 1, 845–867. (18) Gevaert, K.; Van Damme, P.; Ghesquiere, B.; Vandekerckhove, J. Biochim. Biophys. Acta 2006, 1764, 1801–1810. (19) Crimmins, D. L.; Gorka, J.; Thoma, R. S.; Schwartz, B. D. J. Chromatogr. 1988, 443, 63–71. (20) Kawasaki, H.; Imajoh, S.; Suzuki, K. J. Biochem. (Tokyo) 1987, 102, 393– 400. (21) Gevaert, K.; Goethals, M.; Martens, L.; Van Damme, J.; Staes, A.; Thomas, G. R.; Vandekerckhove, J. Nat. Biotechnol. 2003, 21, 566–569. (22) McDonald, L.; Robertson, D. H. L.; Hurst, J. L.; Beynon, R. J. Nat. Methods 2005, 2, 955–957. (23) Betancourt, L.; Gil, J.; Besada, V.; Gonzalez, L. J.; Fernandez-de-Cossio, J.; Garcia, L.; Pajon, R.; Sanchez, A.; Alvarez, F.; Padron, G. J. Proteome Res. 2005, 4, 491–496. (24) Timmer, J. C.; Enoksson, M.; Wildfang, E.; Zhu, W.; Igarashi, Y.; Denault, J. B.; Ma, Y.; Dummitt, B.; Chang, Y. H.; Mast, A. E.; Eroshkin, A.; Smith, J. W.; Tao, W. A.; Salvesen, G. S. Biochem. J. 2007, 407, 41–48. (25) Dormeyer, W.; Mohammed, S.; van Breukelen, B.; Krijgsveld, J.; Heck, A. J. R. J. Proteome Res. 2007, 6, 4634–4645. (26) Yamaguchi, M.; Nakazawa, T.; Kuyama, H.; Obama, T.; Ando, E.; Okamura, T.; Ueyama, N.; Norioka, S. Anal. Chem. 2005, 77, 645–651. (27) Munchbach, M.; Quadroni, M.; Miotto, G.; James, P. Anal. Chem. 2000, 72, 4047–4057. (28) Miyagi, M.; Nakao, M.; Nakazawa, T.; Kato, I.; Tsunasawa, S. Rapid Commun. Mass Spectrom. 1998, 12, 603–608. (29) Wang, D. X.; Kalb, S. R.; Cotter, R. J. Rapid Commun. Mass Spectrom. 2004, 18, 96–102.

not demand for a discriminative modification between the protein N-termini and the lysine side chains. The internal peptides with free R-NH2 groups formed upon digestion can be depleted from the mixture in (i) an unbiased way if separation is based on a R-NH2 tagging approach21,22 or (ii) in a less selective way when the R-NH2 basicity in combination with net overall charge is used as criterion.19,20,31 Good repetitive performance, a major requisite in a biomarker discovery context, of the unbiased methods is challenging as they involve sophisticated experiment setups and/or multiple chemical reactions.21,22 Therefore, from an operational point of view, the simplicity of the indirect N-terminal peptide enrichment method capitalizing on differences in charge between R-NH2-blocked peptides and internal peptides with free R-NH2 groups holds great promise. At the same time it can be argued from an N-teromics perspective that charge (alone) is not the most selective criterion: some classes of true in vivo generated N-terminal peptides will be lost, whereas other classes of peptides, e.g., C-terminal peptides, are coenriched. 18-Crown-6 ethers are well-known to selectively form complexes with ammonium and monoalkylammonium ions by hydrogen bonds between the ammonium hydrogen atoms and the ring oxygen atoms,32 a property that should allow us to target unmodified R-NH2 groups without interference of net overall charge of the parent peptide. However, to the best of our knowledge, application of this unique selectivity to extract peptides from complex peptide mixtures has never been reported. Rather, applications in separation science of this selective complexation are largely found in crown ether-based chiral stationary phases for resolving (drug) enantiomers.33 Recently a few papers reported on the usage of crown ethers in liquid-liquid extractions of small peptides34 or cytochrome c.35 Because it allows one to influence peptide fragmentation behavior, the coordination of 18-crown-6 ethers with primary amines from peptides has found some use in mass spectrometry.36 For our experiments, we used an interesting stationary phase which was developed to enable the resolution of ammonium in the presence of excesses of sodium or potassium in the field of ion analyses. It is a weak cation-exchange column which contains carboxylate and phosphonate functional groups as well as crown ether groups.37,38 This combined weak cationexchange/crown ether (WCX/CrE) column was implemented in an N-terminal peptide enrichment approach to investigate its feasibility for the subselection of peptides in proteomics. Within the scope of this initial study, we have shown for a serum-derived peptide mixture that a simple chromatographic prepurification step (30) Hunt, T.; Huang, Y.; Ross, P.; Pillai, S.; Purkayastha, S.; Pappin, D. Mol. Cell. Proteomics 2004, 3, S286. (31) Staes, A.; Van Damme, P.; Helsens, K.; Demol, H.; Vandekerckhove, J.; Gevaert, K. Proteomics 2008, 8, 1362–1370. (32) Rudiger, V.; Schneider, H. J.; Solov’ev, V. P.; Kazachenko, V. P.; Raevsky, O. A. Eur. J. Org. Chem. 1999, 1847–1856. (33) Hyun, M. H. Bull. Korean Chem. Soc. 2005, 26, 1153–1163. (34) Buschmann, H. J.; Mutihac, L. Anal. Chim. Acta 2002, 466, 101–108. (35) Shimojo, K.; Kamiya, N.; Tani, F.; Naganawa, H.; Naruta, Y.; Goto, M. Anal. Chem. 2006, 78, 7735–7742. (36) Julian, R. R.; Beauchamp, J. L. J. Am. Soc. Mass Spectrom. 2002, 13, 493– 498. (37) Rey, M. A.; Pohl, C. A.; Jagodzinski, J. J.; Kaiser, E. Q.; Riviello, J. M. J. Chromatogr., A 1998, 804, 201–209. (38) Pohl, C.; Rey, M.; Jensen, D.; Kerth, J. J. Chromatogr., A 1999, 850, 239– 245.


