Anal. Chem. 2002, 74, 3190-3198
Quantitation of Neuropeptides in Cpefat/Cpefat Mice Using Differential Isotopic Tags and Mass Spectrometry Fa-yun Che and Lloyd D. Fricker*
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
Neuroendocrine peptides play important roles as intercellular messengers. We previously developed a technique to isolate and identify a large number of neuroendocrine peptides from Cpefat/Cpefat mice (Che, F.; et al. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 9971-6); these mice lack carboxypeptidase E activity and this defect causes an accumulation of neuropeptide intermediates that contain C-terminal Lys or Arg residues (Naggert, J. K.; et al. Nat. Genet. 1995, 10, 135-42). In the present study, we have developed a differential isotopic-labeling technique that can be used to quantitate changes in neuropeptide levels in Cpefat/Cpefat mouse tissues. Samples are treated with either the H6 or the D6 form of acetic anhydride, peptides that contain C-terminal basic amino acids are isolated by affinity chromatography on anhydrotrypsin agarose, and the isolated peptides are analyzed by mass spectrometry. Measurement of the regulation of pituitary peptides in response to dehydration showed a decrease in vasopressin. The general method described in this report should be widely applicable to a large number of neuroendocrine peptides, known and novel, in a variety of regulatory paradigms. A large number of hormones and neurotransmitters are peptides. Numerous studies have investigated the regulation of one or more neuroendocrine peptides in a variety of animal models. However, the typical method of quantitating peptides involves radioimmunoassays, which require a specific antiserum for each peptide to be investigated. Furthermore, unless the antiserum is specific for a single form of the peptide, this technique does not provide information on posttranslational modifications of the peptide. Although radioimmunoassays followed by mass spectrometry can provide some information on the identity of the peptide form,3,4 this method does not rule out the possibility that * Corresponding author: (phone) 718-430-4225; (fax) 718-430-8954; (E-mail)
[email protected]. (1) Che, F.; Yan, L.; Li, H.; Mzhavia, N.; Devi, L.; Fricker, L. D. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 9971-6. (2) Naggert, J. K.; Fricker, L. D.; Varlamov, O.; Nishina, P. M.; Rouille, Y.; Steiner, D. F.; Carroll, R. J.; Paigen, B. J.; Leiter, E. H. Nat. Genet. 1995, 10, 135-42. (3) Mzhavia, N.; Berman, Y.; Che, F.; Fricker, L. D.; Devi, L. A. J. Biol. Chem. 2001, 276, 6207-13. (4) Mzhavia, N.; Qian, Y.; Feng, Y.; Che, F.; Devi, L. A.; Fricker, L. D. Biochem. J. 2002, 361, 67-76.
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another immunoreactive form is present in the same sample and responsible for any observed changes. Proteomics strategies that involve mass spectrometry provide a rapid and sensitive method for identification of proteins and peptides.5 The general methods do not require prior knowledge about a particular protein or peptide, as does a radioimmunoassay, and are also able to provide information on posttranslational processing. Although typical proteomics techniques are not quantitative, there are several techniques that allow for semiquantitative and quantitative analyses. Internal standards can be used to quantitate levels of peptides with mass spectrometry, although this technique is highly accurate only if the internal standards are stable isotope-incorporated peptides that are chemically identical to the naturally occurring peptides.6-8 Differential isotopic labels enable the accurate comparison of a large number of peptide/protein levels in two samples. These isotopic labels are incorporated into the proteins/peptides either in cell culture9,10 or after extraction of the proteins/peptides from cells or animal tissues.11-15 Subsequent analysis of the relative intensity of the mass spectrometric signals of the heavy and light forms of the labeled protein/peptide reveals the relative levels of the substance in the cell or tissue. In most neuroendocrine tissues, the peptide neurotransmitters or hormones represent a small fraction of the total proteins and peptides. Thus, without some method of enrichment of the peptide neurotransmitter or hormone, they would not be easily detected (5) Gygi, S. P.; Aebersold, R. Curr. Opin. Chem. Biol. 2000, 4, 489-94. (6) Jimenez, C. R.; Li, K. W.; Dreisewerd, K.; Mansvelder, H. D.; Brussaard, A. B.; Reinhold, B. B.; Van der Schors, R. C.; Karas, M.; Hillenkamp, F.; Burbach, J. P. H.; Costello, C. E.; Geraerts, W. P. M. