Anal. Chem. 2008, 80, 2874-2880
Detecting D-Amino Acid-Containing Neuropeptides Using Selective Enzymatic Digestion Michael A. Ewing, Jane Wang, Sarah A. Sheeley, and Jonathan V. Sweedler*
Department of Chemistry and the Beckman Institute, University of Illinois, Urbana, Illinois 61801
Neuropeptides, gene products that undergo extensive post-translational modification (PTM), are frequently characterized using mass spectrometry (MS). One PTM in particular, the conversion of an L-amino acid to a D-amino acid, has no associated mass shift. Therefore, this PTM is difficult to evaluate using MS alone, especially in complex peptide mixtures. Here, enzymatic digestion using microsomal alanyl aminopeptidase is combined with MS characterization. This enzyme selectively degrades peptides lacking a D-amino acid in the second position from the N-terminus. By comparing a sample before and after digestion, D-amino acid-containing peptides (DAACPs) present in small quantities in a complex mixture can be identified, even among much larger quantities of other non-DAACPs. Protocols that use microsomal alanyl aminopeptidase as a discovery-enabling agent are described and validated by identifying a known DAACP from the Aplysia californica abdominal ganglion. Mass spectrometry (MS) is widely used in studies related to the identification and sequencing of novel cell-cell signaling peptides, due in large part to its high information content, exquisite sensitivity, and its ability to characterize post-translational modifications (PTMs). In fact, MS-based characterization of tissues has created the field of neuropeptidomics,1-5 the wholesale discovery and characterization of peptides in the brain. However, there are a few chemical modifications less well-suited to traditional MSbased peptide characterization. Notably, research over the last few decades has revealed a surprising modification in peptidebased toxins and neuropeptides from a range of animalssthe chiral conversion of an L-residue to a D-residue. How is such a neuropeptide created? For all neuropeptides, a gene that encodes for the peptide prohormone is expressed. As the prohormone moves through the trans-Golgi complex and is sorted into dense core vesicles, the prohormone is then processed * To whom correspondence should be addressed. Voice: 217-244-7359. Fax: 217-244-8068. E-mail:
[email protected]. (1) Baggerman, G.; Verleyen, P.; Clynen, E.; Huybrechts, J.; De Loof, A.; Schoofs, L. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2004, 803, 3-16. (2) Fricker, L. D.; Lim, J.; Pan, H.; Che, F. Y. Mass Spectrom. Rev. 2006, 25, 327-344. (3) Hummon, A. B.; Amare, A.; Sweedler, J. V. Mass Spectrom. Rev. 2006, 25, 77-98. (4) Parkin, M. C.; Wei, H.; O’Callaghan, J. P.; Kennedy, R. T. Anal. Chem. 2005, 77, 6331-6338. (5) Svensson, M.; Skold, K.; Nilsson, A.; Falth, M.; Svenningsson, P.; Andren, P. E. Biochem. Soc. Trans. 2007, 35, 588-593.
