Selective Isolation of N-Blocked Peptides by Isocyanate-Coupled

Sep 14, 2007 - Kazuhiro Sonomura , Hiroki Kuyama , Ei-ichi Matsuo , Susumu Tsunasawa , Shiroh Futaki , Osamu Nishimura. Analytical Biochemistry 2011 ...
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Anal. Chem. 2007, 79, 7910-7915

Selective Isolation of N-Blocked Peptides by Isocyanate-Coupled Resin Toshiyuki Mikami and Toshifumi Takao*

Laboratory of Protein Profiling and Functional Proteomics, Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan

We developed a method for selective isolation of Nblocked peptides from a complex mixture such as an enzymatic digest of a protein. The approach is based on a newly designed isocyanate-resin (resin-NCO), which specifically reacts with r-amino or imino groups. This method, then, permits the isolation of N-blocked peptides, even those containing Lys, from a peptide mixture as intact forms by trapping N-free peptides via covalent bonding to the resin-NCO. The present study demonstrates the performance of this method for the selective isolation of N-blocked peptides by applying it to several peptide mixtures, including proteolytic digests. During the maturing process of proteins following the translation of mRNA, proteins often undergo various processing in the N- or C-terminal regions, such as cleavage of the initial methionine by aminopeptidase. Removal of signal peptides, which regulate the localization and targeting of proteins, often occurs for secreted and membrane proteins. N-terminal modifications, such as acetylation, pyroglutamylation, myristoylation, etc., also are common post-translational processing events. In mammals, more than 80% of cytoplasmic proteins are acetylated at the N-termini.1 These processes are known to regulate localization and stability of cellular proteins. In addition to these post-translational modifications, N- and C-terminal processing play crucial roles in regulating cellular function. For example, N-terminal processing of chemokines by aminopeptidases and endopeptidases appears to affect receptor specificity, chemotactic properties, and signaling potency.2 N-terminal information helps to discover substrates for orphan proteases.3,4 N-terminal portions of proteins have important features, which are relevant to protein function; thus, characterization of N-terminal peptide regions via proteomic analysis has been an important research focus. Edman degradation of proteins remains useful in elucidating the N-terminal portion of a protein; however, it is unworkable for N-terminally blocked proteins and is limited to high-purity samples, which are unsuitable to the workflow of proteomics. Mass spectrometry has been used as a powerful alternative in protein identification, as well as in peptide sequencing, even for proteins * To whom correspondence should be addressed. E-mail: [email protected]. Phone: +816-6879-4312. Fax: +816-6879-4312. (1) Polevoda, B.; Sherman, F. J. Biol. Chem. 2000, 275, 36479-36482. (2) Proost, P.; Struyf, S.; Van Damme, J. Biochem. Soc. Trans. 2006, 34, 9971001. (3) Overall, C. M.; Blobel, C. P. Nat. Rev. Mol. Cell Biol. 2007, 8, 245-257. (4) Lopez-Otin, C.; Overall, C. M. Nat. Rev. Mol. Cell Biol. 2002, 3, 509-519.