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 9481-6. (7) Desiderio, D. M. J. Chromatogr., B: Biomed. Sci. Appl. 1999, 731, 3-22. (8) Gobom, J.; Kraeuter, K.; Persson, R.; Steen, H.; Roepstorff, P.; Ekman, R. Anal. Chem. 2000, 72, 3320-6. (9) Oda, Y.; Huang, K.; Cross, F. R.; Cowburn, D.; Chait, B. T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6591-6. (10) Conrads, T. P.; Alving, K.; Veenstra, T. D.; Belov, M. E.; Anderson, G. A.; Anderson, D. J.; Lipton, M. S.; Pasa-Tolic, L.; Udseth, H. R.; Chrisler, W. B.; Thrall, B. D.; Smith, R. D. Anal. Chem. 2001, 73, 2132-9. (11) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nature Biotechnol. 1999, 17, 994-9. (12) Wang, S.; Regnier, F. E. J. Chromatogr., A. 2001, 924, 345-57. (13) Goshe, M. B.; Conrads, T. P.; Panisko, E. A.; Angell, N. H.; Veenstra, T. D.; Smith, R. D. Anal. Chem. 2001, 73, 2578-86. (14) Goodlett, D. R.; Keller, A.; Watts, J. D.; Newitt, R.; Yi, E. C.; Purvine, S.; Eng, J. K.; von Haller, P.; Aebersold, R.; Kolker, E. Rapid Commun. Mass Spectrom. 2001, 15, 1214-21. (15) Griffin, T. J.; Gygi, S. P.; Rist, B.; Aebersold, R.; Loboda, A.; Kilkine, A.; Ens, W.; Standing, K. G. Anal. Chem. 2001, 73, 978-86. 10.1021/ac015681a CCC: $22.00
© 2002 American Chemical Society Published on Web 05/11/2002
over the background of other molecules. We have recently developed a method that enables the purification of peptideprocessing intermediates from Cpefat/Cpefat mice;1 these mice lack carboxypeptidase E activity due to a point mutation in the coding region of this gene.2 Carboxypeptidase E functions in the biosynthesis of numerous peptide hormones and neurotransmitters, cleaving the intermediates generated by the action of an endopeptidase on the neuropeptide precursors.16-18 In wild-type mice, these intermediates with C-terminal basic residues are not detectable due to the efficient cleavage by carboxypeptidase E.1 In the absence of active carboxypeptidase E, the neuroendocrine peptide precursors are initially cleaved by endopeptidases at basic amino acid-containing sites, but further cleavage of the C-terminal Lys or Arg residues, or both, is greatly reduced.19 Some C-terminal processing does occur in the Cpefat/Cpefat mice, presumably due to the action of carboxypeptidase D.19,20 However, levels of carboxypeptidase D are not sufficient to cleave the majority of the peptide intermediates in brain and pituitary, and so these peptides are greatly elevated in the Cpefat/Cpefat mice.19,21-23 Peptides with C-terminal basic residues can be readily isolated using anhydrotrypsin agarose, providing a single-step purification of the neuropeptide-processing intermediates.1,24 Using this purification method and subsequent analysis by liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (MS/MS), we previously identified over 100 peptides in neuroendocrine tissues and detected many additional peptides that have not yet been identified.1 In the present report, we describe a procedure that provides the quantitation of levels of neuroendocrine peptides using differential isotopic labeling. This method is sensitive, rapid, and accurate. In addition, this technique provides information on the posttranslational modifications of the peptide; many modifications influence the biological activity of peptides. EXPERIMENTAL SECTION Materials. Acetic anhydride (H6-Ac2O), acetic anhydride-d6 (D6-Ac2O), and R-cyano-4-hydroxycinnamic acid were obtained from Aldrich Chemical Co. (Milwaukee, WI). Des-Arg1-bradykinin, angiotensin I, neurotensin, adrenocorticotropic hormone 1-17 (ACTH 1-17), and ACTH 18-39 (CLIP) were purchased from Sigma (St. Louis, MO). Little SAAS (rat proSAAS 42-59), PEN (rat proSAAS 221-242), and big SAAS (rat proSAAS 34-59) were the generous gifts of Dr. James Douglass (Amgen, Thousand Oaks, CA). (16) Fricker, L. D. Annu. Rev. Physiol. 1988, 50, 309-21. (17) Fricker, L. D. In Handbook of Proteolytic Enzymes; Barrett, A. J., Rawlings, N. D., Woessner, J. F., Eds.; Academic Press: San Diego, 1998; pp 1341-4. (18) Fricker, L. D. In The Enzymes. Volume 23: Co- and posttranslational proteolysis of proteins; Dalbey, R. E., Sigman, D. S., Eds.; Academic Press: San Diego, 2002; pp 421-52. (19) Fricker, L. D.; Berman, Y. L.; Leiter, E. H.; Devi, L. A. J. Biol. Chem. 1996, 271, 30619-24. (20) Fricker, L. D. In Handbook of Proteolytic Enzymes; Barrett, A. J., Rawlings, N. D., Woessner, J. F., Eds.; Academic Press: San Diego, 1998; pp 134951. (21) Rovere, C.; Viale, A.; Nahon, J.; Kitabgi, P. Endocrinology 1996, 137, 29548. (22) Udupi, V.; Gomez, P.; Song, L.; Varlamov, O.; Reed, J. T.; Leiter, E. H.; Fricker, L. D.; Greeley, G. H. J. Endocrinology 1997, 138, 1959-63. (23) Cain, B. M.; Wang, W.; Beinfeld, M. C. Endocrinology 1997, 138, 4034-7. (24) Fricker, L. D.; McKinzie, A. A.; Sun, J.; Curran, E.; Qian, Y.; Yan, L.; Patterson, S. D.; Courchesne, P. L.; Richards, B.; Levin, N.; Mzhavia, N.; Devi, L. A.; Douglass, J. J. Neurosci. 2000, 20, 639-48.