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via a number of enzymatic processing steps, including cleavage at basic and other amino acids. Last, a variety of PTMs are formed to produce the final bioactive neuropeptides. A standard tenet of translation in animals is that L-amino acids are used to create proteins (and hence peptides) in higher organisms.6 The D-amino acid in D-amino acid-containing peptides (DAACPs) is expected to be a PTM.6-9 In some toxins, like those found in cone snails, frog skin, spiders toxins, and platypus venom, the D-amino acid occupies many different positions in the peptide.10-16 However, in neuropeptides and in the amphibian opioid-like peptides, the D-amino acid is almost exclusively found two or three amino acid positions from the N-terminal end of the peptide.13,17-22 This uniform, position-based cleavage pattern suggests that the D-amino acid results from a PTM that takes place after the basic site cleavages occur to form the peptide (and produce the N-terminus). Indeed, additional evidence demonstrates this change is made late in the processing of a prohormone,9 that it occurs in the secretory vesicles containing the (6) Kreil, G. Science 1994, 266, 996-997. (7) Fujii, N. Origins Life Evol. Biosphere 2002, 32, 103-127. (8) Kreil, G. Annu. Rev. Biochem. 1997, 66, 337-345. (9) Soyez, D.; Toullec, J. Y.; Ollivaux, C.; Geraud, G. J. Biol. Chem. 2000, 275, 37870-37875. (10) Auvynet, C.; Seddiki, N.; Dunia, I.; Nicolas, P.; Amiche, M.; Lacombe, C. Eur. J. Cell Biol. 2006, 85, 25-34. (11) Buczek, O.; Yoshikami, D.; Bulaj, G.; Jimenez, E. C.; Olivera, B. M. J. Biol. Chem. 2005, 280, 4247-4253. (12) Buczek, O.; Yoshikami, D.; Watkins, M.; Bulaj, G.; Jimenez, E. C.; Olivera, B. M. FEBS J. 2005, 272, 4178-4188. (13) Kreil, G.; Barra, D.; Simmaco, M.; Erspamer, V.; Erspamer, G. F.; Negri, L.; Severini, C.; Corsi, R.; Melchiorri, P. Eur. J. Pharmacol. 1989, 162, 123128. (14) Torres, A. M.; Menz, I.; Alewood, P. F.; Bansal, P.; Lahnstein, J.; Gallagher, C. H.; Kuchel, P. W. FEBS Lett. 2002, 524, 172-176. (15) Torres, A. M.; Tsampazi, C.; Geraghty, D. P.; Bansal, P. S.; Alewood, P. F.; Kuchel, P. W. Biochem. J. 2005, 391, 215-220. (16) Heck, S. D.; Siok, C. J.; Krapcho, K. J.; Kelbaugh, P. R.; Thadeio, P. F.; Welch, M. J.; Williams, R. D.; Ganong, A. H.; Kelly, M. E.; Lanzetti, A. J.; et al. Science 1994, 266, 1065-1068. (17) Kamatani, Y.; Minakata, H.; Kenny, P. T. M.; Iwashita, T.; Watanabe, K.; Funase, K.; Sun, X. P.; Yongsiri, A.; Kim, K. H.; Novales-Li, P.; Novales, E. T.; Kanapi, C. G.; Takeuchi, H.; Nomoto, K. Biochem. Biophys. Res. Commun. 1989, 160, 1015-1020. (18) Montecucchi, P. C. D. C., R.; Piani, S.; Gozzini, L.; Erspamer, V. Int. J. Pept. Protein Res. 1981, 17, 275-283. (19) Morishita, F.; Nakanishi, Y.; Kaku, S.; Furukawa, Y.; Ohta, S.; Hirata, T.; Ohtani, M.; Fujisawa, Y.; Muneoka, Y.; Matsushima, O. Biochem. Biophys. Res. Commun. 1997, 240, 354-358. (20) Morishita, F.; Sasaki, K.; Kanemaru, K.; Nakanishi, Y.; Matsushima, O.; Furukawa, Y. Peptides 2001, 22, 183-189. (21) Yasuda-Kamatani, Y.; Kobayashi, M. Y., A.; Fujita, T.; Minakata, H.; Nomoto, K.; Nakamura, M.; Sakiyama, F. Peptides 1997, 18, 347-354. (22) Yasuda-Kamatani, Y.; Nakamura, M.; Minakata, H.; Nomoto, K.; Sakiyama, F. J. Neurochem. 1995, 64, 2248-2255. 10.1021/ac7025173 CCC: $40.75
© 2008 American Chemical Society Published on Web 03/15/2008
