Optimization of Neuropeptide Extraction from the Mouse Hypothalamus Fa-Yun Che, Xin Zhang, Iryna Berezniuk, Myrasol Callaway, Jihyeon Lim, and Lloyd D. Fricker* Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461 Received December 21, 2006
Sample preparation for neuropeptidomic studies is a critical issue since protein degradation can produce high levels of peptides that obscure the endogenous neuropeptides. We compared different extraction conditions for the recovery of neuropeptides and the formation of protein breakdown fragments from mouse hypothalami. Sonication and heating in water (70 °C for 20 min) followed by cold acid and centrifugation enabled the efficient extraction of many neuropeptides without the formation of protein degradation fragments seen with hot acid extractions. The hot water/cold acid extraction procedure resulted in the reproducible recovery of many hypothalamic peptides, including several novel peptides. Keywords: peptide processing • carboxypeptidase E • carboxypeptidase D • prohormone convertase
Introduction Neuropeptides perform a variety of physiological roles in cell-cell communication, ranging from feeding and body weight regulation to anxiety, depression, pain perception, and memory.1 Biologically active peptides are typically produced from larger precursors by the selective action of peptidases. Usually, one or more endopeptidases cleave the precursor at sites containing basic amino acids, and then a carboxypeptidase removes the C-terminal basic residues.2–6 In some cases, additional post-translational modifications are required; examples include amidation, sulfation, acetylation, phosphorylation, and glycosylation.7,8 Most studies investigating the relative levels of peptides have used radioimmunoassays (RIAs) to measure peptide levels. RIAs are often sensitive, but it is difficult to know the precise form of the peptide being measured unless the antiserum is highly specific for a single form of the peptide.9 It is common for antisera to react with N- and/or C-terminally extended forms of the peptides or with other post-translationally modified forms. In contrast, mass spectrometry based “peptidomics” techniques can detect and identify the precise form of each peptide.10–20 When differential stable isotopic tags are used, mass spectrometry based methods also provide relative quantification of peptide levels in two distinct samples. A number of isotopic tags have been developed that react with amines, a group common to most peptides.21–25 Of these, the TMAB labels are the most widely used for quantitative peptidomics studies.23,26–30,13,31,32 This label is readily synthesized with either the H9 or d9 form. The TMAB molecule contains a quaternary amine adjacent to the isotopic label which reduces the formation of hydrogen or deuterium bonds, and as a result, the heavy * Corresponding author. Lloyd Fricker, Ph.D., Professor, Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: 718-430-4225. Fax: 718-430-8954. E-mail:
[email protected]. 10.1021/pr060690r CCC: $37.00
2007 American Chemical Society
and light forms show excellent coelution from reverse-phase columns.22,23 A limitation in mass spectrometry based approaches to detect neuropeptides is the large amount of protein degradation fragments present in brain extracts. This degradation is greatly reduced when mice are sacrificed by microwave irradiation.14,33 Alternatively, protein degradation can be substantially reduced if the brain is microwave irradiated in a conventional microwave oven immediately after decapitation.28 However, extracts of brain regions prepared using either of these microwave techniques still contained protein degradation fragments. In a recent study, we noted that many appear to result from cleavage of Asp-Pro bonds,28 which are typically acid labile.34 In the present study, we first tested several extraction conditions to optimize the reproducible recovery of peptides and the minimization of protein degradation fragments. Then, we used the optimal method to examine peptide levels in replicates of wild type mouse hypothalamic extracts. Altogether, 104 peptides were identified. Of these, the majority corresponded to known neuropeptides, possible neuropeptides, or other fragments of secretory pathway proteins. A minority of the identified peptides corresponded to fragments of cytosolic proteins. Thus, while the sample preparation techniques described in the present study can greatly reduce the degradation of some proteins, other protein fragments appear to be endogenous to the brain.