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modified with N-terminal blocking groups.5 However, proteins are generally digested into small peptides, thus making it difficult to find the original N-terminal peptide among the complex mixture of digested peptides. Several approaches have been developed to isolate or identify N-terminal peptides of original proteins,6-10 even in mixtures that include N-terminally blocked proteins.7-9 Recently, Yamaguchi et al. developed a high-throughput method for sequencing N-terminal peptides of proteins, except for N-terminally blocked proteins. In this method, N-terminal amino groups are coupled with biotinylcysteic acid, followed by isolation of the modified N-terminal peptides using the biotin-avidin technique.6 McDonald et al. reported an approach to isolate N-terminal peptides by trapping internal peptides in an avidin column.7 Kuhn et al. used an amine-scavenging resin to capture non-N-terminal peptides.8 Gevaert et al. developed a method to isolate N-terminal peptides: the hydrophobicity of internal peptides is modulated, followed by combinational fractional diagonal chromatography (COFRADIC). This method has been applied to global N-terminal proteomic analysis.9,11,12 For N-terminally blocked proteins, Akiyama et al. reported a method for selective isolation. N-blocked peptides are recovered in the flow-through from a cyanogen bromide-activated Sepharose column when all the -amino groups of Lys residues are succinylated prior to enzymatic digestion or chemical cleavage.10 These methods are based on chemical modification of the amino groups of a protein, followed by digestion into peptides. All of the resultant peptides, except for the N-terminal peptide that has been natively blocked or chemically modified, have newly formed R-amino groups. Therefore, these internal peptides could be segregated from the blocked N-terminal peptide by various chemical tags that can link to R-amino groups. The present paper describes a method for isolating N-blocked peptides from a complex peptide mixture. The method involves a (5) Kokame, K.; Fukada, Y.; Yoshizawa, T.; Takao, T.; Shimonishi, Y. Nature 1992, 359, 749-752. (6) Yamaguchi, M.; Nakazawa, T.; Kuyama, H.; Obama, T.; Ando, E.; Okamura, T. A.; Ueyama, N.; Norioka, S. Anal. Chem. 2005, 77, 645-651. (7) McDonald, L.; Robertson, D. H.; Hurst, J. L.; Beynon, R. J. Nat. Methods 2005, 2, 955-957. (8) Kuhn, K.; Thompson, A.; Prinz, T.; Muller, J.; Baumann, C.; Schmidt, G.; Neumann, T.; Hamon, C. J. Proteome Res. 2003, 2, 598-609. (9) Gevaert, K.; Goethals, M.; Martens, L.; Van Damme, J.; Staes, A.; Thomas, G. R.; Vandekerckhove, J. Nat. Biotechnol. 2003, 21, 566-569. (10) Akiyama, T. H.; Sasagawa, T.; Suzuki, M.; Titani, K. Anal. Biochem. 1994, 222, 210-216. (11) Van Damme, P.; Martens, L.; Van Damme, J.; Hugelier, K.; Staes, A.; Vandekerckhove, J.; Gevaert, K. Nat. Methods 2005, 2, 771-777. (12) Falb, M.; Aivaliotis, M.; Garcia-Rizo, C.; Bisle, B.; Tebbe, A.; Klein, C.; Konstantinidis, K.; Siedler, F.; Pfeiffer, F.; Oesterhelt, D. J. Mol. Biol. 2006, 362, 915-924. 10.1021/ac071294a CCC: $37.00