Labeling of Standard Peptides with H6-Ac2O and D6-Ac2O. Approximately 2.5 µg of des-Arg1-bradykinin, angiotensin I, CLIP, little SAAS, big SAAS, and PEN were mixed in 210 µL of 0.2 M NH4HCO3, pH 7.5. A 100-µL sample of this standard peptide mixture was added to each of two microfuge tubes, and 1 µL of H6-Ac2O or D6-Ac2O was added. After 10 min, 2 µL of 20% ammonium hydroxide (v/v) was added to maintain the pH at 7.5. This procedure was repeated three times, for a total of 4 µL of the Ac2O solution and 8 µL of ammonium hydroxide (a 9000-fold molar excess of Ac2O over the total concentration of peptide). The reactions were lyophilized and resuspended in 200 µL of 0.1% trifluoroacetic acid. The two labeled peptide mixtures were combined in 4:1, 2:1, 1:1, 1:2, and 4:1 molar ratios. Each of these mixtures was concentrated to 20-30 µL and desalted with a ZipTip C18 (Millipore Co., Bedford, MA). The peptides were eluted with 20 µL of 50% acetonitrile/0.1% trifluoroacetic acid, and 0.5-µL aliquots were used for MALDI-TOF-MS analysis. Animal Treatment. Three pairs of breeder mice heterozygous for the Cpefat mutation in the C57BLKS/J strain were obtained from The Jackson Laboratory (Bar Harbor, ME). These breeders were used to establish a colony of homozygous Cpefat/Cpefat mice. For the treatments, pairs of age- and sex-matched mice were randomly split into two groups. One group received food and water, the other group only food. Two separate experiments were performed. The first used 16 mice aged 9-10 weeks of which half were Cpefat/Cpefat and the others were wild-type littermates. For this experiment, the period of water deprivation lasted for 48 h, and individual pituitary glands were extracted as per the scheme in Figure 1. In the other experiment, 20 mice aged 9-12 weeks were used of which 12 were Cpefat/Cpefat and the remainder were wild-type littermates. For this experiment, the period of water deprivation lasted for 24 h, and pairs of age- and sex-matched pituitaries were pooled for extraction. For both experiments, animals were euthanized with carbon dioxide and the pituitaries were removed. Tissue was stored at -80 °C until analysis. Extraction, Labeling, and Purification of Peptides from Mice. Typically 100 µL of boiling 10 mM HCl was added to a microfuge tube that contained either a single pituitary gland or pools of two glands from similarly treated animals (age-, sex-, and genotype-matched). After incubation in a boiling water bath for 10 min, the tissue was sonicated (Heat systems-Ultrasonics, Inc., Farmingdale, NY) for 8 s with 50% duty cycle and neutralized with 1 µL of 1.0 M NaOH. The sonication tip was washed twice with 100 µL of 0.2 M NH4HCO3, pH 7.5, and these washings were combined with the HCl extract. A 3-µL aliquot of H6-Ac2O or D6Ac2O was added to the homogenate. After 10 min, 6 µL of 20% ammonium hydroxide (v/v) was added to maintain the pH at 7.5. This acetylation procedure (3 µL of Ac2O followed by 6 µL of ammonium hydroxide) was repeated three times, and the reaction was quenched by the addition of 85 µL of 2.5 M glycine. After 40 min, the H6-Ac2O-labeled samples and the D6-Ac2O-labeled samples from age- and sex-matched animals were combined and centrifuged at 50000g for 30 min at 4 °C. The supernatant was removed and filtered through a Centriplus-10 membrane (Amicon). The filtrate was combined with 1.0 M sodium acetate, pH 5.0, 5% CHAPS, and 1.0 M CaCl2 to give a final concentration of 50 mM, 0.25%, and 20 mM, respectively. The resulting mixture was applied to a column containing 0.2 mL of anhydrotrypsin agarose (60-90 Analytical Chemistry, Vol. 74, No. 13, July 1, 2002
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Figure 1. General scheme for the quantitative analysis of neuroendocrine peptides in Cpefat/Cpefat mice. In this approach, individual tissues (or tissue pools from several mice) are extracted and labeled with either H6-Ac2O or D6-Ac2O. The labeled extracts are combined and the peptides with C-terminal Lys or Arg groups isolated by chromatography on anhydrotrypsin agarose. Following LC/MS analysis, the spectra obtained for the Cpefat/Cpefat mouse tissues are compared to the spectra obtained from wild-type mice and only those ions substantially enriched in the Cpefat/Cpefat mouse tissues are further pursued. For those ions that show enough of a mass separation between the H3-Ac and D3-Aclabeled forms, the relative intensities of the signals are quantitated to provide a ratio of the peptide in the two groups. To control for animalto-animal variations and for potential errors with the labeling, this scheme should be performed with multiple groups of animals and with some of the groups “reverse” labeled so that the treated group is H-acetylated and the untreated group is D-acetylated.