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using a WCX/CrE column results in an enrichment of R-NH2blocked peptides in the column’s flow through. Evidently, the coavailability of the WCX functionality prevents one to perform true unbiased N-teromics, yet this limitation issfor this pilot studysconsidered as a predictable artifact. Furthermore, a favorable coenrichment of N-glycosylated peptides was demonstrated. This ability to enrich several peptide subclasses using the same analytical setup opens up a variety of alternative analytical schemes to complement the current proteomics tools. EXPERIMENTAL SECTION Sample Preparation. An amount of 70 µL of a crude human serum sample (healthy male) was diluted 1:4 in proprietary “buffer A”, part of the multiple affinity removal system (MARS) (Agilent, Santa Clara, CA). “Buffer” was supplemented with protease inhibitor tablets (Roche, Basel, Switzerland) in a ratio of 1:300 mL of buffer. After filtration (0.22 µm, 14 000 rpm; Costar Spin-X centrifuge tube filters) (Cole-Parmer, Vernon Hills, IL) the sample was depleted in two consecutive runs on a human MARS human-6 column (Agilent) per the manufacturer’s instruction, effectively removing six high-abundant serum proteins. Albumin and IgG depletion efficiency was tested via Western blotting. The two flow throughs (2 × 1 mL) were pooled, and the protein concentration was roughly determined to be ∼0.5 mg/mL by measuring the absorbance at 280 nm (Evolution 3000, Thermo Electron, Waltham, MA). The protein sample was subsequently concentrated 3× using a Vivaspin filter with a molecular weight cutoff (MWCO) of 3000 Da (Vivascience, Littleton, MA) to yield a protein concentration of ∼1.5 mg/mL for the sample, as confirmed by bicinchoninic acid (BCA) test assay (Pierce, Rockford, IL). Protein denaturation was effected by adding guanidine hydrochloride (Merck, Whitehouse Station, NJ) up to a final concentration of 3 M. The resulting protein mixture was reduced during a 10 min incubation step at 30 °C with a 25 molar excess of tris(2carboxyethyl)phosphine (Pierce). Subsequently, the protein cysteinyl residues were alkylated for 60 min with a 50 molar excess of iodoacetamide (Sigma-Aldrich, Buchs SG, Switzerland) at 30 °C. A buffer exchange to 1.4 M guanidinium chloride in 50 mM sodium phosphate pH ) 8 was performed on a PD10 column (Amersham Biosciences, Uppsala, Sweden) according to the manufacturer’s guidelines. The resulting 3.5 mL effluent volume was concentrated to 2.0 mL by vacuum centrifugation (Centrivap Concentrator, Labconco, Kansas City, MO). Then, the protein content was acetylated for 90 min at 30 °C with a 75 molar excess of sulfo-N-hydroxysuccinimidyl acetate (Pierce). A deacetylation, for 20 min at room temperature, with a 3.5 molar excess hydroxylamine compared to sulfo-N-hydroxysuccinimidyl acetate, was performed to deprotect acetylated serine, threonine, and tyrosine side chains. Following the reverse acetylation step, the sample was again desalted on a PD10 column and captured in a 20 mM NaHCO3 pH ) 8 buffer. The final protein concentration was measured to be 0.49 mg/mL (BCA). The modified protein material was then heated for 5 min at 99 °C and digested overnight at 37 °C after adding trypsin (Promega Corporation, Madison, WI) in a substrate/trypsin ratio of 50:1 (w/w). Before storage at -20 °C the sample volume was reduced by vacuum concentration to obtain a concentration of ∼2 mg/mL (extrapolation of the BCA results) and an associated buffer concentration of 80 mM NaHCO3 (pH ) 8). 2458


For the actual experiments, two aliquots of 125 µL (∼250 µg peptide material) were taken and concentrated to dryness by means of vacuum centrifugation and stored at -20 °C until sorting. After WCX/CrE sorting (vide infra) one-third of the four flow through fractions was combined into one sample, which was dried by evaporation under reduced pressure (vacuum centrifuge). One of the two combined flow throughs was further processed: after reconstitution in 100 µL of 0.02 M sodium phosphate pH ) 8, 1 unit of PNGase F was added, followed by an overnight incubation at 37 °C. After deglycosylation the sample was again dried by vacuum centrifugation. This sample will be referred to as “PNGase F POST” in the remainder of the text. The other nondeglycosylated sample will be termed “reference”. Weak Cation-Exchange/Crown Ether Operation (Sorting). The WCX/CrE column used was an IonPac CS 15 cation-exchange column (Dionex, Amsterdam, The Netherlands) of 2 mm i.d. × 250 mm length, containing an 8 µm particulate resin of 55% crosslinked ethylvinylbenzene/divinylbenzene, functionalized with phosphonate, carboxylate, and crown ether groups. The WCX/CrE separations were performed on an Agilent 1100 series highperformance liquid chromatography (HPLC) system (Agilent Technologies, Waldbronn, Germany) equipped with a multiple wavelength detector and an 1100 series fraction collector. Operation, data collection, and analysis were done using the Chemstation software (Agilent). An additional 400 µL stainless steel seat capillary (Agilent) was mounted on the injector allowing for largevolume injections (500 µL). All chromatographic steps were performed at room temperature with a flow rate of 100 µL/min, and the UV absorbances at 214 and 280 nm were recorded. All solvents, i.e., water (H2O) and acetonitrile (ACN), and additives, i.e., formic acid (FA) and trifluoroacetic acid (TFA), for WCX/CrE operation and nano-LC operation (vide infra) were purchased from the same supplier in the appropriate grade (Biosolve, Valkenswaard, The Netherlands). The samples, i.e., “reference” and “PNGase F POST”, were reconstituted in 510 µL of the loading solvent (0.025:49.975:50 (v/v) FA/H2O/ACN). Three discrete chromatographic steps were implemented: (i) A sample loading step comprising a 500 µL in-flow injection of the sample followed by an isocratic 45 min of the loading solvent. Sample flow throughs were collected in four fractions: 4.5-14.5 min (1000 µL), 14.5-24.5 min (1000 µL), 24.5-29.5 min (500 µL), and 29.5-39.5 min (1000 µL). (ii) An elution step of 90 min in which the bound peptide fraction was released from the WCX/CrE column by a 70 min linear gradient from 100% 50:50 (v/v) H2O/ACN (A) to 100% 1:49:50 (v/v) TFA/H2O/ACN (B) followed by a 10 min isocratic section of 100% B and a 10 min reverse gradient to 100% A. (iii) A 3 h equilibration step using the loading solvent (0.025: 49.975:50 (v/v) FA/H2O/ACN), after which the next sample could be applied. Following this sorting step one-third of the four flow through fractions of each run was pooled. One pool was dried completely (vacuum centrifugation; “reference”), and the other pool was processed further as described above (“PNGase F POST”). RPLC-MS/MS Analysis. An Agilent 1200 series nano-HPLC system (Agilent) was fitted with a column-switching setup comprising a 300 µm i.d. × 5 mm C18 reversed-phase (RP) (5