peptides,10 and that it is enzymatically mediated.23 In several cases, researchers have shown that the normal DNA codon for L-amino acids is present in positions where the D-amino acids occur in the final DAACP.24-27 Thus, considerable evidence exists to support that the D-amino acid occurs as a result of posttranslational modification of the peptide. D-Amino acid-containing neuropeptides and opioid-like peptides have been found in a variety of animals during the past several decades, starting with the opioid peptides from frogs.13,18,28 Later, various neuropeptides were characterized in the molluscan central nervous system (CNS) including achatin-I and the fulicin generelated peptides from the snail Achatina fulica and NdWFa from the sea hare Aplysia kurodai, with several of these initial reports accompanied by bioactivity tests.17,19-22,29,30 In addition, the crustacean hyperglycemic hormone contains a D-Phe in the third residue from the N-terminal end with the biological activity of the neurohormone depending on residue confirmation.31,32 In mammals, the natriuretic peptide from platypus includes a D-amino acid.14,15 It appears that such peptides are found in animals from a number of phyla. Interestingly, in the cases of the platypus and spider Agelenopsis aperta venoms, the isomerase enzymes have been characterized.16,33-35 Even with these prior studies, the prevalence of D-amino acids in neuropeptides is not currently known. Research to identify new D-amino acid-containing neuropeptides has relied upon observing the differences in biological activity or chromatographic retention time between the purified native peptides and the synthesized all L-amino acid-containing peptides (LAACPs). However, most peptides when discovered are not associated with a robust bioactivity assay and so are difficult to evaluate in this manner. We expect that there may be many additional DAACPs to be characterized in a variety of animals. This is supported by the normal protocol for testing the bioactivity of peptides in many systems; when a peptide is characterized in a location that indicates further bioactivity testing is worthwhile, the peptide is synthesized and the activity of the synthetic peptide measured. In Aplysia californica, for example, a peptide found in a particular neuron known (23) Jilek, A.; Mollay, C.; Tippelt, C.; Grassi, J.; Mignogna, G.; Mullegger, J.; Sander, V.; Fehrer, C.; Barra, D.; Kreil, G. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 4235-4239. (24) Erspamer, V.; Melchiorri, P.; Falconieri-Erspamer, G.; Negri, L.; Corsi, R.; Severini, C.; Barra, D.; Simmaco, M.; Kreil, G. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 5188-5192. (25) Mignogna, G.; Simmaco, M.; Kreil, G.; Barra, D. EMBO J. 1993, 12, 48294832. (26) Richter, K.; Egger, R.; Kreil, G. Science 1987, 238, 200-202. (27) Satake, H.; Yasuda-Kamatani, Y.; Takuwa, K.; Nomoto, K.; Minakata, H.; Nagahama, T.; Nakabayashi, K.; Matsushima, O. Eur. J. Biochem. 1999, 261, 130-136. (28) Mor, A.; Delfour, A.; Nicolas, P. J. Biol. Chem. 1991, 266, 6264-6270. (29) Kanemaru, K.; Morishita, F.; Matsushima, O.; Furukawa, Y. Peptides 2002, 23, 1991-1998. (30) Morishita, F.; Minakata, H.; Sasaki, K.; Tada, K.; Furukawa, Y.; Matsushima, O.; Mukai, S. T.; Saleuddin, A. S. Peptides 2003, 24, 1533-1544. (31) Gallois, D.; Brisorgueil, M. J.; Conrath, M.; Mailly, P.; Soyez, D. Eur. J. Cell Biol. 2003, 82, 431-440. (32) Soyez, D.; Van Herp, F.; Rossier, J.; Le Caer, J. P.; Tensen, C. P.; Lafont, R. J. Biol. Chem. 1994, 269, 18295-18298. (33) Torres, A. M.; Tsampazi, M.; Kennett, E. C.; Belov, K.; Geraghty, D. P.; Bansal, P. S.; Alewood, P. F.; Kuchel, P. W. Amino Acids 2007, 32, 63-68. (34) Torres, A. M.; Tsampazi, M.; Tsampazi, C.; Kennett, E. C.; Belov, K.; Geraghty, D. P.; Bansal, P. S.; Alewood, P. F.; Kuchel, P. W. FEBS Lett. 2006, 580, 1587-1591. (35) Heck, S. D.; Faraci, W. S.; Kelbaugh, P. R.; Saccomano, N. A.; Thadeio, P. F.; Volkmann, R. A. Proc. Natl. Acad. Sci. U.S.A 1996, 93, 4036-4039.