nmo of protein/mL of resin; Panvera, Madison, WI). The column was washed with 0.25% CHAPS in 50 mM NaAc buffer, pH 5.0, that contained 20 mM CaCl2 and 0.5 M NaCl. To remove the detergent and NaCl, the column was subsequently rinsed with 10 mM NaAc, pH 5.0, that contained 10 mM CaCl2. Peptides were eluted first with 3 mL of water, followed by 6 mL of 6 mM HCl. The column eluates were concentrated to ∼40 µL using a vacuum centrifuge. Matrix-Assisted Laser Desorption/Ionization-Time-ofFlight-Mass Spectrometry (MALDI-TOF-MS). Aliquots (0.5 µL) of sample solution were mixed with 1 µL of R-cyano-4-hydroxycinnamic acid saturated in 45% acetonitrile and 0.1% trifluoroacetic acid, and the mixture was loaded onto the MALDI-TOF-MS sample stage. MALDI-TOF-MS analysis was done in the delayed-extraction linear positive mode on a Voyager-DE STR mass spectrometer (PerSeptive Biosystems, Framingham, MA). For each sample, the spectra produced from 120 laser shots were accumulated. External multipoint mass calibration was performed with des-Arg1-bradykinin ([M + H]+ 904.4681), angiotensin I ([M + H]+ 1296.6853), neurotensin ([M + H]+ 1672.9175), ACTH 1-17 ([M + H]+ 2094.4600), and ACTH 18-39 ([M + H]+ 2466.7200). 3192 Analytical Chemistry, Vol. 74, No. 13, July 1, 2002
Electrospray Ionization-Time-of-Flight-Mass Spectrometry (ESI-TOF-MS) and Electrospray Ionization-Ion Trap Mass Spectrometry (ESI-ion trap MS). Aliquots (∼50%) of each sample were analyzed using high-performance liquid chromatography (HPLC) coupled on-line with either an ESI-TOF Mariner mass spectrometer (PerSeptive Biosystems) or an ESI-ion trap mass spectrometer (Finnigan LCQ, San Jose, CA). For both analyses, a C18 capillary column (Hypersil C18 BDS, 3 µm, i.d. 300 µm, LC Packings, San Francisco, CA) was used with a gradient from 2% to 15% solvent B over 5 min and from 15% to 75% B over 60 min at a flow rate of 5 µL/min (solvent A: 5% acetonitrile, 0.1% formic acid in water. solvent B: 95% acetonitrile, 0.1% formic acid in water). The ESI-ion trap MS was performed so that the most intense ion in each MS scan was selected to generate collisioninduced dissociated fragmentation (MS/MS spectra). MS Data Analysis. Relative levels of peptides between two samples were quantified by the ratio of peak intensity of the H3Ac and D3-Ac-labeled peptide pairs. For all peptides, the first three peaks of each ion were averaged; these peaks correspond to the monoisotopic peak and the peaks with one and two 13C atoms. For those peptides with a significant contribution from the three
Figure 2. MALDI-TOF-MS quantitative analysis of a standard peptide mixture labeled with H6-Ac2O or D6-Ac2O. (A) A peak pair of diacetylated angiotensin I combined with a H/D molar ratio of 2:1. The peak at 1380.74 Da corresponds to the di-H3-Ac form of the singly protonated monoisotopic peptide (theoretical mass, 1380.71 Da), the peak at 1386.76 Da corresponds to the di-D3-Ac form of the singly protonated monoisotopic peptide (theoretical mass, 1386.75 Da). The additional peaks that are 1 and 2 mass units larger than the monoisotopic peak correspond to the peptides with one and two atoms of 13C, respectively. To calculate peak intensity, the height of the monoisotopic peak was added to the heights of the peaks with one and two atoms of 13C. The H/D ratio of the combined peak intensities was 2.03. (B) A peak pair of diacetylated angiotensin I combined with a H/D molar ratio of 1:2. The ratio of the combined peak intensities was 0.53. (C) Comparison of the observed ratio of the peak intensities for six standard peptides added in H/D molar ratios of 1:4, 1:2, 1:1, 2:1, and 4:1. Circles, monoacetylated des-Arg1-bradykinin; squares, diacetylated angiotensin I; triangles, monoacetylated little-SAAS; inverted triangles, monoacetylated PEN; diamonds, diacetylated and triacetylated CLIP; hexagons, diacetylated big-SAAS.
or four 13C-containing forms, these peaks were included in the average. Peptides were identified by first comparing the parent masses (after subtraction of the added acetyl groups) obtained from MALDI-TOF-MS or ESI-TOF-MS data to those of peptides identified in our previous work.1 The identification was confirmed by comparing MS/MS data from the ESI-ion trap MS with the predicted b-and y-series of fragments calculated by using the SHERPA computer program. For those peptides that could not be identified using this strategy, parent masses and fragments observed in MS/MS spectra were used to search the NCBInr or pdbEST mouse database using the MS-TAG or MS-SEQ programs. RESULTS Principle of the Neuropeptide Quantitation Method. In the previous method for proteomics of peptide hormones and neurotransmitters, the peptides extracted from Cpefat/Cpefat mouse tissues were purified on anhydrotrypsin agarose and then analyzed by LC/MS.1 The parallel processing and analysis of tissues from Cpefat/Cpefat and wild-type littermates was performed, and the two data sets were compared; only those ions that were substantially elevated in the Cpefat/Cpefat mouse samples represented neuroendocrine peptides.1 To quantitate changes in the relative levels of neuropeptides in two groups of Cpefat/Cpefat mice, the extracted peptides are labeled with either the heavy (D6) or light (H6) form of acetic anhydride under conditions that preferentially label amines (Figure 1). Pairs of samples from the two groups of Cpefat/ Cpefat mice and pairs from similarly treated wild-type mice are combined and purified on anhydrotrypsin agarose (Figure 1). The affinity column eluates are analyzed first by LC/MS. A comparison of the signals from the Cpefat/Cpefat and the wild-type mice reveals those peptides that are substantially increased in the mutant mice. Only the peptides that are present in the Cpefat/Cpefat mouse extracts and that are not detectable in the wild-type mouse extracts are further investigated. The intensities of the signals from the