µm, 300SB Zorbax) precolumn (Agilent) and a custom-made 100 µm i.d. × 120 cm analytical nano-RP column (C18, 5 µm, 300SB Zorbax).5 The samples were reconstituted in 100 µL of 0.1% (v/v) FA in H2O of which 40 µL was injected. The precolumn was loaded at 20 µL/min with 0.1:99.9 (v/v) FA/H2O. After 5 min, the sample was transferred onto the nano-RP column. The analytical chromatography involved a binary solvent system, i.e., 0.1:99.9 (v/v) FA/H2O (solvent A) and 0.1:19.9:80 (v/v) FA/H2O/ACN (solvent B), and a flow rate of 350 nL/min was used. Peptide elution was achieved by applying a linear gradient from 10% B to 65% B in 400 min (initialized at sample injection), followed by a rinsing (65% B to 90% B (400-401 min), 90% B (401-416 min)), and a re-equilibration section (90% B to 10% B (416-417 min) and 10% B (417-480 min)). The column was directly joined to a PicoTip electrospray ionization (ESI) emitter (silica, distal-coated, 360/20 µm o.d., 10 µm i.d.) (New objective, Woburn, MA) by means of a stainless steel zero-dead-volume connection (Agilent), via which the electrospray voltage was applied to the column effluent. The emitter assembly was fitted on a Nanospray stage (Applied Biosystems/MDS SCIEX, Foster City, CA) mounted on a QSTAR Elite Hybrid LCMS/MS system (Applied Biosystems/MDS SCIEX). The mass spectrometer was operated in the information dependent analysis (IDA) mode. The following instrument parameters were used: a positive ESI voltage of +2000 V, a declustering potential of 55 V, and a curtain gas pressure of 20 psi. The IDA criteria adopted for precursor ion selection were a m/z range of 300-1500, a 1 s accumulation time, and selection of the two most intense 2+ or 3+ charged signals per scan for fragmentation, if exceeding a set threshold of 40 counts/s. Selected precursor ion masses were then excluded for 600 s. For the product ions spectra acquisitions a m/z range of 70-1500 was set. Optimal collision energy values were automatically determined as well as spectrum quality: automatic MS/MS accumulation was enabled with a maximum of 3 s. Mass spectrometric data was collected during the entire nanoLC run. MS/MS Data Analysis and Peptide Categorization. The collected MS/MS spectral data were converted to Mascot generic files (mgf) using the Analyst QS 2.0 software plug-in (mascot.dll; Matrix Science/Applied Biosystems/MDS SCIEX). The Mascot search algorithm (Matrix Science Inc., Boston, MA) was run with Swiss-Prot 54.2 as database, holding 17 170 human protein sequences. To accommodate the extensive protein processing encountered in serum samples, the spectra were searched using no-enzyme search settings. All real database searches were complemented with a search against its random counterpart to calculate the false discovery rate (FDR). MS and MS/MS tolerance was set to 0.1 Da, and charges up to 3+ were allowed. In total, nine modifications were preset: one fixed, i.e., carbamidomethylation of cysteine, and eight variable, i.e., acetylation of lysine and the N-terminus, deamidation of asparagine and glutamine, oxidation of methionine to its sulfoxide derivative, pyrocarbamidomethyl formation on N-terminal cysteine (pyro-cmc), and pyroglutamate formation of N-terminal glutamine and glutamic acid (pyro-glu Q/E). Only peptides ranking no. 1 with scores above the identity probability threshold were withheld. Spectra that had multiple peptide hits above the probability threshold were

regarded as unidentified. A protein is reported only if it was represented by at least one unequivocally assigned peptide. For all identified peptides titration curves were calculated according to the Shimura algorithm,39 using an automated (perl) script providing pI information as well as the net charge for all pH values of 0-14 in increments of 0.1. Typical amino acid pKa’s were used (http://www.innovagen.se/custom-peptide-synthesis/ peptide-property-calculator/peptide-property-calculator-notes. asp, Innovagen, Lund, Sweden), except where modifications affect the value (pyroglutamate formation on N-terminal glutamic acid and glutamine, deamidation of asparagine and glutamine, acetylation of lysine, N-terminal acetylation). To classify a peptide as an N-glycopeptide, the peptide had to contain the consensus motif N-X-S/T, with X not being a proline, together with the PNGase F mediated conversion of asparagine to aspartic acid (cf., deamidation of N to D). Visualization of the data was done by means of Spotfire DecisionSite 9.0 (Tibco Spotfire, Go ¨teborg, Sweden). RESULTS Prediction of the Experimental Outcome. An indirect N-terminal peptide enrichment approach requires a specific series of sample preparation steps. A protein mixture is first denatured, next the disulfide bridges are reductively cleaved and the released cysteine side chains are alkylated, and finally, all primary NH2 groups13 are selectively blocked, e.g., by acetylation (which requires a reverse acetylation step to deacetylate hydroxyl groups). Upon digestion with trypsin the resulting peptide pool theoretically constitutes (i) N-terminal peptides with acetylated R-NH2 groups, (ii) an excess of internal peptides with free R-NH2 groups, (iii) C-terminal peptides with free R-NH2 groups, and (iv) a group of (internal) peptides of which the N-terminal amino acid underwent an intramolecular cyclization reaction to a pyroglutamyl or a pyrocarbamidomethyl residue (from carbamidomethylcysteine).40 It is noteworthy that this peptide pool is enriched in tryptic peptides with a C-terminal arginine because the acetylation of lysines impairs trypsin activity C-terminal of lysine. The starting point of this research is the premise that it should be possible to selectively enrich Nterminally blocked peptides from such a highly complex peptide mixture with a WCX/CrE column at pH ∼ 3 conditions. To enable an in-depth assessment of the WCX/CrE approach the impact on the separation of each of the constituents of the mixed stationary phase, i.e., the weak cation-exchange characteristics (WCX) and the 18-crown-6 ether functionality (CrE), was first analyzed. In a digest obtained as described in the previous paragraph, the peptide protonation sites are restricted to (C-terminal) arginine, histidine, and free R-NH2 groups of internal peptides. At pH ∼ 3 the carboxyl functionalities of aspartic acid and glutamic acid are (mostly) uncharged (undissociated acid) as opposed to the peptides’ C-terminal carboxylate moiety (lower pKa, dissociated acid at pH ∼ 3). Peptides whose charge is only determined by the C-terminal arginine, e.g., N-terminally blocked tryptic peptides, have a net charge of zero and will be (39) Shimura, K.; Kamiya, K.; Matsumoto, H.; Kasai, K. Anal. Chem. 2002, 74, 1046–1053. (40) Geoghegan, K. F.; Hoth, L. R.; Tan, D. H.; Borzillerl, K. A.; Withka, J. M.; Boyd, J. G. J. Proteome Res. 2002, 1, 181–187.