Experimental Section Reagents. Constant boiling sequanal grade hydrochloric acid (6 N) was from Pierce (Rockford, Illinois). Disodium phosphate, sodium hydroxide, hydroxylamine, glycine, and DMSO were purchased from Aldrich Chemicals (Milwaukee, WI). Acetonitrile, TFA, and formic acid were obtained from Fisher Scientific (Fair Lawn, NJ). The 4-trimethylammoniumbutyryl (TMAB) stable isotopic labeling reagents, H9 and d9 forms of 3-(2,5Journal of Proteome Research 2007, 6, 4667–4676 4667 Published on Web 11/03/2007
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Che et al. on ice for 30 min and centrifuged at 50 000g for 40 min at 4 °C. The supernatant was frozen and concentrated to 600 µL. The pH was adjusted to 9.5 by the addition of 200 µL of 0.4 M phosphate buffer, pH 9.5. The samples were then labeled with H9-TMAB or d9-TMAB as described below. In experiment 2, the tissue was sonicated in water and split into two aliquots, and one tube was incubated at 70 °C for 20 min while the other was kept on ice. After cooling the heated sample, both aliquots were acidified with ice-cold 0.1 M HCl to a final concentration of 10 mM and processed as described for experiment 1. For experiment 3, the tissue pools were sonicated in water, heated to 70 °C for 20 min, cooled in ice, combined with ice-cold HCl to a final concentration of 10 mM, and processed as above.
Figure 1. Experimental design. Pools of mouse hypothalami (hypo) from 4-6 animals were sonicated in water and then either split into two equal parts before extraction (experiments 1 and 2) or extracted and labeled separately (experiment 3). Extractions were performed in either hot acid (10 mM HCl for 20 min at 70 °C), hot water (20 min at 70 °C), or cold water (20 min in an ice bath). After cooling and addition of dilute HCl (or water, for experiment 1), the samples were centrifuged and the supernatant labeled with H9- or d9-TMAB, as indicated. Following quenching of the labeling reagent, samples were pooled, fractionated by microfiltration, desalted on C18 resin, and analyzed by reversephase liquid chromatography and mass spectrometry (LC/MS), as described in the Experimental Section.
dioxopyrrolidin-1-yloxycarbony)propyl trimethylammonium chloride, were synthesized as described.22,23 Animals. Mice (C57BKS/j) were bred in the Albert Einstein College of Medicine barrier facility. Both males and females were used. All mice were adults (12 weeks or older), and the two groups that were subsequently pooled were age-matched and of a single gender. Three experiments were carried out with different groups of mice as shown in Figure 1. Six mice were used for experiment 1 and six for experiment 2. For experiment 3, a total of 26 mice were divided into two groups of 5 female mice/group (Figure 1, “run 3”), two groups of 4 female mice/ group (“run 4”), and two groups of 4 male mice/group (“run 5”). After decapitation, the mouse head was immediately placed in a microwave oven (General Electric, 1.38 kW) and irradiated for 8 s at full power. This was shown previously to raise the temperature of the brain to 80 °C.28 After cooling, the brain was removed and the hypothalamus collected and pooled as described above. Tissues were frozen in dry ice and stored at –70 °C until processing. Peptide Extraction and Differential Isotopic Labeling. The tissue pool was sonicated three times for 5 s in 0.9–1.5 mL of ice-cold H2O, depending on the number of hypothalami in the pool (300 µL per hypothalamus). The aim of experiments 1 and 2 was to study the effects of different peptide extraction methods on the recovery of neuropeptides and the formation of protein degradation fragments. In experiment 1, hot acid and hot water extraction methods were compared, while in experiment 2, hot water and cold water extraction methods were tested (Figure 1). In experiment 1, the homogenate was split equally into two parts, and one aliquot was acidified with 0.1 M HCl to a final concentration of 10 mM HCl. The two samples were incubated in a 70 °C water bath for 20 min and cooled on ice. The tube containing only water was combined with ice-cold 0.1 M HCl so that both tubes contained a final concentration of 10 mM HCl. The homogenates were placed 4668