to activate the feeding network would be tested for its ability to activate feeding.36 If the peptide is not bioactive, then other peptides from the same prohormone are synthesized and tested. How many native peptides of interest that contain an uncharacterized D-amino acid have led to the testing of a synthetic peptide that was not the endogenous molecule and therefore lacked bioactivity? Our goal is to prevent such occurrences by creating improved methods to scan for this PTM. How have D-amino acids in peptides been characterized previously? The peptides can be acid-hydrolyzed and the chirality of the resulting amino acids determined using liquid chromatography (LC)37 or capillary electrophoresis (CE).38,39 Degradation using chirally selective enzymes such as D-amino acid oxidase,24 N-terminal and C-terminal sequencing,37 and CE or LC separations when comparing migration times of peptides to standards40-42 also have been used for chiral determinations. In fact, CE has confirmed the presence of NdWFa in an individual neuron.40 Czerwenka and Lindner43 have described methods for stereoselective peptide analysis in an excellent recent review that emphasizes separation-based approaches. MS alone can be used to characterize peptides with a D-amino acid, as peptides with chiral amino acid substitutions often display different collisionally activated dissociation or electron capture dissociation patterns.44-46 However, such MS-based approaches normally require rather pure peptide fractions and oftentimes require standards to validate the fragmentation information. Although the MS- and LC-based methods offer impressive performance, they require characterization of the peptide. When performing a peptidomics experiment, one often detects hundreds of unknown putative peptide peaks. Our goal is to develop a protocol to determine, without full characterization, which peptides may possess a D-amino acid. Peptides containing a D-amino acid may resist degradation by endogenous amino- or carboxypeptidase enzymes, affecting the peptide lifetime after release. This is likely because the D-amino acid changes the peptide backbone conformation and thus affects how the peptide fits within the active site of the enzyme. Here, this difference in degradation is exploited for chirality determination. In particular, we demonstrate that the conversion of an L-amino acid residue to a D-amino acid residue in the first several (from the N-terminus) positions changes the efficacy of enzymatic digestion. Both standards and a crude cell lysate are used to compare peaks present with and without (36) Sweedler, J. V.; Li, L.; Rubakhin, S. S.; Alexeeva, V.; Dembrow, N. C.; Dowling, O.; Jing, J.; Weiss, K. R.; Vilim, F. S. J. Neurosci. 2002, 22, 77977808. (37) Iida, T.; Santa, T.; Toriba, A.; Imai, K. Biomed. Chromatogr. 2001, 15, 319327. (38) Liu, J. P.; Hsieh, Y. Z.; Weiesler, D.; Novotny, M. Anal. Chem. 1991, 63, 408-412. (39) Liu, Y. M.; Schneider, M.; Sticha, C. M.; Toyooka, T.; Sweedler, J. V. J. Chromatogr., A 1998, 800, 345-354. (40) Sheeley, S. A.; Miao, H.; Ewing, M. A.; Rubakhin, S. S.; Sweedler, J. V. Analyst 2005, 130, 1198-1203. (41) Czerwenka, C.; Lindner, W. Rapid Commun. Mass Spectrom. 2004, 18, 2713-2718. (42) Czerwenka, C.; Maier, N. M.; Lindner, W. Anal. Bioanal. Chem. 2004, 379, 1039-1044. (43) Czerwenka, C.; Lindner, W. Anal. Bioanal. Chem. 2005, 382, 599-638. (44) Serafin, S. V.; Maranan, R.; Zhang, K.; Morton, T. H. Anal. Chem. 2005, 77, 5480-5487. (45) Adams, C. M.; Zubarev, R. A. Anal. Chem. 2005, 77, 4571-4580. (46) Grigorean, G.; Gronert, S.; Lebrilla, C. B. Int. J. Mass Spectrom. 2002, 219, 79-87.