light and heavy forms of the acetylated peptides are compared; this analysis provides an indication of the relative abundance of each peptide. Subsequent analysis is performed using MS/MS to fragment the peptide and to provide conclusive identification. Quantitation of Standard Peptides and Cpefat/Cpefat Mouse Pituitary Peptides. The linearity of the measurement of the isotopic ratios by mass spectrometry was assessed by acetylating a mixture of six synthetic peptides using either H6- or D6-Ac2O and combining the two mixtures in 4:1, 2:1, 1:1, 1:2, or 1:4 molar ratios. Mass spectrometry was performed with both MALDI-TOF and Mariner ESI-TOF mass spectrometers; similar results were obtained. All peptides were acetylated by the treatment and in most cases existed in two acetylation states (i.e., with one and two or with two and three acetyl groups). The number of acetyl groups incorporated was apparent from the mass difference of the two peaks (based on the difference of 3 mass units per acetyl group). The peak intensities of the heavy and light forms of the peptides reflected the added molar ratio of the two forms (Figure 2, panels A and B, and data not shown). Quantitation of the six standard peptides showed the observed H/D ratio of the peak intensities to be within 2% of 1.00 when equal amounts of the two peptide mixtures were added (Figure 2C). The 1:4, 1:2, 2:1, and 4:1 molar ratios produced signals that were within 10% of the expected value (Figure 2C). To test whether this technique works with endogenous peptides in the Cpefat/Cpefat mice, and whether the acetylated peptides can be purified on the anhydrotrypsin affinity column, five pituitaries from these mice were homogenized and their peptides extracted and pooled so that the reproducibility of the technique could be examined without the complication of interanimal variations. Twenty percent of this extract (representing one pituitary) was placed into each of two tubes; one tube was labeled with H6-Ac2O, the other tube with D6-Ac2O, and the two Analytical Chemistry, Vol. 74, No. 13, July 1, 2002
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Figure 3. MALDI-TOF-MS analysis of endogenous peptides extracted from pituitaries of Cpefat/Cpefat mice, labeled with H6- or D6-Ac2O, and combined with a H/D molar ratio of 1:1 (panel A) or 1:2 (panel B). The peaks at 1510.60 (A) and 1510.82 Da (B) correspond to the di-H3-Ac form of singly protonated monoisotopic vasopressin-GKR (theoretical mass, 1510.70 Da). The peaks at 1516.66 (A) and 1516.84 Da (B) correspond to the di-D3-Ac form of the singly protonated monoisotopic peptide (theoretical mass, 1516.70 Da). The peak intensities were determined by combining the height of the monoisotopic peak and the peaks with one and two atoms of 13C. The observed H/D ratios of the total peak intensities were 0.99 (A) and 0.52 (B). Table 1. Quantitation of Peptides That Were Extracted from CpeFat/CpeFat Pituitaries, Purified on an Anhydrotrypsin Agarose Column, and Analyzed by MALDI-TOF (MH+) masses theoretical
observed
theoretical
observed
peptidea
2 H-Ac
2 H-Ac
2 D-Ac
2 D-Ac
1:1b
ratio D/H 2:1c
oxytocin-GKR vasopressin-GKR R-MSH-GKKR CLIP-KR
1433.68 1510.70 2220.41 2876.23
1433.63 1510.60 2220.12 2875.98
1439.68 1516.70 2226.41 2882.23
1439.69 1516.66 2226.12 2881.73
1.02 1.00 1.01 1.02
1.95 2.06 1.94 2.02
a Abbreviations: R-MSH, R melanocyte-stimulating hormone; CLIP, corticotropin-like immunoreactive peptide; G, glycine; K, lysine; R, arginine. The identity of these four peptides was confirmed by MS/MS sequence analysis, as shown in Table 2 (note that the masses indicated in Table 1 are MH+ for the acetylated peptide, while Table 2 shows the unprotonated mass after subtraction of the mass contributed by the acetyl groups). b Equal amounts of extract were labeled with either H -Ac O or D -Ac O. c The reaction with D -Ac O contained twice as much pituitary extract as 6 2 6 2 6 2 used for the reaction with H6-Ac2O.
extracts were combined and purified on the anhydrotrypsin column. Two other tubes contained either 20% or 40% of the extract; these tubes were also labeled with H6- and D6-Ac2O, respectively, and the extracts were combined and purified on the anhydrotrypsin column. MALDI-TOF analysis revealed a number of peptides with masses that correspond to the previously detected pituitary peptides containing C-terminal basic residues and with the extra mass of one, two, or three acetyl groups; these peptides were detected only in the Cpefat/Cpefat mouse extracts and not in the wild-type mouse extracts. When equal amounts of extract were used for the D6-Ac2O and H6-Ac2O labeling, the resulting intensities of the heavy and light peaks were comparable (Figure 3A). The samples with twice the amount of extract in the D-labeling reaction had peak intensities of the D-labeled peptides that were twice that of the H-labeled peptides (Figure 3B). Quantitation of the peak heights of the doubly acetylated form of four major peptides, which ranged in molecular mass from 1433 to 2882 Da, showed an observed D/H ratio within 2% of the expected value for the 1:1 molar ratio and within 3% of the expected value for the 2:1 molar ratio (Table 1). Application of the Method to the Quantitative Analysis of Neuroendocrine Peptide Levels in Pituitary Following Dehydration. The quantitative proteomics method was used to 3194 Analytical Chemistry, Vol. 74, No. 13, July 1, 2002
examine neuropeptide levels in mouse pituitary following water deprivation. Dehydration has been shown to alter vasopressin mRNA levels in mouse and rat hypothalami, and vasopressin peptide levels in mouse and rat pituitary.6,25-30 Pituitaries from water-deprived and from nondeprived Cpefat/Cpefat and wild-type mice were extracted and treated with acetic anhydride as per the scheme in Figure 1; for one pair of mice, the labeling was reversed so that the untreated mice received the heavy form of acetic anhydride and the water-deprived mice the light form. After labeling, the samples from pairs of age-, sex-, and weight-matched mice were pooled. To compare levels of vasopressin, aliquots were analyzed by MALDI-TOF mass spectrometry prior to purification on the anhydrotrypsin column. The intensity of the signal for diacetylated vasopressin in wild-type mice deprived of water for 2 days is ∼80% of the signal of wild-type mice that had normal access (25) Tracer, H. L.; Loh, Y. P. Neuropeptides 1993, 25, 161-7. (26) Hollt, V.; Haarmann, I.; Seizinger, B. R.; Herz, A. Neuroendocrinology 1981, 33, 333-9. (27) Aguilera, G.; Lightman, S. L.; Kiss, A. Endocrinology 1993, 132, 241-8. (28) Zamir, N.; Zamir, D.; Eiden, L. E.; Palkovits, M.; Brownstein, M. J.; Eskay, R. L.; Weber, E.; Faden, A. I.; Feuerstein, G. Science 1985, 228, 606-8. (29) Negro-Vilar, A.; Samson, W. K. Brain Res. 1979, 169, 585-9. (30) Morris, M.; Means, S.; Oliverio, M. I.; Coffman, T. M. Am. J. Physiol. Regulat. Integr. Comp. Physiol. 2001, 280, R1177-1184.