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enriched in the flow through of a cation-exchange column at pH ∼ 3. Peptides with functionalities which add an extra positive charge to the equation, e.g., N-terminally blocked tryptic peptides containing histidine, peptides starting with a proline, or internal tryptic peptides starting with a free R-NH2 group, will be trapped on the column, whereas, e.g., nontryptic peptides containing maximal one basic functionality (arginine, histidine, a free R-NH2, or an N-terminal proline) are expected to be coenriched in the flow through. The charge-based selectivity at pH ∼ 3 of the WCX component of the WCX/CrE column can be predicted by the following rules: A peptide is expected in a WCX flow through if no. of Arg + no. of His + no. of free R-NH2 + no. of N-terminal Pro e 1 (cf. C-terminal carboxylate) A peptide is expected to be trapped on a WCX column if no. of Arg + no. of His + no. of free R-NH2 + no. of N-terminal Pro > 1 (cf. C-terminal carboxylate) Peptide titration curves essentially give the same information as a classification based on amino acid residue counting but are more sophisticated because they allow us to evaluate whether a peptide will be enriched in a charge-based experiment in function of the applied pH conditions. Therefore, some in-house tools were also developed to be able to calculate the titration curves of any identified peptide. The crown ether functionality is expected to solely interact with freely available (monoalkyl)ammonium groups. This complexation requires spatial proximity, implicating that steric effects could interfere with the retention mechanism, but in principle, at pH ∼ 3, the crown ether functionality will retain all peptides with a free (R/ε)-NH2, whereas all N-terminally blocked peptides and peptides starting with proline (cf., secondary amine) are expected in the flow through. For the working hypothesis it was assumed that the mode of action of the CrE component of the column was predictable by the following: A peptide is expected in a CrE flow through if no. of free (R/ε)-NH2 ) 0 A peptide is expected to be trapped on a CrE column if no. of free (R/ε)-NH2 > 0 From the above, prefractionation by the combined WCX/CrE column can theoretically be formalized in the following rules: A peptide is expected in a WCX/CrE flow through if no. of Arg + no. of His + no. of N-terminal Pro e 1 A peptide is expected to be trapped on a WCX/CrE column if no. of Arg + no. of His + no. of N-terminal Pro > 1 and/or no. of free (R/ε)-NH2 > 0 This working hypothesis was developed prior to the sorting experiments with the purpose to expose any artifacts more easily and to enable a better understanding of the true mode of action of the WCX/CrE stationary phase. 2460


Analysis of a WCX/CrE Flow Through (Reference Experiment). A three-step procedure for the prepurification of chemically modified peptide mixtures on the WCX/CrE column was pursued: (i) sample injection and collection of the nonretained peptide fraction in the column flow through, followed by (ii) an elution step whereby all peptides retained on the column are at once eluted and (iii) a regenerative step preparing the column for the next injection. This requires a good on/off sorting performance where the flow through containing the peptides of interest and the retained fraction are well resolved. Furthermore, the elution step needs to be comprehensive to avoid crosscontamination and guarantee experimental reproducibility. (i) Because the WCX/CrE column under test was originally developed for ion analyses, the chromatographic conditions suggested by the manufacturer had to be adapted. To limit competitive cation binding and to prevent any aspecific peptide retention by the underlying ethylvinylbenzene/divinylbenzene support, an aqueous nonbuffered 0.05% FA (v/v) solution (pH ∼ 2.6) supplemented with 50% ACN (v/v) (final FA concentration 0.025% (v/v)) (loading solvent) was used. The added ACN brings the effective pH to ca. 2.941 which is the optimal pH for WCX (SCX). Because both potassium and ammonium exhibit high affinities for 18-crown-6 ethers,37 the tryptic digest was performed in a sodium bicarbonate buffer of low molarity. Since no resolution within the flow through is needed and the chromatography is performed in an on/off fashion, largevolume injections were applied, enabling an extra dilution of the sample and its salt content. So, the residue of a dried tryptic digest of a serum protein sample (cf., the previous section and the Experimental Section) was reconstituted in 510 µL of loading buffer and injected (500 µL) onto the WCX/CrE column under isocratic loading solvent conditions. Injection was followed by a 45 min isocratic run during which the nonretained peptide fraction was collected in four discrete fractions. The corresponding UV traces at 214 and 280 nm are shown in Figure 1, panels A and B, respectively. (ii) Designed for the strongly acidic conditions typical for ion analyses, the WCX/CrE column permitted the use of stringent elution conditions to release the retained peptide fraction. During a 70 min linear gradient from 50:50 H2O/ACN (v/v) to 1:49:50 TFA/H2O/ACN (v/v) the retained fraction was eluted by hydronium (H3O+) competition for the active sites. Exemplary UV traces are also shown in Figure 1C. (iii) After a fast reversed gradient with the elution mobile phases the column was regenerated for 3 h with the loading solvent (0.025% FA). Repeats of this three-step chromatographic procedure with blanks (loading solvent) have shown that the application of 1% TFA eluted all retained peptides at once, as opposed to salt gradients (data not shown). One-third of each of the four collected flow through fractions (see Figure 1) was pooled, dried, reconstituted, and further analyzed. Although a comprehensive analysis of the serum proteome as such was not intended with this study, a high-level RPLC-ESI-MS/MS setup was chosen to allow for sufficient peptide identification capacity as the complexity of the WCX/CrE flow through was anticipated to be still very high. From this (41) Gagliardi, L. G.; Castells, C. B.; Rafols, C.; Roses, M.; Bosch, E. Anal. Chem. 2007, 79, 3180–3187.