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enzymatic digestion; those resistant to digestion are considered as putative DAACPs that require follow-up testing. We use microsomal alanyl aminopeptidase (mAAP; also known as aminopeptidase N, aminopeptidase M; membrane alanyl aminopeptidase, EC 3.4.11.2),47 an enzyme that has been used for sequential digestion and MS sequencing,48 and document the degradation rate difference quantitatively. The difference in peptide degradation rates consisting entirely of L-amino acids, and those containing a D-amino acid in the penultimate positions, can then be used in conjunction with LC coupled to electrospray ionization (ESI) MS to search for novel peptides containing a D-amino acid. As shown in the results, the lifetime of DAACPs in the presence of this enzyme is more than 1 order of magnitude longer than peptides containing no PTMs. For example, most peptides are digested within 1 or 2 h, whereas DAACPs can be stable on a time scale of days. The method also can be applied to degradation time-course experiments to search for other lowabundance peptides containing unusual N-terminal PTMs that resist enzyme digestion. EXPERIMENTAL SECTION When listing peptides containing D-amino acids, the standard single capital letter for each specific amino acid is used; in addition, a “d” indicates that the following amino acid is in the Dconfiguration, and an “a” indicates an amidation of the C-terminus. Chemicals. The standard peptides used were YAELFa, YdAEFLa (fulyal), NWFa, NdWFa, R-BCP(1-8), angiotensin I, and bradykinin, typically present in 100 µg/mL volumes. YAELFa and YdAEFLa were synthesized by the Protein Sciences Facility, University of Illinois (UrbanasChampaign, IL). The peptides NWFa, NdWFa, and R-BCP(1-8) were purchased from American Peptide Co. (Sunnyvale, CA). Trifluoroacetic acid (TFA), glacial acetic acid, ammonium acetate, angiotensin I, bradykinin, and microsomal alanyl aminopeptidase (mAAP; leucine aminopeptidase, microsomal from porcine kidney, type IV-S ammonium sulfate suspension) were purchased from Sigma-Aldrich (St. Louis, MO). HPLC-grade acetonitrile (ACN) was obtained from Fisher. MilliQ filtered water (Millipore, Bedford, MA) was used for LC solvents. Liquid Chromatography-Electrospray Ionization-Mass Spectrometry. For ESI-MS, the analyte solution was fed directly into an LCQ Deca ESI mass spectrometer (Thermo-Finnigan, San Jose, CA) at 3 µL/min using the built-in syringe pump. The needle was held at 4.30 kV. The triple play mode (full scan, ZoomScan, CID with 35% collisional energy), data-dependent dynamic exclusion method was used. For LC, a Magic 2002 (Biochrom BioResources, Auburn, CA) system was used with two solvents: A (95% H2O, 5% ACN, 0.02% TFA, 0.01% acetic acid) and B (90% ACN, 10% H2O, 0.014% TFA, and 0.08% acetic acid). A Vydac (Hesperia, CA) reversed-phase column (2.1 × 150 mm) with 5-µm particles and 30-nm pores at a uniform flow rate of 100 µL/min was employed. (The polymer column is ranked between a C8 and C18 silica-based column in terms of hydrophobicity.) The gradient was linear between the following points: 5% B at 0 min, 10% B at 5 min, 40% B at 50 min, 80% B at 58 min, and 5% B at 60 min. The appropriate peptide (47) Wachsmuth, E. D.; Fritze, I.; Pfleiderer, G. Biochemistry 1966, 5, 169174. (48) Doucette, A.; Li, L. Proteomics 2001, 1, 987-1000.
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mixtures (10 µL) were injected onto the column. The detection used both a dual-wavelength UV absorbance detector set to 200 and 220 nm, as well as the LCQ Deca mass spectrometer. LC-MS of Standards. Solutions containing 100 µL of 0.1 mg/ mL combinations of NWFa, NdWFa, YAEFLa, and YdAEFLa in 20 mM ammonium acetate (pH 7) were injected onto a smallmolecule trap inline with the injection loop before being injected onto the same LC system, column, and solvents described earlier. For the time-course study, 49 µL of peptide standard solutions were prepared of 0.1 mg/mL NWFa/NdWFa, fulyal, and angiotensin I in 20 mM ammonium acetate, pH 7.5; 1 µL of enzyme digestion solution, containing 0.068 units of enzyme, 1.75 M (NH4)2SO4, and 5 mM MgCl2 was added. After incubation at room temperature for the desired length of time, 10 µL of concentrated HCl was added to the digested peptide mixture. For the 0-min digestion, the concentrated HCl was added just prior to the addition of the enzyme. For the other enzyme digestions not in the time-course