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The TMAB differential isotopic labeling was performed as shown in Figure 1. The labeling procedure was described previously.23 Briefly, 7 µL of 350 µg/µL H9-TMAB or d9-TMAB in DMSO was added to each tissue extract. After 10 min of incubation at room temperature, an appropriate volume of 1.0 M NaOH was added to adjust the pH back to 9.5, and the sample was further incubated for another 10 min The addition of TMAB and NaOH was repeated six times to ensure all peptides were completely labeled. To quench the remaining TMAB, 60 µL of 2.5 M glycine was added, and the mixture was incubated for 40 min The H- and d-labeled samples were combined and filtered through a Microcon YM-10 unit (Millipore) to remove proteins >10 kDa. To remove TMAB tags from Tyr residues, the filtrate was adjusted to pH 9.0 with 1 M NaOH, combined with 5 µL of 2.0 M hydroxylamine, and incubated for 10 min; this was repeated two more times for a total of 15 µL of hydroxylamine and 30 min of incubation. After desalting with a PepClean C18 spin column (Pierce), peptides were eluted with 100 µL of 70% acetonitrile and 0.1% trifluoroacetic acid in water, frozen, and dried to about 20 µL in a vacuum centrifuge. Aliquots of the sample were analyzed by nano-LC/ MS/MS. Nano-LC/MS/MS Analysis. Nano-LC and tandem mass spectrometry analysis were performed on a Q-Star Pulsar-i quadrupole time-of-flight mass spectrometer (Applied Biosystems/MDS Sciex) equipped with a nanoelectrospray ionization source. An UltiMate capillary/nano-LC system connected to a FAMOS microautosampler was coupled to the mass spectrometer. The sample was loaded on a PepMap C18 trapping column (5 µm, 100 Å, 300 µm inner diameter × 5 mm, LC Packings) and desalted for 30 min with 5% acetonitrile in 0.1% formic acid. Peptides were separated on a Vydac MS C18 capillary column (3 µm, 100 Å, 75 µm inner diameter × 150 mm) by gradient elution using solvents A and B at a flow rate of 0.25 µL/min. Solvent A was 2% acetonitrile/0.1% formic acid in water, and solvent B was 80% acetonitrile/0.1% formic acid in water. The gradient used was 5-45% solvent B in 50 min. The most intense ions in MS scans were fragmented to generate MS/MS spectra. The dynamic exclusion time for fragmented ions was 240 s. The collision energy was in the 20–45 eV range and dynamically changed based on the m/z value and charge state of the ion. The relative levels of peptides in the experimental and control groups of mice were measured by the ratio of peak intensity of the H9- and d9-TMAB-labeled peptide pairs as described previously.23,26–28 All MS/MS spectra were manually interpreted, and the Mascot program was also used for database searching. The following criteria were used to consider a peptide as identified: (a) the peptide mass had to be within 40 ppm (preferably 20 ppm) of the theoretical mass; (b) the
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Peptidomics of the Mouse Hypothalamus observed number of TMAB tags on the peptide matched the number of free amines available (i.e., N-terminus and lysine residues); (c) the observed charge state(s) of the peptide was consistent with the expected number of positive charges; (d) 80% or more of the major fragments observed in MS/MS matched predicted fragmentation ions; (e) there were at least five matches (within 40 ppm) between observed MS/MS fragments and b- and/or y-series ions. Representative spectra of the peptides listed in Table 1 are included in the Supporting Information. For many peptides, Mascot search results are also included in the Supporting Information. In most cases, Mascot correctly identified the peptide as the number 1 hit, although in some cases Mascot ranked the peptide slightly lower or failed to find it; for these peptides, only the manual interpretation is shown (see Supporting Information).
Results In our previous study of peptides extracted from microwaveirradiated mouse brain, a disproportionate number of protein degradation fragments resulted from cleavage C-terminal to an Asp and in many cases between Asp and Pro. Because the AspPro linkage is especially sensitive to acid,34 we first tested whether the short extraction at 70 °C in dilute 10 mM HCl contributed to the formation of protein degradation fragments. Hypothalami from microwave-irradiated mouse brains were sonicated in water and split into two parts: one was heated for 20 min in 10 mM HCl at 70 °C, and the other part was heated in water for the same time (Figure 1, experiment 1). After labeling of the supernatant with H9- or d9-TMAB and LC/MS/ MS analysis (Figure 1), a total of 95 peptides were identified from the MS/MS spectra. Representative MS and MS/MS spectra are shown in Figure 2. Of these, 64 were neuropeptides or other peptides derived from secretory pathway proteins (Table 