Figure 1. Traces A and B depict the 45 min WCX/CrE chromatography obtained under isocratic loading solvent conditions (0.025:49.975:50 (v/v) FA/H2O/ACN) following serum digest injection, monitored at, respectively, (A) 214 nm and (B) 280 nm, whereby the nonretained peptides are collected in four fractions. In panel C the gradient elution step is illustrated in which the retained peptides are released by means of hydronium displacement. The blue and red traces depict the UV absorbances at 214 and 280 nm (normalized), respectively; the green trace shows the applied gradient program in terms of % A (A, 50:50 (v/v) H2O/ACN; B, 1:49:50 (v/v) TFA/H2O/ACN).

analysis, 341 peptides were identified with an adopted database search strategy which was tailored to (i) account for the specific chemistry applied and (ii) to deal with in vivo processing events typical for the serum proteome (cf., the Experimental Section). The peptides were classified based on the number of basic residues, their N-terminal modification, their pI value, and the net charge at pH ) 3 (Figure 2A-C). The observed grouping of the peptides in discrete pI values (vertical lines) agrees with the fact that the formulas used to calculate the titration curves entail a count of the aforementioned acidic and basic groups, giving rise to a limited number of combinations. The group at pI ) 0, for instance, exclusively contains sequences without basic functionality and with a nonlimiting number of acidic groups (Asp, Glu, Tyr, C-terminal carboxylate). With respect to the calculated net charges at pH ) 3 it was demonstrated in independent SCX data sets (not shown) that all sequences with a calculated charge 0 e x e 0.5 have to be considered neutral and as a result are truly sorted when it comes to charge. This deviation can be attributed to inaccuracies in the set of pKa values employed to calculate the titration curves. In view of the WCX/CrE retention mechanism under study, one can discern four relevant subpopulations, indicated by boxes

i-iv in Figure 2, panels B and C. Box i contains 96 (96/341; 28.15%) peptides without basic functionality which are negatively charged under the applied pH ∼ 3 conditions. Due to this negative charge they are subjected to an ion exclusion mechanism on the WCX/CrE column as the latter is also negatively charged (cf., phosphonate; the carboxyl moiety is undissociated at the given pH). It is interesting to note that the peptides constituting this group are all N-terminally blocked nontryptic peptides. The 135 (39.59%) peptides in box iii have a blocked N-terminus (no. of R-NH2 ) 0) and only one basic group. However, it is interesting to note that 28 (20.74%) out of these 135 sequences contain a nonacetylated lysine which should have been targeted by the CrE functionality. These 28 modified sequences are presented in Table 1. All but one (peptide 15) have a nonacetylated C-terminal lysine. Intramolecular ion pair formation between the dissociated C-terminal carboxylate and the free ε-NH2 group would prohibit the [crown ether · · · H3N+-group] complexation, impairing chromatographic retention of such sequences. The 87 (25.51%) modified sequences in box ii do agree with the prediction rules derived for the WCX contribution of the WCX/CrE as the net overall charge ) 0. However, due to the availability of a free R-NH2, these peptides should have been Analytical Chemistry, Vol. 81, No. 7, April 1, 2009

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Figure 2. In panel A the N-terminal modifications of all 384 selected peptides of the “reference” experiment are plotted vs their calculated pI values. Furthermore, the symbol shapes indicate the net charge at pH ) 3 as calculated for each of the modified sequences (0.5 charge unit bins). An extract of all sequences with no charged Arg, His, or underivatized Lys residues is given in panel B. The peptides in box i are N-terminally blocked by acetylation or intramolecular cyclization (pyro-glu or pyro-cmc). Box ii contains modified sequences which still have a free R-NH2 at the N-terminus. Similarly, panel C depicts the peptides that do contain one charged residue, i.e., Arg, His, or underivatized Lys. The modified sequences in box iii are N-terminally blocked, whereas those in box iv have an additional free R-NH2.

retained by the crown ether component of the column. An amino acid frequency analysis on the first 10 amino acids of the sequences in box iii by means of the web-based application Weblogo (http://weblogo.berkeley.edu/)42 revealed that generally the N-terminal part (up to residue 6) of these peptides is rich in acidic aspartic acid (D) and glutamic acid (E) residues (Figure 3). Probably neighboring amino acids influence the [crown ether · · · H3N+-group] complexation, especially when they have acidic side chains. The latter locally lead to a negative charge in the N-terminal part of the sequence which may impair complexation by charge exclusion with the WCX component. Intramolecular ion pair formation effects may also have an influence. Different from the peptide fraction in box iii, no sequences with underivatized lysine residues are present. On (42) Crooks, G. E.; Hon, G.; Chandonia, J. M.; Brenner, S. E. Genome Res. 2004, 14, 1188–1190.