© 2007 American Chemical Society Published on Web 09/14/2007

newly developed isocyanate-coupled resin (resin-NCO) and the use of reaction conditions specific to R-amino groups. The method, thus, allows peptides with R-amino groups to be captured by the resin; whereas, N-blocked peptides, irrespective of the presence of Lys residue(s), remain intact in the supernatant of the resin reaction mixture. In the context of emerging methodologies for N-terminome,3,11,12 this capability is especially useful, as it allows for isolation of N-terminal blocked peptides of proteins and N-blocked bioactive peptides in biological samples. This study evaluates the performance and application of the method using synthetic Lys-containing peptides and complex peptide mixtures, including enzymatic digests of N-terminally blocked proteins. EXPERIMENTAL SECTION Materials. Angiotensin I, dynorphin, ACTH (1-24), β-endorphin, neurotensin, and β-neoendorphin were purchased from the Peptide Institute (Osaka, Japan). Sequencing-grade modifiedtrypsin was purchased from Promega (Madison, WI). Bovine serum albumin (BSA), cytochrome c, calmodulin, fibrinopeptide B, chymotrypsin, R-cyano-4-hydroxycinnamic acid (CHCA), and acetonitrile were obtained from Sigma (St. Louis, MO). Methylenediphenyl 4,4′-diisocyanate (MDI) was purchased from Wako Pure Chemical (Osaka, Japan). Phosphoric acid and triethylamine (TEA) were obtained from Nacalai Tesque (Kyoto, Japan). Water18O (98 atom % 18O) was purchased from Taiyo Nippon Sanso (Tokyo, Japan). Poros 20 NH Amine activated packing was purchased from Perseptive BioSystems (Framingham, MA). Peptides R86-1 (Ac-YGGFLSYPLK) and R86-2 (YGGFLSYPLK) were synthesized by BioSynthesis Inc. (Lewisville, TX). Caution: MDI is highly toxic and care should be taken in handling, analysis, and disposal of this substance. Protective glove and safety glass should be used and operation should be done under adequate ventilation. Preparation of Isocyanate-Coupled Resin (Resin-NCO). Poros 20 NH (50 mg) was washed with acetonitrile and then suspended in 400 µL of acetonitrile. The suspension of Poros 20 NH was mixed with 400 µL of 0.5 M MDI in acetonitrile at room temperature for 60 min. After the reaction was complete, the resin was extensively washed with acetonitrile and dried in a vacuum. The product was stored in argon at -20 °C until use. Trypsin-Catalyzed C-Terminal 18O Incorporation of Peptides. Peptides (10 nmol) were dissolved in 45 µL of 50 mM NH4HCO3 in H218O and 5 µL of acetonitrile, and the resulting solution was transferred to a tube containing 100 pmol of trypsin. The solution was incubated at 37 °C for 24 h. Peptide labeling with 18O was confirmed by mass spectrometry. Isolation of N-Blocked Peptides. Resin-NCO resin was washed and suspended with acetonitrile before use. The resin (1.8 mg) was suspended in 55 µL of acetonitrile. A mixture of peptides was dissolved in 0.05 M phosphoric acid-TEA buffer (pH 3.5), and the solution (5∼8 µL), containing up to ∼1 nmol of peptides, was mixed with the resin for 20 min at room temperature. The resin was then spun down, and the supernatant, which contained the N-blocked peptides, was directly analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDITOFMS) (see below). For model proteins, each protein was first digested using trypsin or chymotrypsin in 50 mM NH4HCO3. The digests were then dried, reconstituted in 0.05 M phosphoric acidTEA buffer (pH 3.5), and reacted with resin-NCO, as described above. After centrifugation, the supernatant was subjected to mass spectrometry.

Figure 1. General scheme for selective isolation of N-blocked peptides. Poros 20 NH resin coupled with an isocyanate group (resinNCO) can specifically react with R-amino groups under weak acidic conditions (see Experimental Section). N-blocked peptides are recovered in the nonreactive materials in the supernatant of the resin reaction mixture. In the case of an N-terminally blocked protein (A), cysteine residues are first reduced and S-alkylated. Then, the protein is digested with a protease, such as trypsin, chymotrypsin, or V8 protease. The digest is further incubated with resin-NCO, allowing all internal peptides to be trapped by the resin-NCO and the N-blocked peptide to be recovered from the supernatant of the reaction mixture. As for N-terminally free proteins (B), all amino groups are acylated before proteolysis to form chemically N-blocked proteins. The rest of the process is the same as that described in part A. In combination with protease-catalyzed 18O labeling at the C-termini of peptides, comparative proteomic analysis focused on N-terminal regions might be possible.13,14,16

The reaction efficiency of resin-NCO with R-amino groups was dependent on reaction pH. Solutions with a pH less than 3 resulted in half-finished reactions. The optimal pH for selective reaction with R-amino groups was between 3.5 and 4.0. In addition, excess water in solution (g20%) significantly reduced the reaction yield because isocyante gradually decomposed in water. Thus, the reaction was carried out at higher acetonitrile concentrations, 85-90%, in the aqueous buffer, which also reduced nonspecific hydrophobic adsorption of peptides to the resin. Mass Spectrometry. MALDI mass spectra were acquired on a Micromass MALDI time-of-flight mass spectrometer (Manchester, UK). The matrix was a saturated solution of CHCA in a 50% aqueous solution of acetonitrile and 0.1% TFA. An aliquot (0.5 µL) of the sample solution was mixed with 0.5 µL of the matrix solution on the MALDI plate. The instrument was calibrated with a mixture of peptides (angiotensin I (m/z 1296.6), dynorphin (m/z 1604.0), ACTH (1-24) (m/z 2932.6), and β-endorphin (m/z 3463.8)). RESULTS AND DISCUSSION Strategy for Isolation of N-Blocked Peptides from a Peptide Mixture Using Isocyanate-Resin (Resin-NCO). Since amino-reactive reagent reacts with both R- and -amino groups of peptides, protection of -amino groups prior to reaction has been required,6,7,9 taking into account that N-blocked peptides may contain lysine residues. However, slight differences in pKa between the R-amino (9.06) and -amino (10.54) groups of Lys would permit the amino-reactive reagent to react specifically with R-amino groups by adjusting the pH and/or solvent. Among several aminoreactive reagents, we found that isocyanate compounds were most suitable for selective reaction with R-amino groups under weak Analytical Chemistry, Vol. 79, No. 20, October 15, 2007