1), while 31 were protein degradation fragments (Table 2). With one exception, all of the peptides derived from secretory pathway proteins were present in the hot acid and hot water extracts in roughly comparable levels (Table 1, Figure 3). The one exception was oxytocin, which was 2–3-fold more abundant in the hot acid extracts than in the hot water extract (Table 1). In contrast, the protein degradation fragments showed much more variability among the hot acid and hot water extracts (Table 2, Figure 3). All protein degradation fragments that resulted from cleavage C-terminal to an Asp residue were present at higher levels in the hot acid extracts (Table 2, Figure 3). The majority of protein degradation fragments that arose from cleavage at residues other than Asp showed generally similar levels between the two extraction conditions (Table 2, Figure 3). Two notable exceptions, both N-terminal fragments of β-tubulin, contained an acidic group (the C-terminus of glutathione) located near the acid labile bonds (Table 2). To investigate whether heating in water contributed to the protein breakdown or was necessary for the extraction of peptides, the hypothalamic extract was divided into two parts, and one was heated while the other was kept on ice. Following cooling, acidification, and subsequent analysis, 49 peptides were identified; 12 of these were protein degradation fragments, and 37 peptides were derived from secretory pathway proteins (Figure 3). In contrast to the large variability for protein fragments seen in hot acid versus hot water, the variability between levels of protein fragments in hot and cold water extracts was much smaller (Figure 3). Thus, hot water does not appear to induce the degradation of proteins as does the hot
acid treatment. In addition to the experiments shown in Figure 1, additional experiments tested whether methanol extracts resulted in additional peptides not detected in the aqueous acid extracts. Few peptides were detected in the methanol extracts, and none were more abundant in the methanol extracts than in the aqueous extracts (data not shown). Because many previous procedures for extraction of peptides from tissue used heat,35–41 and it was not detrimental providing that acid was avoided, further studies employed extraction in hot water (Figure 1, experiment 3). The extraction procedure was tested with replicates of the mouse hypothalamus to determine whether the technique was reproducible. Altogether, 67 secretory pathway peptides were identified by MS/MS sequencing (Table 1). Nearly all of the secretory pathway protein-derived peptides showed average variations less than 25%, with relative ratios between 0.75 and 1.25 (Table 1 and Figure 4). Protein degradation fragments were also detected but showed higher variability among replicates, with relative ratios ranging from 0.86 to 2.0 (Table 2 and Figure 4).
Discussion A problem central to nearly all research is that of experimenter-induced changes. For studies that involve extraction of materials from tissue, it is important that the form of the material in the extract is identical to that in the original sample. Microwave irradiation, either to sacrifice the animals or immediately postdecapitation, can eliminate many of the enzymecatalyzed postmortem changes in the brain.42 For example, the phosphorylation state of proteins is higher when animals are sacrificed by microwave irradiation than by other methods,43 and the recovery of neuropeptides is enhanced.28,38–40 In the case of mass spectrometry based peptidomics analyses, microwave irradiation or rapid heating of brain tissue is necessary to prevent the postmortem degradation of proteins that would overwhelm the weaker signals from endogenous neuropeptides.14,28,33,18 Still, protein degradation fragments were detected in the microwave-irradiated brain extracts, and it was not clear if these were normally present in the tissue or created by either the microwave irradiation or subsequent extraction procedures. It is reasonable to expect that some protein degradation fragments will normally be present in all tissues; proteins are in a constant flux and are typically degraded into peptides by proteasomes.44 Many peptide extraction protocols involve heating the tissue homogenate under acidic conditions to inactivate proteases and solubilize the peptides.35–40 Because the microwave irradiation step in our current procedure serves the purpose of inactivating proteases, the extraction procedure needs to merely solubilize peptides without