to water (Figure 4A and B). Identical results were obtained regardless of whether the light or heavy form of acetic anhydride was used for the treated group (compare Figure 4A and B). Similar analysis of Cpefat/Cpefat mice showed a comparable 20% decrease in the pituitary vasopressin level after 2 days of water deprivation (Figure 4C). The level of vasopressin precursor (i.e., vasopressinGKR) was also decreased by ∼20% upon water deprivation for 2 days (Figure 4D). Purification of the GKR-extended vasopressin on the anhydrotrypsin column resulted in an identical H/D ratio as found for this peptide prior to the column (Figure 4E). Thus, the Cpefat/Cpefat mice respond similarly to the wild-type mice of this strain in their regulation of pituitary vasopressin levels by water deprivation. In addition, the change in vasopressin is paralleled by a comparable change in the vasopressin-processing intermediate (i.e., vasopressin-GKR), and the purification on the anhydrotrypsin column does not alter the ratio of the heavy and light forms of the labeled peptide. MALDI-TOF analysis typically reveals only a subset of the peptides present in a complex mixture. To examine additional neuroendocrine peptides, samples from a second experiment were analyzed by LC/MS. For this second experiment, a total of 12 Cpefat/Cpefat mice and 8 wild-type mice were divided into two groups and the water was removed from one of these groups 24 h before sacrifice. The pituitary glands were extracted and labeled with acetic anhydride, as shown in the general scheme (Figure 1) except that two glands from each treatment group were initially combined in order to have more sample for analysis. Altogether, this procedure resulted in three pools of Cpefat/Cpefat mouse tissue and two pools of wild-type mouse tissue; each pool contained the extract of two treated and two untreated animals. For one pool, the labeling was reversed from that of the other two pools to reduce the chances of error due to the labeling procedure. Upon LC/MS analysis, 44 different m/z ions were detected in the Cpefat/ Cpefat extracts that were not present in the wild-type extracts and that showed sufficient resolution of the heavy and light labels to permit quantitation. After consideration of multiple charge or acetylation states, these 44 different m/z ions were found to represent 23 distinct peptides (Table 2). In general, these multiple signals showed a similar magnitude of change upon water deprivation and so the relative ratios of the various ions were averaged together for each peptide. The ratio of the relative abundance of the various peptides in the dehydrated mice versus the untreated Cpefat/Cpefat mouse pituitaries shows an overall average of 1.06 with a standard deviation of 0.11. Except for the peptide with an unacetylated monoisotopic mass of 1425.75 Da, none of the observed peptides differed significantly from the average of all peptides. The 1425.75-Da peptide decreased by 14% after 1 day of water deprivation; this decrease was statistically significant (p < 0.001) using Student’s t-test. To confirm the identity of the 1425.75-Da peptide as vasopressin with a Gly-Lys-Arg C-terminal extension (expected monoisotopic mass 1425.65 Da) and to determine the identity of the other peptides, the samples were reanalyzed by LC/MS and MS/MS fragmentation data were obtained. A representative MS/MS spectrum is shown in Figure 5. A peptide was considered identified if the observed parent mass (after subtracting the mass of the added acetyl groups) was within 100 ppm of the predicted mass and if >80% of the MS/MS fragments matched with expected b-
or y-series fragment ions. Using these criteria, 14 of the 23 peptides were identified (Table 2), 12 of which were previously identified in Cpefat/Cpefat mouse pituitary.1 The two peptides not previously identified but found in the present study represent fragments of provasopressin (GFFRLTR) and of proopiomelanocortin (GSPEPSPREGKR). In addition to these 14 identified peptides, several of the other peptides had parent masses that were within 100 ppm of peptides that were previously identified in Cpefat/Cpefat mouse pituitary. However, either MS/MS data were not obtained for these peptides or the majority of the MS/MS ions could not be defined, and so these putative matches remain “unidentified” (Table 2). DISCUSSION The method for the quantitation of neuroendocrine peptides described in this report has several advantages over previous methods of peptide quantitation, which have generally involved either radioimmunoassays or radioreceptor assays. One major advantage of the present method is that the precise form of the neuropeptide is usually measured. If there are multiple endogenous forms of a peptide that differ by their posttranslational modification, then these forms will be detected as separate entities by the MS-based technique (although it is conceivable that a peptide exists with the same modification in two different positions, and if these peptides coelute on HPLC then they would be indistinguishable in the present method). In contrast, radioimmunoassays and radioreceptor assays detect all forms of the peptide that react with the antibody or receptor and do not provide any information on the precise molecular entity. Another drawback of the radioimmunoassay method is that it requires advance knowledge of the peptide and generation of the appropriate antiserum, which is an expensive and time-consuming step. The current method takes only a few days for the extraction, labeling, and quantitative analysis of specific peptides present in the samples. Furthermore, it can detect a