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the other hand, 18 sequences of box ii have an acetylated lysine as C-terminal amino acid. All other sequences of box ii are nontryptic. The 23 (6.75%) peptides in box iv possess a free R-NH2 group or an N-terminal proline and a C-terminal arginine, implicating these peptides are tryptic. It is obvious that these peptides do not comply with the working hypothesis, which is also reflected in their calculated net charge at pH ) 3 which ranges from 0.5 < x e 1.5. The according peptides are presented in Table 2. It is striking that the sequences of this group are rich in glutamic acid and aspartic residues, located in the N-terminal part and/or in close proximity of each other. In calculations of the net charge and pI, the contributions of each of the residues are assumed to be independent of each other. However, in reality (proximal) amino acids affect each others pKa values, and important deviations between calculated and observed pI values

Table 1. List of Identified Sequences from the “Reference” Experiment Which Have a Blocked N-Terminus, i.e., Acetylated or Intramolecularly Cyclized, and One Nonacetylated Lysine Residue, Together with Their Respective Mascot Scoresa

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 a

modified sequence

Mascot score

〈acetyl (N-term)〉EEEMLENVSLVCPK 〈acetyl (N-term)〉VSPTDCSAVEPEAEK 〈acetyl (N-term)〉K〈acetyl (K)〉VTYTSQEDLVEK 〈pyro-glu (N-term Q)〉QNPASPPEEGSPDPDSTGALVEEEDPFFK 〈pyro-glu (N-term Q)〉QTVTDYGK〈acetyl (K)〉DLMEK〈acetyl (K)〉VK〈acetyl (K)〉SPELQAEAK 〈acetyl (N-term)〉DQTVSDNELQEMSNQGSK 〈acetyl (N-term)〉DQTVSDNELQEMSNQGSK〈acetyl (K)〉YVNK 〈acetyl (N-term)〉DQTVSDNELQEMSNQGSK〈acetyl (K)〉YVNK〈acetyl (K)〉EIQNAVN〈deamidation (NQ)〉GVK 〈acetyl (N-term)〉DQTVSDNELQEMSNQGSK〈acetyl (K)〉YVNK〈acetyl (K)〉EIQNAVN〈deamidation (NQ)〉GVK〈acetyl (K)〉QIK 〈acetyl (N-term)〉EPLDDYVNTQGASLFSVTK〈acetyl (K)〉K 〈acetyl (N-term)〉TPDVSSALDK〈acetyl (K)〉LK 〈acetyl (N-term)〉TPDVSSALDK〈acetyl (K)〉LK〈acetyl (K)〉EFGN〈deamidation (NQ)〉TLEDK 〈pyro-cmC (N-term camC)〉CSYTEDAQCIDGTIEVPK 〈pyro-glu (N-term Q)〉QELN〈deamidation (NQ)〉GNTK〈acetyl (K)〉SK 〈acetyl (N-term)〉SK〈acetyl (K)〉DLEEVKAK〈acetyl (K)〉 〈acetyl (N-term)〉SQESVTEQDSK 〈acetyl (N-term)〉DVSSALDK 〈acetyl (N-term)〉DVSSALDK〈acetyl (K)〉LK〈acetyl (K)〉EFGN〈deamidation (NQ)〉TLEDK 〈acetyl (N-term)〉DVSSALDK〈acetyl (K)〉LK〈acetyl (K)〉EFGNTLEDK 〈pyro-glu (N-term Q)〉QAK〈acetyl (K)〉EPCVESLVSQYFQTVTDYGK〈acetyl (K)〉DLM〈oxidation (M)〉EK〈acetyl (K)〉VK 〈pyro-glu (N-term Q)〉QEM〈oxidation (M)〉SK〈acetyl (K)〉DLEEVK 〈pyro-glu (N-term Q)〉QEM〈oxidation (M)〉SK〈acetyl (K)〉DLEEVK〈acetyl (K)〉AK 〈pyro-glu (N-term Q)〉QEM〈oxidation (M)〉SK〈acetyl (K)〉DLEEVK〈acetyl (K)〉AK〈acetyl (K)〉VQPYLDDFQK 〈pyro-glu (N-term Q)〉QEMSK〈acetyl (K)〉DLEEVK〈acetyl (K)〉AK〈acetyl (K)〉VQPYLDDFQK 〈acetyl (N-term)〉SLPEGVAN〈deamidation (NQ)〉GIEVYSTK 〈acetyl (N-term)〉EDDIIIATK〈acetyl (K)〉N〈deamidation (NQ)〉GK 〈pyro-glu (N-term Q)〉QESQSEEIDCNDK〈acetyl (K)〉DLFK 〈pyro-glu (N-term Q)〉QESQSEEIDCNDK〈acetyl (K)〉DLFK〈acetyl (K)〉AVDAALK

95.31 69.64 84.44 67.25 63.69 83.97 77.19 79.08 64.07 70.31 91.27 67.69 61.46 71.12 65.64 64.15 56.98 72.82 81.64 72.14 65.48 63.19 64.57 63.78 79.98 76.97 72.64 78.82

Except for the N-terminal modifications, the indicated modifications (between brackets) allude to the preceding amino acid residue.

Figure 3. Relative frequency of each amino acid in the 10 first amino acids of 87 (out of 94) sequences in which the (sole) basic functionality is a free R-NH2. The amino acids are colored according to their chemical properties: polar amino acids (G, S, T, Y, C, Q, N) are green, basic (K, R, H) are blue, acidic (D, E) are red, and hydrophobic (A, V, L, I, P, W, F, M) are black. The remainder seven sequences contain 90% of these sequences, with calculated net charges e0.5 at pH ) 3, fitted the predicted WCX outcome. The presence of some data points which do not fit the

Figure 4. Total ion current (TIC) of the combined MS/MS experiments and the therefrom extracted ion chromatograms (XIC) corresponding to the diagnostic glycopeptide oxonium ions N-acetylhexosamine (m/z 204) and dehydrated N-acetylneuraminic acid (m/z 274) are plotted for both the “reference” and the “PNGase F POST” experiment. To assist comparison between the two experiments lines are drawn at equal intensities.

WCX prediction rules can be rationalized by flaws in the net charge calculation formula applied,39 because the calculations fail to account for deviations of the theoretical acid/base properties of amino acid residues due to particular neighboring amino acid effects. For example, local “densities” of glutamic acid and aspartic acid residues are suggested to shift the effective net charge to neutrality precluding the parent peptides to be trapped by WCX. Because an experimental setup which targets peptides which are not retained by the WCX/CrE column coenriches for the sorting artifacts, beneficial sorting effects attributable to the crown ether functionality will be better reflected in the retained fraction. However, an analysis of the retentate was not considered instruc-

tive because it contains all internal tryptic peptides and has a complexity similar to the initial protein digest. Instead, an indirect analysis of the crown ether contribution to the sorting was done by comparing the flow throughs of a WCX/CrE experiment as described above and an SCX experiment as described by Staes et al.31 The flow through of the latter experiment should coincide largely with the WCX contribution in the WCX/CrE experiment because the same selection criteria apply. The WCX/CrE and SCX flow throughs of equal parts of a processed serum digest were subsequently resolved by the same RPLC separation. From the normalized UV profiles at 214 nm in Figure 6 it is clear that the WCX/CrE flow through contains far less peptide material. This Analytical Chemistry, Vol. 81, No. 7, April 1, 2009