study, the appropriate volume of the digestion buffer, consisting of 35 mM (NH4)2SO4 and 10 mM MgCl2, with 0.5 unit of enzyme/3.6 mL, was added to an equivalent amount of analyte solution and allowed to digest at room temperature for periods up to 48 h. After digestion, the mixture was injected first onto a smallmolecule trap inline with the injection loop and then injected onto the same LC system, column, and solvents as described earlier. MS was performed on the eluant using the LCQ Deca with a triple play as stated above. Preparation of Biological Samples. A. californica were obtained from the Aplysia research facility (Miami, FL) and kept in an aquarium containing continuously circulating, aerated and filtered artificial seawater (ASW: 460 mM NaCl, 10 mM KCl, 10 mM CaCl2, 22 mM MgCl2, 6 mM MgSO4, 10 mM HEPES, pH 7.8) at 14-15 °C until used. Animals were anesthetized by injection of isotonic MgCl2 (∼30-50% of body weight) into the body cavity. Aplysia were dissected and abdominal ganglia placed in ASW. The sheath was digested enzymatically by incubating the ganglia in ASW-antibiotic solution (ASW with 100 units/mL penicillin G, 100 mg/mL streptomycin, and 100 mg/mL gentamicin) containing 1%f protease (type IX, Bacterial; Sigma-Aldrich) at 36 °C for 1 h. Next, the ganglia were washed in fresh ASW and the abdominal ganglion was desheathed. Using 0.38-µm-diameter tungsten wire (WPI, Sarasota, FL), the ganglia were pinned to a silicone elastomer layer (Sylgard, Dow Corning, Midland, MI) in a dissection chamber containing 3-4 mL of ASW-antibiotic media. Abdominal ganglia were homogenized in 50 µL of acidified acetone (40 parts acetone, 6 parts H2O, 1 part HCl) and 50 µL of 20 mM ammonium acetate (pH 7) was mixed with the homogenate. The homogenate was centrifuged (Biofuge 17R; Baxter Scientific, Deerfield, IL) at 12 000 rpm for 5 min. Supernatant was transferred to a different vial and then evaporated using a Speed Vac (ThermoSavant, Holbrook, NY) for 1 h; ∼1 µL of NH4OH was added to bring the pH up to 7.8. The solution was split into two vials of ∼120 µL apiece. The first vial was immediately analyzed using LC-MS and 7.2 µL of 3.5 mM (NH4)2SO4 and 10 mM MgCl2, and 1 unit of mAAP was added to the other vial. This second sample was allowed to digest for 24 h before being analyzed with LC-MS. Both LC-MS runs were performed in the same manner as the standards.
Figure 1. Time-course series showing mass spectra from a peptide mixture indicating the times mAAP was added to the mixture. The mixture included NWFa (465.1 Da), R-BCP(1-8) (1009.6 Da), and angiotensin I (1296.8 Da), with doubly charged forms observed for several peptides. In (A), the phenylalanine group in the NWFa was in the L-form (NWFa), and in (B), it was in the D-form (NdWFa). NdWFa is resistant to mAAP degradation for several days.
RESULTS AND DISCUSSION Does mAAP digest peptides containing a D-amino acid near the N-terminal of the peptide? In order to test whether combining mAAP with LC-MS facilitates the search for DAACPs, the rate of enzymatic degradation of various standard peptides was tested first, some containing D-amino acids and some containing none. Specifically, two D/L-peptide pairs were used that could reasonably be expected to be observed in the Aplysia brain, NdWFa and YdAEFLa. Two samples were prepared for analysis, the first containing three peptide standards consisting of all L-amino acids: NWFa (Asn-Trp-Phe-NH2), R-BCP(1-8) (Ala-Pro-Arg-LeuArg-Phe-Tyr-Ser) and angiotensin I (Asp-Arg-Val-Tyr-Ile-His-ProPhe-His-Leu). The second sample was identical to the first, except that it contained NdWFa (Asn-D-Trp-Phe-NH2) in place of NWFa.
All samples were prepared in 20 mM ammonium acetate buffer at pH 7, the optimum pH for enzymatic activity. Next, mAAP was added to each vial, and the vials were incubated at 37 °C. As expected, NWFa was quickly consumed by mAAP (Figure 1A). Both R-BCP(1-8) and angiotensin I, although containing amino acids exclusively in the L-configuration, were detectable for ∼60 min. This stability can be explained by the presence of proline residues in both of these peptides as mAAP digests amino acids in the following approximate order: Ala > Phe > Tyr > Leu > Arg > Thr > Trp > Lys > Ser > Asp > His > Val. Proline and R- or γ-glutamic acid are digested even more slowly.49 Regardless, (49) Turner, A. J. In Handbook of Proteolytic Enzymes; Barrett, A. J., Rawlings, N. D., Eds.; Academic Press: San Diego, 1998; pp 996-1000.