large number of neuroendocrine peptides, both known and novel. In addition, the MS-based method is extremely sensitive; depending on the mass spectrometer used for the analysis, femtomoles or less of peptide can be detected. Most importantly, this method is accurate. The variation between the added and observed ratios of standard peptides was less than 3% when peptides were present in 1:1 molar ratios (Figure 2C and Table 1); this variation is much smaller than the typical animal to animal variations in peptide levels (Table 2). Along with the advantages of the method described in this report, there are also some limitations. For example, the present technique only provides a measure of the relative changes in the peptide levels and does not provide a true quantitation of the peptide amount. Another concern is that the peptides to be quantitated must be detectable by MS after the acetylation step. Low-abundance peptides can be difficult to detect above the background from other substances within the extract. An advantage of the Cpefat/Cpefat mice used in the present study is that the anhydrotrypsin affinity chromatography column provides a singlestep purification of a large number of neuroendocrine peptides, thus reducing the background signal from nonneuroendocrine peptides. The use of these mice is not an absolute requirement if the peptides are abundant enough to detect without purification, as are several pituitary peptides, or if an alternative method of purification is used. For example, isotopically coded affinity labels Analytical Chemistry, Vol. 74, No. 13, July 1, 2002
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Figure 4. MALDI-TOF-MS analysis of peptides extracted from pituitaries of wild-type mice (panels A and B) or Cpefat/Cpefat mice (panels C and D), comparing untreated mice with those subjected to 2 days of water deprivation. (A, C, and D) Peptides from mice given water were labeled with H6-Ac2O, and peptides from water-deprived mice were labeled with D6-Ac2O. (B) Reverse labeling: Peptides from mice given water were labeled with D6-Ac2O, and peptides from water-deprived mice were labeled with H6-Ac2O. In panels A-C, the peaks at ∼1168.5 Da correspond to the di-H3-Ac form of singly protonated monoisotopic vasopressin (theoretical mass, 1168.45 Da), and the peaks at ∼1174.5 Da correspond to the di-D3-Ac form of singly protonated monoisotopic vasopressin (theoretical mass, 1174.49 Da). In panel D, the peak at 1510.74 Da corresponds to the di-H3-Ac form of singly protonated monoisotopic vasopressin-GKR (theoretical mass, 1510.70 Da), and the peak at 1516.76 Da corresponds to the di-D3-Ac form of the singly protonated monoisotopic peptide (theoretical mass, 1516.70 Da). (E) Quantitation of the ratio of peptide levels in three pairs of pituitaries. One set of pituitaries was reverse labeled, and the H/D ratio from these data combined with the D/H ratio from the other sets so that the resulting ratio corresponded to (peptide in the dehydrated animals)/(peptide in animals that received water). Error bars show the standard error of the mean (n ) 3). Column 1: vasopressin in extracts of wild-type mice (analyzed prior to purification on the anhydrotrypsin affinity column, which does not bind vasopressin). Column 2: vasopressin in extracts of Cpefat/Cpefat mice (also analyzed prior to the anhydrotrypsin column). Column 3: vasopressin-GKR in extracts of Cpefat/Cpefat mice (analyzed prior to the anhydrotrypsin column). Column 4: vasopressin-GKR in extracts of Cpefat/Cpefat mice (analyzed after purification on the anhydrotrypsin column). For vasopressin, data represent the average of the mono- and diacetylated forms of the peptide. For vasopressin-GKR, data represent the average of the mono-, di-, and triacetylated forms. 3196 Analytical Chemistry, Vol. 74, No. 13, July 1, 2002
Table 2. Peptides Detected in CpeFat/CpeFat Mouse Pituitary Extracts That Were Not Present in Wild-Type Mouse Pituitary Extracts and That Gave Sufficient Resolution of the Heavy- and Light-Tagged Forms To Permit Quantitation mass w/o Acc precursor (peptide)a chromogranin A proVP/NP proSAAS proVP/NP POMC prooxytocin (oxytocin-GKR) unidentified provasopressin unidentified unidentified proVP/NP (vasopressin-GKR) unidentified unidentified unidentified unidentified POMC (Ac-RMSH-GKKR) POMC (ox.Ac-RMSH-GKKR) POMC (diAc-RMSH-GKKR) POMC (ox. diAc-RMSH-GKKR) unidentified POMC unidentified POMC (CLIP-KR)
sequenceb PGPQLRR GFFRLTR ARPVKEPR GPARALLLR GSPEPSPREGKR CYIQNCPLGGKR AGTRESVDSAKPR CYFQNCPRGGKR
AcSYSMEHFRWGKPVGKKR AcSYSM*EHFRWGKPVGKKRb DiAcSYSMEHFRWGKPVGKKR DiAcSYSM*EHFRWGKPVGKKRb GPYRVEHFRWSNPPKDKR RPVKVYPNVAENESAEAFPLEFKR
no. of Ac
obs
theor
D/Ud
1 1 2 1 2 1,2 2 1 1,2 2 1,2 2 3 2,3 3 3 2,3 2,3 2,3 2 2,3 2,3 2
822.54 895.57 951.64 965.70 1295.77 1348.74 1370.71 1372.81 1386.70 1402.66 1425.75 1463.69 1493.88 1699.09 1737.05 2134.26 2150.28 2176.25 2192.32 2235.80 2268.33 2737.66 2789.62
822.48 895.50 951.56 965.61 1295.67 1348.64
1.00 ( 0.10 0.99 ( 0.12 0.99 ( 0.19 1.07 ( 0.07 1.01 ( 0.13 1.06 ( 0.09 1.12 ( 0.13 1.11 ( 0.37 1.00 ( 0.17 0.95 ( 0.17 0.86 ( 0.06 0.88 ( 0.07 1.11 ( 0.18 1.15 ( 0.40 1.02 ( 0.25 1.19 ( 0.35 1.26 ( 0.48 1.13 ( 0.31 1.07 ( 0.19 1.06 ( 0.24 0.97 ( 0.10 1.13 ( 0.12 0.97 ( 0.06
1372.71 1425.65
2134.10 2150.08 2176.12 2192.12 2268.17 2789.45
a Abbreviations are as defined in Table 1; and POMC, proopiomelanocortin; proVP/NP, provasopressin/neurophysin. b M* indicates oxidized methionine. c All values represent the observed (obs) or theoretical (theor) monoisotopic masses of the unprotonated and unacetylated (w/o Ac) peptide. d D/U, dehydrated/untreated ((SD).