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Figure 2. MALDI-TOFMS spectra from an equimolar mixture of the synthetic peptides (R86-1 and R86-2) prior to (A) and after (B) incubation with resin-NCO and spectra from a mixture of 5 pmol of R86-1 and 1 nmol of R86-2 (C) and those treated with 2.7 mg (D), 1.8 mg (E), and 0.9 mg (F) of resin-NCO.

acidic conditions. Thus, isocyanate-coupled resin (resin-NCO) was prepared by reacting a divalent isocyanate, methylenediphenyl 4,4′-diisocyanate (MDI), with aminopropyl resin (poros-NH), as a support medium. Because resin-NCO specifically reacts with R-amino groups of N-free peptides and not with -amino groups of Lys under weak acidic conditions (pH 3∼6), N-blocked peptides can be recovered from the supernatant of the reaction mixture. The method involves: (1) incubation of a peptide mixture with the resin-NCO in weak acidic solution for 20 min; (2) centrifugation and collection of the supernatant; and (3) characterization of peptides in the supernatant by MS. Figure 1 shows an overall workflow for selective isolation of N-blocked peptides from a peptide mixture or a protein digest with the resin-NCO. Peptides in a mixture can be classified into two types: N-free and N-blocked peptides. In the case of peptides or proteins that contain Cys residues, they should be first reduced and S-alkylated. When the N-terminal R-amino groups of proteins are free, they should be modified by, for example, an acetyl group prior to cleavage into peptides. The method is rapid and simple for selective isolation 7912

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of N-blocked peptides without laborious steps, such as protection of Lys side-chains. Evaluation of Resin-NCO. The synthetic peptide (YGGFLSYPLK, R86-2) and its NR-acetylated form (R86-1) were first used to optimize specific reaction conditions between resin-NCO and certain R-amino groups. After incubation of the mixture of these peptides with resin-NCO in 0.05 M phosphoric acid-TEA (pH 3.5)/acetonitrile ) 5/45 (see Experimental Section), the signal at m/z 1144, which corresponded to the N-free peptide, disappeared; while another signal at m/z 1186 (N-blocked peptide) was distinctly observed (Figure 2A,B). It is worth mentioning that the two peaks observed in Figure 2A remained unchanged after treatment of the above peptide mixture with poros-NH resin, which is the foundation of resin-NCO, under the reaction conditions described above. The results suggest that resin-NCO specifically reacts with R- but not with -amino groups. Therefore, selective capture of N-free peptides with the resin-NCO permits exclusive isolation of N-blocked peptides without protecting the -amino groups.

Figure 3. MALDI-TOFMS spectra from the N-blocked peptide (R86-1) after treatment with resin-NCO. R86-1 (5 pmol in total (100 fmol/µL) in parts A and B; 1 pmol (20 fmol/µL) in parts C and D) was mixed with a known amount of the 18O-labeled peptide (see Experimental Section) as an internal standard prior to MALDI-TOFMS analysis. The N-free peptide, R86-2 (1 nmol) and the resin (1.8 mg) were added to the above R86-1 peptide solutions and incubated (parts B and D), and each resultant supernatant was measured.