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Figure 2. Peak intensity from the ESI-MS data. (A) The peak heights of angiotensin I, YdAEFLa, and NdWFA relative to the amount of time digested with mAAP. (B) The ratio of intensity of the YdAEFLa to NdWFa and of angiotensin 1/NdWFa peaks as a function of time of incubation with mAAP, showing that the LAACPs degrade faster than the fastest degrading DAACP.
Table 1. Time Constants and Goodness of Fit for the Degradation of YdAEFLa and Angiotensin I as Measured by LC-ESI-MS and Direct Infusion ESI-MS LC
time const (min-1) R2
YdAEFLa 2
3
359
427
454
0.93 -
ESI-MS
time const (min-1) R2
Angiotensin I
1
0.97
overall 430
0.96
0.94
1
2 513
0.88
2
3
118
146
115
0.95
YdAEFLa
442
1
0.98
0.99
overall 123 0.94
Angiotensin I overall 474
0.88
0.87
both peptides were eventually consumed. NdWFa was resistant to mAAP(Figure 1B); in fact, in some experiments, NdWFa was still present 10 days later. The performance of the enzyme has been optimized in terms of temperature and pH. Not surprisingly, the higher the temperature, the faster mAAP digests peptide standards, with appreciable activity seen at temperatures as low as 10 °C. The selectivity toward LAACPs over DAACPs did not vary much with temperature, but the optimum enzyme digestion appeared to be at room temperature. The digestion buffer solution and pH were also varied, demonstrating that a pH of ∼7.5 is optimum, with the makeup of the digestion buffer having little impact. Once it was determined that mAAP does indeed preferentially degrade L-amino acids, the next step was to ascertain whether mAAP degrades DAACPs if given enough time (i.e., is there an 2878 Analytical Chemistry, Vol. 80, No. 8, April 15, 2008
1 98 0.71
2 127 0.69
overall 110 0.68
incubation time that is too long?) This could become a concern when working with cellular extracts, where additional digestion might cause the peptide to be present below an instrument’s detection limit. We focused on two known DAACPs and used the peptides YAEFLa/YdAEFLa and NWFa/NdWFa, as well as the slowest degrading LAACP tested, angiotensin I. Figure 2 shows ESI-MS data demonstrating that angiotensin I degraded more quickly than YdAEFLa, measured as a ratio of the amount of NdWFa, a species we showed does not degrade. In order to improve this comparison when using LC-MS, we compared peptide intensities from different spectra obtained within a single LC run. Table 1 summarizes this using the LC-MS, with the direct ESI data showing similar degradation results. These data demonstrate that a mixture of L- and D-amino acid-containing peptides can be distinguished by running differential LC-MS
Figure 3. Acidified extract of the Aplysia abdominal ganglion. Half of the extract was used in (A) without mAAP and in (B) with 1.0 units of mAAP incubated for 24 h. In both cases, the LC base peak chromatogram intensity from 465 and 466 m/z is shown. (C) Extracted mass spectra showing the NWFa/NdWFa peaks are barely detectable above the noise, but (D) after digestion, the NdWFa peak is clearly visible.