inducing protein degradation. Most peptide bonds are stable for several days at elevated temperatures in 70% formic acid; this treatment is used to partially fragment proteins as Asp-Pro bonds, and other peptide bonds generally remain intact.34 Therefore, it was not expected that a brief 20 min exposure of the peptides to 10 mM HCl at 70 °C would cause much protein degradation, especially at bonds other than Asp-Pro. However, from our present results, it is clear that all procedures that use acid, even dilute acid, should avoid elevated temperatures. Some protocols for peptide extraction use organic solvents, often in combination with acid. In addition to the experiments described in the present study, methanol extracts of microwaveirradiated mouse hypothalami were also tested in comparison to hot water and hot acid extracts, and the methanol extracts Journal of Proteome Research • Vol. 6, No. 12, 2007 4669
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prohormone convertase 1 prohormone convertase 2 prohormone convertase 2 promelanin concentrating hormone proneuropeptide Y
proneurotensin proneurotensin pronociceptin/ orphanin FQ
619-628
procholecystokinin procholecystokinin prodynorphin prodynorphin prodynorphin prodynorphin proenkephalin proenkephalin Proenkephalin proenkephalin proenkephalin proenkephalin proenkephalin proenkephalin
C-terminal region neuromedin N neurotensin nociceptin
neuropeptide EI
fragment from pro region C-terminal fragment
46-62 46-63 dynorphin A8 R-neoendorphin 183-199 177-199 heptapeptide octapeptide 221-228 197-208 218-228 114-133 238-261 phosphorylated 238-261
preprotachykinin B
33-50 33-52 cerebellin 1-15 cerebellin
peptide name
392-402 358-371 438-446 588-597 600-613 438-453 438-454 516-535 substance P C-terminal flanking peptide neurokinin B
CART CART cerebellin 1 precursor cerebellin 1 precursor chromogranin A chromogranin A chromogranin B chromogranin B chromogranin B chromogranin B chromogranin B chromogranin B preprotachykinin A preprotachykinin A
precursor
KIPYIL pyroE-LYENKPRRPYIL FGGFTGARKSARKLANQ
745.47 1671.93 1807.99
3432.79
1446.68
2610.33
pyroE-ELEEELDEAEERSLQSILRKN EIGDEENSAKFPIamide SSPETLISDLLMKESTENAPRTRLEDPSMW
1278.60
1102.54
1807.06 1878.07 980.50 1227.68 1847.72 2509.04 876.39 929.44 1153.44 1385.66 1465.65 2235.97 2497.11 2577.11
1209.53
IKMALQQEGFD
DMHDFFVGLMamide AVLRTDGEPRARLGALL AVLRTDGEPRARLGALLA YGGFLRRI YGGFLRKYPK DEDGGQDGDQVGHEDLY SSEMARDEDGGQDGDQVGHEDLY YGGFMRF YGGFMRSL PEWWMDYQ SPQLEDEAKELQ VGRPEWWMDYQ MDELYPMEPEEEANGGEILA FAESLPSDEEGENYSKEVPEIE FAESLP-phosphoSDEEGENYSKEVPEIE GVEKMVNVVE
1219.58 1676.86 1041.51 1116.58 1662.81 1839.95 1910.93 2400.17 1346.73 1844.85
1631.78
SGSAKVAFSAIRSTNH AYGFRDPGPQL WSRMDQLAKELTAE LLDEGHYPV SFARAPQLDL QYDGVAELDQLLHY LLDEGHYPVRESPIDT LLDEGHYPVRESPIDTA LGALFNPYFDPLQWKNSDFE RPKPQQFFGLM-amide ALNSVAYERSAMQNYE
1951.08 2166.29 1494.73
obsd mass
APGAMLQIEALQEVLKKL APGAMLQIEALQEVLKKLKS SGSAKVAFSAIRSTN
peptide sequence
Table 1. Neuropeptides and Peptides Derived from Secretory Pathway Proteinsa
745.47 1671.91 1807.98
3432.65
1446.68
2610.31
1278.64
1102.57
1807.04 1878.08 980.53 1227.68 1847.71 2508.99 876.40 929.45 1153.45 1385.67 1465.65 2235.96 2497.10 2577.06
1209.52
1219.60 1676.82 1041.52 1116.59 1662.79 1839.90 1910.93 2400.15 1346.74 1844.84
1631.84
1951.12 2166.25 1494.78
theor. mass
60 68 61 62 76 60 60 83 62 60
-19 25 -14 -9 12 25 2 8 -6 4
79 62 60 54
38 -2 11 6
59
-1
62
-30
88
58
-27
7
61 61 62 56 55 55 63 62 70 57 66 71 65 66
10 -7 -32 2 5 19 -8 -7 -15 -7 2 8 5 18
76
54
-38
12
84 79 55
elute (min)
-21 20 -32
diff (ppm)
2 2,3 4,5
4,5
2
3
2
2
4 4 3 4 2 3 2 2 1 2,3 2,3 2 2,3 2,3
2
2 3 2 2 2 3 3 2,3 2,3 2,3
4
3 4 3
z
2 1 3
2
2
1
2
2
1 1 1 3 1 1 1 1 1 2 1 1 2 2
1
1 2 1 1 1 1 1 2 2 1
2
3 4 2
#T
0.98 0.94 1.15
0.95
1.00
1.19
0.98
1.01
0.75 1.00 n/d 1.01 n/d 0.69 0.96 1.02 1.28 1.02 0.96 0.87 1.10 0.85
0.90
1.02 0.88 1.04 1.10 0.81 0.97 1.06 0.91 1.00 0.90
0.84
1.11 0.88 1.43
hot acid: water
0.78 (1) 0.93 ( 0.06 (3) n/d
1.09 ( 0.36 (2)
0.87 ( 0.10 (2)
0.92 ( 0.01 (2)
0.83 ( 0.14 (3)
0.97 (1)
1.21 ( 0.11 (2) 0.90 (1) 1.01 (1) 1.07 (1) 1.11 ( 0.27 (3) 0.77 ( 0.09 (2) 0.86 ( 0.01 (2) 0.79 ( 0.11 (2) 0.93 ( 0.27 (3) 0.81 ( 0.16 (2) 0.77 ( 0.23 (2) 0.83 ( 0.10 (3) 1.03 ( 0.28 (3) 1.05 ( 0.32 (3)
1.09 ( 0.44 (2)
0.80 ( 0.16 (2) 0.83 ( 0.30 (2) 1.11 ( 0.37 (2) 0.87 ( 0.01 (2) 0.82 ( 0.22 (2) 0.88 ( 0.17 (2) 0.91 ( 0.06 (3) 0.79 ( 0.13 (2) 0.89 ( 0.13 (3) 1.08 ( 0.30 (3)
1.01 (1)
1.02 ( 0.30 (2) 0.94 ( 0.06 (2) 0.83 ( 0.20 (3)
WT:WT ( sd(n)
research articles Che et al.