Next, reaction capacity of resin-NCO was examined using the peptide mixture described above, except for addition of a 200fold excess of the N-free peptide (R86-2). The signal corresponding to R86-2 (m/z 1144) was exclusively observed; R86-1 could not be detected due to the 200-fold excess of R86-2 (Figure 2C). In MALDI experiments, minor components in a mixture are often suppressed by abundant components. After incubating the same peptide mixture with resin-NCO (2.7 mg, 1.8 mg), the R86-1 peptide peak was distinctly observed, while the R86-2 signal almost disappeared (Figure 2D). Capture of a 1 nmol-equivalent R-amino group of the peptide R86-2 (Figure 2E) was still possible when the amount of resin-NCO was reduced to 1.8 mg, while 0.9 mg of resin was not sufficient to make the minor component (R86-1) detectable (Figure 2F). This result indicated that the resin-NCO prepared in this study (see Experimental Section) had an estimated 1 nmol-equivalent isocyanate group per 1.8 mg of resin that specifically reacted with the R-amino groups. Recovery of an N-blocked peptide from the resin-NCO was tested using the above peptide mixture. To quantify the peptide (R86-1) using MS, an 18O-labeled peptide was prepared according to a previously published method13,14 and was used to spike the reaction mixture. Figure 3A shows the mass spectrum of R86-1 (5 pmol of R86-1 dissolved in 50 µL of the reaction solvent) mixed with 18O-labeled peptide (5 pmol). 18O-labeled R86-1 was observed at m/z 1190, which showed 4 Da increments compared with the original non-labeled R86-1. Incubation of peptides (5 pmol of R86-1 and 1 nmol of R86-2 in 50 µL of the reaction solvent) with resinNCO (1.8 mg) allowed for the detection of R86-1 and its 18O-labeled species (5 pmol) (Figure 3B). On the basis of the ratio of peak intensities, i.e., peak intensity at m/z 1186 to peak intensity at

m/z 1190, the recovery of R86-1 in this experiment was calculated to be 80%. When the amount of R86-1 was reduced to 1 pmol, such that the amount of R86-1 was 1/1000 that of R86-2, the recovery of R86-1 was 70% (Figure 3C,D). However, the recovery of an N-blocked peptide from the resin-NCO might be dependent on the nature of peptides (acidity, hydrophobicity, etc.) and the reaction conditions (pH, solvent, resin amount, etc.). To confirm the effectiveness of the method, three kinds of peptides (β-neoendorphin, fibrinopeptide B, and neurotensin) were mixed with a BSA tryptic digest and used in experiments with resin-NCO. The N-blocked peptides (fibrinopeptide and neurotensin) were selectively isolated from the complex mixture as a result of specific capture of all N-free peptides (BSA tryptic peptides and β-neoendorphin) by resin-NCO (Figure 4A,B). Therefore, these results indicate that the present method allows selective isolation of N-blocked peptides, even those containing Lys residues, using this one-step batchwise procedure that does not require protection of the -amino groups on Lys. Figure 5A shows the MALDI spectrum of the tryptic digest of calmodulin, which is originally acetylated at the N terminus. The signal at m/z 1563, which corresponds to the N-terminal tryptic peptide, containing a Lys at the C terminus, was the only peak observed after one-step treatment with resin-NCO (Figure 5B). Figure 6 shows another experiment where cytochrome c, which is originally acetylated at the N-terminus, was digested with chymotrypsin. The N-terminal peptide, which contains three Lys resi(13) Heller, M.; Mattou, H.; Menzel, C.; Yao, X. J. Am. Soc. Mass Spectrom. 2003, 14, 704-718. (14) Wang, Y. K.; Ma, Z.; Quinn, D. F.; Fu, E. W. Anal. Chem. 2001, 73, 37423750.

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Figure 4. MALDI-TOFMS spectra from N-blocked peptides in a complex peptide mixture. Three synthetic peptides (40 pmol of β-neoendorphin (YGGFLRKYP), 40 pmol of neurotensin (Pyr-LYENKPRRPYIL), and 20 pmol of fibrinopeptide-B (Pyr-GVNDNEEGFFSAR)) mixed with a BSA tryptic digest (15 pmol) were dissolved in 8 µL of 0.05 M phosphoric acid-TEA (pH 3.5) and 45 µL of acetonitrile. An aliquot of the solution (0.5 µL) was subjected to MALDI-TOFMS prior to (A) and after (B) incubation with 1.8 mg of the resin-NCO.