experiments before and after incubation of the sample solution with enzyme solution. Next, to assess whether mAAP degradation could be used to identify DAACPs in a complex mixture, we used the Aplysia abdominal ganglia as our test case. As indicated previously, NdWFa is known to be present in specific neurons of the abdominal ganglion.20,30,40 Using a homogenate of the abdominal ganglion, as shown in Figure 3, we compare the resulting spectra of mAAP degradation to a control sample without mAAP. This comparison shows that the NdWFa in the sample does not degrade. Without knowing the peak’s identity, its resistance to degradation indicates a putative DAACP, even though the sample contained hundreds of other peptides at higher concentrations. Tandem MS experiments demonstrate fragmentation consistent with the sequence of NdWFa, thereby confirming the identity of the peak. Because only a few cells out of thousands produce NdWFa, these data help to validate the approach. While both CE and matrix-assisted laser desorption/ionization MS of carefully identified neurons have been used to probe for NdWFa,40 to our knowledge, this is the first application of LC-MS to a CNS extract for the identification of NdWFa. This approach succeeds because mAAP reduces the complexity of a CNS extract, particularly because we are able to focus on the peaks remaining after mAAP digestion from among all those peaks observed before degradation. While these results demonstrate that DAACPs are detected, there is another related question; what other peptides are resistant to degradation? As shown in Figure 4, in an extract from a different brain region, the cerebral ganglion, we see that only a few peaks resist degradation with an elution time between 10-20 min in the predigestion LC-MS. We also see that a number of new peaks have appeared with mAAP. As they do not match previously seen peaks without mAAP, these may not be DAACPs and more likely are partial degradation products of previously seen peptides. Among other notable features seen in Figure 4A is the cleanliness of the LC base peak spectra and the mass spectra of samples after treatment with mAAP. After degradation, the number of peptides remaining for further analysis is greatly reduced. We observe that one peptide, Aplysia insulin D, which has not been reported to contain a D-amino acid, degrades slowly under the effects of mAAP. This is a peptide we previously identified from the Aplysia insulin prohormone50 that contains a γ-carboxyglutamic acid residue.51 We are not sure whether this residue is responsible for its resistance to degradation. In order to examine
Figure 4. LC-MS chromatogram of cerebral ganglion homogenates. (A) Chromatogram prior to treatment with mAAP, with the inset showing Aplysia insulin D at 882.5 m/z as a minor peak eluting from 11.7 to 14.0 min (B) Chromatogram after treatment with mAAP, with the inset showing a mass spectrum with Aplysia insulin D as the major peak, in this case averaged from 13.0 to 16.0 min. In both cases, tandem MS was used to confirm the identity of the Aplysia insulin D-peptide.
this issue, we synthesized the γ-carboxyglutamate D-peptide and found it does not degrade quickly under the effects of mAAP, even without a D-amino acid; data to support this observation are presented in Figure 4B. In addition, we sometimes detect Nterminal-modified peptides such as pGlu-containing peptides as being resistant to digestion. FUTURE DIRECTIONS We have shown that the enzyme mAAP efficiently degrades neuropeptides without PTMs near the N-terminal. D-amino acids, especially those in the second position from the N-terminus, cause peptides to be particularly resistant to degradation by mAAP. Thus, the lack of degradation during incubation with mAAP strongly increases the likelihood that the peptide contains a D-amino acid. (50) Garden, R. W.; Shippy, S. A.; Li, L.; Moroz, T. P.; Sweedler, J. V. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3972-3977. (51) Jakubowski, J. A.; Hatcher, N. G.; Xie, F.; Sweedler, J. V. Neurochem. Int. 2006, 49, 223-229.
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As noted above, peptides containing PTMs such as pGlu located at the N-terminus of the peptide also make the peptide resistant to digestion. Recently, a mammalian aminopeptidase has been characterized that can digest L, L/D-peptides.52 In order to screen out DAACPs from degradation-resistant peptides due to the presence of other PTMs, it may be possible to digest a homogenate with mAAP and this recently discovered enzyme. Peptides that are resistant to mAAP, but are digested by the aminopeptidase that degrades the L/D-peptides, become the most likely DAACP candidates. These approaches will be used to screen several model systems for new DAACPs. (52) Krstanovic, M.; Brgles, M.; Halassy, B.; Frkanec, R.; Vrdoljak, A.; Branovic, K.; Tomasic, J.; Benedetti, F. Prep. Biochem. Biotechnol. 2006, 36, 175195.
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ACKNOWLEDGMENT This material is based upon work supported by the following agencies: the National Science Foundation under Award CHE 0400768, the National Institutes of Health under Award NS 031609, and the National Institute on Drug Abuse under Award DA 018310 to the UIUC Neuroproteomics Center. In addition, a Howard Hughes Undergraduate Research fellowship to J.W. and an NSF predoctoral fellowship to M.A.E. are gratefully acknowledged.
Received for review December 11, 2007. Accepted February 1, 2008. AC7025173