Figure 5. Tandem mass spectrum obtained by collision-induced dissociation of the (M + 3H)3+ precursor, m/z 959.54. The parent mass matches with the predicted average mass (2875.23 Da) of di(H)acetylated CLIP-KR. Most of the observed major fragment ions match with the predicted b- or y-series fragment ions of this modified peptide. One of the acetyl groups is attached to the N-terminal residue, the other acetyl group is attached to Lys4.
that react with SH groups on proteins have been designed with biotin so that the resulting tryptic peptides can be purified on an avidin affinity column.11,15 However, these labels would only detect the small subset of peptides that contain Cys and would not distinguish between neuroendocrine peptides and other peptides
present in the tissue (such as degradation fragments of proteins). The relative specificity of the present technique for neuroendocrine peptides is a major advantage of this method. Although the increased body weight of mature Cpefat/Cpefat mice may complicate the dosing of drugs or other treatments, these mice do not become Analytical Chemistry, Vol. 74, No. 13, July 1, 2002
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fat until 12-16 weeks of age. Thus, the use of adolescent mice eliminates the potential problems of obesity in the experimental design. Another limitation of the method described in this report is that the number of acetyl groups incorporated must be sufficient so that the mass difference in H- and D-labeled peptides is clearly distinct. The list of peptides shown in Table 2 represents about half of all the observed Cpefat/Cpefat mouse pituitary-specific peptides (i.e., those peptides that were found in the affinity-purified Cpefat/Cpefat mouse extracts that were not present in the wildtype mouse extracts); the other Cpefat/Cpefat mouse pituitaryspecific peptides had overlapping masses of the H- and D-labeled peptides and could not be accurately quantitated. Reagents that label amines are ideal because many neuroendocrine peptides contain a free N-terminal amine group, and many also contain one or more internal Lys residues. Although the C-terminal residue needs to be basic to bind to the anhydrotrypsin column, the vast majority of neuropeptide-processing intermediates that accumulate in the Cpefat/Cpefat mice end with either ArgArg or Lys-Arg. Thus, modification of the C-terminal Lys residues will not eliminate very many neuropeptides from the analysis. Another advantage of labeling the amines is that, for many peptides, acetylation of the N-terminus and the Lys side chains reduces the charge state of the parent peptide, thus facilitating the interpretation of MS/MS data. The peptides detected by LC/ MS after acetylation ranged from 2+ to 5+, whereas a previous study that did not acetylate the peptides found many of the larger peptides to exist with charge states of 8+ or more.1 Interpretation of the MS/MS data for ions with high-charge states is often impossible, and so the reduction of the charge state by acetylation should help with the data analysis. Another advantage of acetylation is that many of the software programs for interpretation of MS data allow for this modification because it is naturally occurring. Native acetyl groups within the peptide are not a problem for the data interpretation because only the mass difference between the added acetyl groups is compared for quantitation of peak intensity. Changes in the levels of vasopressin were smaller than those found in previous studies that investigated the regulation of this
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peptide in pituitary using water deprivation or salt-loading paradigms. In one study that involved rats, water deprivation for 3 days decreased pituitary vasopressin by 50%.29 A study on mice (129xC57Bl/6) reported a decrease in pituitary vasopressin of ∼50% with water deprivation for either 1 or 2 days.30 It is possible that the strain of mouse used in the present study (C57BLKS/J) responds less robustly to the absence of water than the hybrid strain used in the other study. Based on the finding that pituitary vasopressin levels in wild-type C57BLKS/J mice decrease to the same extent as vasopressin in the Cpefat/Cpefat C57BLKS/J mice, it is not likely that the weaker response is due to the fat mutation. Also, the similar change in vasopressin and vasopressin-GKR in the Cpefat/Cpefat mice suggests that the measurement of the LysArg-extended peptides is a valid reflection on the levels of the mature forms of the peptides. In summary, the general technique described in the present study should be useful to measure changes in brain and pituitary peptides under a range of conditions such as food deprivation or various drug treatments. In addition to detecting known peptides, the present method allows for the detection of novel peptides and known peptides with novel modifications. Such information will likely lead to a better understanding of the roles of various peptides in neuroendocrine function. ACKNOWLEDGMENT This work was supported primarily by National Institutes of Health Grant R01 DA-04494 and also by grant K02 DA-00194 (L.D.F.). Mass spectrometry was performed in the Laboratory for Macromolecular Analysis of the Albert Einstein College of Medicine, which is supported in part by the Cancer Center Core, Grant CA13330 and by the Diabetes Research Training Center Core, Grant DK20541. We thank Dr. Haiteng Deng for assistance with the Mariner mass spectrometer and Galina Sidyelyeva for maintaining the Cpefat/Cpefat mouse colony.
Received for review November 9, 2001. Accepted April 2, 2002. AC015681A