Figure 5. MALDI-TOFMS spectra from an N-terminally blocked protein, calmodulin, treated with trypsin. A tryptic digest of calmodulin (30 pmol) was dissolved in 7 µL of 0.05 M phosphoric acid-TEA (pH 3.5) and 45 µL of acetonitrile. An aliquot (0.5 µL) of the solution was subjected to MALDI-TOFMS prior to (A) and after (B) incubation with resin-NCO (0.9 mg). The peak observed at m/z 1563 is assigned to the N-terminal peptide (Ac-ADQLTEEQIAEFK) derived from calmodulin. 7914 Analytical Chemistry, Vol. 79, No. 20, October 15, 2007

Figure 6. MALDI-TOFMS spectra from an N-terminally blocked protein, cytochrome c, treated with chymotrypsin. A chymotryptic digest of cytochrome c (40 pmol) was dissolved in 7 µL of 0.05 M phosphoric acid-TEA (pH 3.5) and 45 µL of acetonitrile. An aliquot (0.5 µL) of the solution was subjected to MALDI-TOFMS prior to (A) and after (B) incubation with resin-NCO (1.8 mg). The peak observed at m/z 1162 is assigned to the N-terminal peptide (Ac-GDVEKGKKIF) derived from cytochrome c, which contains three Lys.

dues, was predominantly observed at m/z 1162 after treatment with resin-NCO (Figure 6B); the MS/MS clearly gave the N-terminal sequence (Ac-GDVEKGKKIF). The method allowed for selective isolation of blocked N-terminal peptides of proteins, even proteins containing multiple Lys residues. CONCLUSIONS The present study demonstrates a facile method for selective isolation of N-blocked peptides using an isocyanate-coupled resin (resin-NCO). Resin-NCO specifically captured N-free peptides via linkages with R-amino groups under acidic conditions (see Experimental Section), omitting the step of protecting the -amino groups on Lys. The method allows for rapid isolation not only of N-blocked peptides but also of N-terminal peptides of proteins, when N-terminal R-amino groups are blocked or chemically modified prior to cleavage into peptides (see Figure 1). In addition, the use of a stable-isotope labeled reagent for modification, such as a mixture of acetic anhydride-d0/d6, makes it possible to distinguish N-blocked peptides from those derived from N-free proteins, based on the stable-isotope labeling. The amounts of resin used in the present study, ranging from 1.8 to 2.7 mg, were enough to capture approximately 1 nmol of N-free peptides, from which 1 pmol of N-blocked peptides, 1/1000th of the total peptide charged to the resin, was recovered in relatively high yield (70%, Figure 3D). The reaction capacity of the resin could be scaled up or down, according to the sample size. However, minimal amounts should be used to avoid sample loss due to nonspecific binding (15) Felth, M.; Skold, K.; Norrman, M.; Svensson, M.; Fenyo, D.; Andren, P. E. Mol. Cell. Proteomics 2006, 5, 998-1005. (16) Yao, X.; Freas, A.; Ramirez, J.; Demirev, P. A.; Fenselau, C. Anal. Chem. 2001, 73, 2836-2842.

to the resin, sespecially when small amounts of target N-blocked peptides are to be recovered from the resin. The method could be applied to global proteomic surveys of N-terminal proteolytic peptides (N-terminome).7,9 In these experiments, proteins were chemically acetylated at all amino groups, treated with trypsin, and the resulting tryptic peptides (except for the original N-terminal peptide) then were biotinylated at the newly produced amino groups and captured using streptavidincolumns7 or separated using combinational fractional diagonal chromatography9 to segregate the tryptic peptides from the original N-terminal peptides. At this step, the use of resin-NCO could improve the speed and efficiency of the analysis. By virtue of no requirement of prior chemical modification, the present method could be only a way for the survey of in vivo N-blocked peptides and their isolation, as intact forms, from biological samples, a major fraction of which have been identified as bioactive peptides.15 For example, identification of N-blocked peptides in an extract of the pituitary gland, which is rich in bioactive peptides, is now underway. ACKNOWLEDGMENT This study was supported by a Grant-in-Aid for Creative Scientific Research (Grant 15GS0320 to T.T.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a project on ‘‘Research on Proteomics” from the Ministry of Health, Labour and Welfare of Japan. Received for review June 19, 2007. Accepted July 26, 2007. AC071294A Analytical Chemistry, Vol. 79, No. 20, October 15, 2007

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