LLEAAQEEGAVTPDLPGLEKVQVRPE ISSSISEDPVPI VQLAGTRESVDSAKPRVY SGQLGLPDEEN ESKDQLSEDASKVITYL TNEIVEEQYTPQSLATLESVFQELGKLTGPSNQ
N-terminal fragment
111-122
151-end 287-297 300-316 secretoneurin (184-216) 489-507 NAPPEPVPPPRAAPAPTHV
EEKEEDVEAEERGDLGEVGAWRPH
83-106
1913.99
1975.09 1157.53 1925.00 3649.87
1242.64
2787.53
2765.24
2677.21
1184.63 1437.76 1453.81 1542.82 1754.98 1812.01 2061.06 2316.24 2476.37 2531.38 2745.56 2954.68 1197.49
1006.43 1639.76 951.51
2505.29 2585.24
1939.88
1679.81
1663.81
1621.75
obsd mass
1914.01
1975.05 1157.52 1924.96 3649.80
1242.63
2787.48
2765.25
2677.19
1184.63 1437.76 1453.81 1542.81 1754.98 1812.01 2061.06 2316.23 2476.37 2531.31 2745.55 2954.58 1197.50
1006.44 1639.77 951.53
2505.26 2585.23
1939.86
1679.80
1663.79
1621.77
theor. mass
55 57 70 87 54
-11
65
84
60
72
22 10 19 18
7
15
-6
8
61 58 64 65 60 67 54 81 60 85 62 82 68
60 59 67
-14 -6 -20 0 0 -5 2 2 0 0 3 -1 26 3 33 -10
67 70
57
55
11 5
13
7
61
58
-10 12
elute (min)
diff (ppm)
3
3,4 2 3 3
2
3,4
4,5
2,3
1,2 2 2 1,2 3 2 2 2,3 4 4 4,5 4,5 1,2
1,2 2 2
3 3
2,3
2
2,3
4
z
1
2 1 3 2
1
2
2
1
1 1 1 1 1 1 1 1 2 1 2 1 1
1 1 1
2 2
1
1
1
2
#T
hot acid: water
1.33
0.83 n/d 0.79 0.95
0.73
0.94
0.84
1.06
0.95 n/d 1.01 0.96 0.97 0.96 1.02 0.97 0.92 0.82 0.86 1.05 1.02
2.34 0.90 0.99
0.89 1.22
1.02
n/d
1.03
0.93
WT:WT ( sd(n)
0.93 ( 0.11 (2)
0.95 ( 0.31 (2) 1.20 (1) 0.92 ( 0.19 (3) 0.85 ( 0.29 (2)
1.13 ( 0.09 (2)
1.00 ( 0.17 (3)
1.02 ( 0.19 (3)
0.93 ( 0.21 (3)
0.88 ( 0.12 (3) 0.85 (1) 0.94 ( 0.13 (3) 0.86 ( 0.08 (2) 0.87 ( 0.15 (3) 0.90 ( 0.17 (3) 0.82 ( 0.12 (3) 0.91 ( 0.04 (3) 0.93 ( 0.26 (2) 0.95 ( 0.22 (2) 1.08 ( 0.21 (2) 0.94 ( 0.26 (2) 0.81 ( 0.17 (3)
1.37 (1) 1.01 ( 0.11 (3) 0.95 ( 0.16 (3)
0.89 ( 0.12 (2) 0.98 ( 0.54 (3)
1.20 ( 0.87 (3)
0.95 ( 0.12 (2)
0.84 ( 0.31(2)
1.16 ( 0.23 (2)
Peptides identified by MS/MS sequencing also matched the following criteria: a small difference (less than 40 parts per million, or ppm) between the theoretical (theor.) and observed (obsd) peptide mass, an observed charge state (z) that reflects the theoretical number of charged groups, and the observed number of isotopic tags (#T) incorporated equal to the number of free amines (N-terminus and Lys residues). All masses are monoisotopic. The relative levels of peptides were calculated by the ratio of peak intensity of the H9-TMAB- and d9-TMAB-labeled peptide pairs. Hot Acid:Water indicates the ratio of peptide in hot acid extracts relative to hot water extracts (Figure 1, run 1). WT:WT indicates the variation of peptide