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Figure 5. In panel A the N-terminal modifications of all 207 nonglycopeptide sequences of the “PNGase F POST” experiment are plotted vs their calculated pI values. The symbol shapes correspond to the net charge at pH ) 3 as calculated for each of the sequences (0.5 charge unit bins). In panel B the same representation is given for the 201 sequences of the “PNGase F POST” experiment which are considered to be N-glycopeptides. Hereto, the identified sequence has to contain the N-glycopeptide N-X-S/T consensus sequence, together with the PNGase F induced Asn to Asp deamidation at the same locus. It is of importance to note that for the glycopeptides the actual net charge during the WCX/CRrE prepurification steps is even higher than the calculated values in panel B. Because the PNGase F induced conversion of Asn to Asp leads to an additional Asp residue after sorting, bringing an extra acidic group into the calculation, the net charges are theoretically 0.13 charge units higher.

outcome supports the starting idea that, indeed, the crown ether functionality adds to the global sorting performance of the WCX/CrE column. The above presented data demonstrate that the [crown ether · · · H3N+-group] complexation within the used WCX/CrE column is influenced by the nature of neighboring amino acids. In part these effects are accentuated by the coavailability of the WCX functionality. Acidic residues in the N-terminal part of sequences will impair crown ether complexation of the R-NH2 group due to local charge repulsion effects with the WCX component. But the crown ether affinity is clearly also impeded when intramolecular ion pair formation involving the targeted amine functionality is more favorable. This is nicely demonstrated by the presence of several peptides in the flow through with an underivatized lysine at the C-terminus which indicates that the ε-NH3+ group preferably interacts with the nearby C-terminal carboxylate. Because little is known about the peptide binding function of the crown ether ligand, solution NMR spectroscopy was applied to elucidate the complexation interaction at the molecular level. The interaction and affinity of 18-crown-6 ether with three dipeptides, i.e., H2N-Ala-Ala-CONH2, Ac-NH-Ala-AlaCONH2, and H2N-Asp-Ala-CONH2, was studied. These experiments confirmed that the 18-crown-6 ethers indeed form tight complexes with peptides when protonated R-NH2 groups are available. Acetylation or deprotonation of the R-NH2 groups 2466


abolished this interaction with the 18-crown-6 ether. H2N-AspAla-CONH2 was included in the NMR study to assess the effect of the presence of proximal acidic residues. Hereto two distinct charge states of the latter dipeptide, i.e., (i) protonated R-NH2 plus undissociated aspartic acid and (ii) protonated R-NH2 plus (partially) dissociated aspartic acid, were titrated with crown-6 ether to determine their affinity constants. It showed that the presence of the negative charge from the dissociated aspartic acid weakened the complexation compared to the dipeptide charge state wherein the aspartic acid is still undissociated. These NMR data are in good agreement with the earlier deduced/speculated sensitivity of the [crown ether · · · peptide] complexation to intramolecular ion formation effects exerted by neighborhood amino acids (/sialic acids). A more elaborate description of the NMR experiments can be found in the Supporting Information. The surprisingly effective coenrichment of N-glycopeptides in the WCX/CrE flow through can also be rationalized by the presence of the crown ether functionality. The crown ether gives an extra reduction of the final complexity of the flow through by retaining internal and C-terminal peptides with free R-NH2 groups which would not have been retained on a column solely exhibiting WCX or SCX properties (see Figure 6). At the same time it does not retain (sialo-)glycopeptides due to both steric effects and/or internal salt formation with available sialic acid residues. This selective reduction of the complexity probably

Figure 6. Traces in panel A depict the normalized UV traces at 214 nm of the RPLC analyses performed on the WCX/CrE flow through (green) and the SCX flow through (red) from the same processed serum digest. Equal sample amounts were used for the respective prepurification steps. Panel B shows a blow-up of the relevant chromatographic window to aid comparison.

accounts for the relative glycopeptide enrichment. From this, it was envisioned that a similar experimental setup without the N-terminal blocking step (acetylation) would lead to a highly effective N-glycopeptide enrichment. Indeed, in a preliminary test experiment 77% (71/92) of the retrieved sequences were Nglycopeptides, whereas most of the remainder sequences corresponded to in vivo acetylated protein N-termini (16/21). It is noteworthy that in the “PNGase F POST” experiment 11.06% of the identified sequences in the nonglycopeptide fraction and 29.85% in the glycopeptide fraction contained a nonacetylated lysine side chain. The significantly higher number in the glyco-

peptide fraction is in good agreement with the underlying sorting mechanism. It might also point to impaired acetylation efficiency as a result of local glycan shielding effects. Possibly a protein deglycosylation step prior to the acetylation could lead to a more efficient N-teromics analytical scheme. Biological Considerations. This study was not intended as a comprehensive serum proteome analysis. This would require a more efficient LC-MS setup to achieve sufficient separation capacity and to limit undersampling effects. Yet, in view of the peptide signature inset of this investigation, some interesting information was nevertheless obtained. In total, the “reference” Analytical Chemistry, Vol. 81, No. 7, April 1, 2009

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Figure 7. For the combined data of the “reference” and the “PNGase F POST” experiment the location of the identified sequences within the proteins is shown (binned per 5). The location is determined by the position of the starting amino acid of the identified peptide. The bar graphs correspond to the number of unique sequences observed. The color reflects whether the sequence is N-terminally acetylated (red) or not (blue). Panel A summarizes the data for all unique modified sequences which are considered not to be glycopeptides, whereas panel B plots the same information for the glycopeptides.

and the “PNGase F Post” experiment led to the identification of 102 different protein groups, of which 37 were identified in both experiments. A total of 39 proteins were unique to the N-teromics data (“reference” experiment and the nonglycopeptide fraction of the “PNGase F POST” experiment), whereas 26 were only represented in the glycopeptide fraction of the “PNGase F POST” experiment. In Figure 7 the position of the identified sequences in their parent proteins is summarized for the nonglycopeptides and the glycopeptides (based on the N-terminal amino acid position; up to position 200). It is clear from panel A in Figure 7 that many of the acetylated nonglycopeptides are positioned at the beginning of their proteins. Most of these peptides are not located at position 1 because the loss of any signal peptides is not corrected for. This confirms the merits of a WCX/CrE flow through analysis in an N-teromics context: a preferential enrichment of the true N-termini of proteins is obtained. The acetylated sequences retrieved from other positions in the proteins point to in vivo processing events, information that is also considered relevant in a biomarker context. The glycopeptides showsas expectedsa more uniform distribution in terms of their positions within the proteins (Figure 7B). As argued above, N-terminally acetylated peptides are indicative for in vivo processing of the serum proteins. Often such in 2468


vivo processing involves aminopeptidase activity45 resulting in N-terminally ragged sequences. The N-teromics approach here applied exposes such processing events because it selects for N-terminally acetylated peptides. An example is given in Table 3. Surprisingly, within the glycopeptide enriched fraction, groups of unacetylated N-terminally ragged sequences were also identified (Table 3). This could indicate that the presence of glycan modifications close to free R-NH2 groups (in vivo) impairs the acetylation reaction. Therefore, a slightly adapted N-teromics workflow, wherein prior to acetylation the proteins are deglycosylated, could improve the acetylation efficiency. At the same time, these data show that glycopeptide (co)enrichment can expose in vivo protein processing events. The significance of this finding with regard to the well-accepted model in which glycosylation is said to protect serum proteins against processing events remains unclear. CONCLUSION A WCX/CrE column, initially developed for inorganic ion analyses, was applied in a proteomics work flow. The coavailability of the 18-crown-6 ether functionality was expected to improve the (45) Sanderink, G. J.; Artur, Y.; Schiele, F.; Gueguen, R.; Siest, G. Clin. Chem. 1988, 34, 1422–1426.

Table 3. Exemplary N-Terminally Acetylated Sequences of r-1-Acid Glycoprotein 2 [Precursor] (Swiss-Prot Entry P19652) and a Set of Sequences Derived from the Platelet Basic Protein [Precursor] (Swiss-Prot Entry P02775)a R-1-acid glycoprotein 2 [precursor] position 44, 45,..., 62 Ac-NLAKGKEESLDSDLYAELR Ac-LAKGKEESLDSDLYAELR Ac-AKGKEESLDSDLYAELR Ac-GKEESLDSDLYAELR Ac-EESLDSDLYAELR

platelet basic protein [precursor] position 56, 59,..., 81 NH2-SVQEIQATFFYFTPNKTEDTIFLR NH2-EIQATFFYFTPNKTEDTIFLR NH2-ATFFYFTPNKTEDTIFLR NH2-TFFYFTPNKTEDTIFLR NH2-FFYFTPNKTEDTIFLR NH2-FYFTPNKTEDTIFLR NH2-YFTPNKTEDTIFLR NH2-FTPNKTEDTIFLR NH2-TPNKTEDTIFLR

a The left column shows some exemplary N-terminally acetylated sequences of R-1-acid glycoprotein 2 [precursor] (Swiss-Prot entry P19652). Compliant with the applied N-teromics approach the observed N-terminal ragging is indicative for some in vivo aminopeptidase activity. Within the right column a set of sequences derived from the platelet basic protein [precursor] (Swiss-Prot entry P02775) is given. These sequences demonstrate no N-terminal acetylation, yet they were coenriched within the WCX/CrE flow through because they were N-glycosylated during the sorting step. Interestingly, some N-terminal ragging is also apparent in these sequences. To the best of our knowledge no protocol related reasons account for this N-terminal ragging, implicating these sequences also reflect some in vivo processing events.

depletion of peptides containing unmodified primary amino groups from a complex mixture. This premise was put to test in an indirect N-teromics approach, whereby a negative subselection of peptides in which all amino functionalities were blocked by acetylation was pursued. Prior to the sorting experiments, a theoretical model was developed in which the impact on the sorting of each of the column constituents, i.e., WCX and CrE, was predicted. Analysis of the identified peptides in the WCX/CrE flow through confirmed the enrichment of the targeted peptides and revealed that the sorting performance of the WCX component is robust and agrees well with the prediction model. The sorting performance of the crown ether component was found to be less predictable because its complexation of monoalkylammonium groups is sensitive to steric and local charge/amino acid effects. From the experimental data it wasscautiouslysconcluded that the crown ether indeed adds to the global sorting performance, when compared to a pure

WCX or SCX column. Interestingly, the WCX/CrE column also led to a favorable coenrichment of glycopeptides. In view of the current biomarker efforts, the WCX/CrE column holds considerable promise: the presented workflow targets peptide subpopulations with high biomarker potential. Furthermore, one can easily envision a more generic platform design which can be modulated to target different peptide subpopulations. Such platform would be based on a crown ether sorting step followed by an in-depth LC-MS analysis of the nonretained fraction. Versatility of the platform would be achieved by adding/ removing the acetylation step in the sample preparation procedure and by the timing of the deglycosylation step. This way, one could solely target N-terminally acetylated peptides, or N-terminally acetylated peptides and glycopeptides, or glycopeptides and in vivo acetylated peptides only. In conclusion, these initial findings justify more research to probe the potential of crown ethers for proteome subselection. In particular, it would be extremely interesting to analyze the sorting behavior of a pure crown ether column. ACKNOWLEDGMENT The authors thank Dionex for kindly providing the IonPac CS 15 cation-exchange column. K.G. is grateful to the Research Council of UGent for a Ph.D. Grant (GOA project 01.G015.07). J.C.M. thanks the SBO program of the IWT for a postdoctoral Research Grant. J.C.M. acknowledges the Fund for Scientific Research-Flanders (FWO-Vlaanderen) for various Equipment Grants (G.0365.03, G.0064.07). The 700 MHz equipment of the Interuniversitary NMR Facility was financed by Ghent University, the Free University of Brussels (VUB), and the University of Antwerp through the “Zware Apparatuur” Incentive of the Flemish Government. SUPPORTING INFORMATION AVAILABLE Additional description of the NMR experiments and data as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review September 18, 2008. Accepted February 3, 2009. AC801975B


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Crown Ether

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