in different groups of wild type mice (Figure 1, runs 3–5). For those peptides observed in more than one run in experiment 3, the error range indicates standard deviation for n replicates. Abbreviations: CART, cocaine and amphetamine-regulated transcript; MSH, melanocyte-stimulating hormone; J-peptide, joining peptide; CLIP, corticotropin-like intermediate lobe peptide; LPH, lipotropin; PACAP, pituitary adenylate cyclase-activating polypeptide; and n/d, not detected. Note that SAAS, LEN, PEN, GAV, and VGF are names, not abbreviations.
a
VGF
oxytocin 111-128 cleaved pro peptide
prooxytocin ProPACAP propeptidyl-amidatingmonooxygenase ProSAAS ProSAAS ProSAAS ProSAAS ProSAAS ProSAAS ProSAAS ProSAAS ProSAAS ProSAAS ProSAAS ProSAAS prothyrotropin releasing hormone prothyrotropin releasing hormone prothyrotropin releasing hormone prothyrotropin releasing hormone ProVasoactive intestinal peptide provasopressin secretogranin II secretogranin II secretogranin II FIDPELQRSWEETEGEEGGLMPE
CLIP phosphorylated CLIP
proopiomelanocortin proopiomelanocortin
178-200
J-peptide
proopiomelanocortin
ASAPLVETSTPL AVPRGEAAGAVQELA ASAPLVETSTPLRL SLSAASAPLVETSTPL LENPSPQAPARRLLPP SLSAASAPLVETSTPLRL SVDQDLGPEVPPENVLGALL SVDQDLGPEVPPENVLGALLRV ARPVKEPRSLSAASAPLVETSTPL GEAAGAVQELARALAHLLEAERQE ARPVKEPRSLSAASAPLVETSTPLRL AVPRGEAAGAVQELARALAHLLEAERQE SFPWMESDVT
R-MSH (oxidized)
proopiomelanocortin
peptide sequence
SYSMEHFRWGKPVamide Ac-SYSMEHFRWGKPVamide Ac-SYS-MoxEHFRWGKPV-amide AEEEAVWGDGSPEPSPREamide RPVKVYPNVAENESAEAFPLEF RPVKVYPNVAENE-phosphoSAEAFPLEF CYIQNCPLG-amide GAGENLGGSAVDDPAPLT FRSPLSVF
little SAAS 5-16 GAV 1-15 little SAAS 5-18 little SAAS 1-16 big LEN little SAAS PEN 1-20 PEN big SAAS 1-24 GAV 5-28 big SAAS GAV 160-169
R-MSH
proopiomelanocortin
peptide name
des-acetyl-MSH
proopiomelanocortin
precursor
Table 1. Continued
Peptidomics of the Mouse Hypothalamus
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Figure 2. Representative MS and MS/MS spectra. (A) Ions at m/z 597.97 and 600.99 (monoisotopic peaks) correspond to mono-H9- and d9-TMAB-labeled R-melanocyte stimulating hormones (R-MSH). (B) Ions at m/z 921.41 and 927.44 (monoisotopic peaks) correspond to the di-H9- and d9-TMAB-labeled N-terminal fragment of Dynein light chain 2. (C) MS/MS of mono-H9-TMAB-labeled R-MSH. (D) MS/MS of the di-H9-TMAB-labeled N-terminal fragment of Dynein light chain 2. Note that upon collision-induced dissociation the neutral loss of H9-trimethylamine (mass ) 59 Da) or d9-trimethylamine (mass ) 68 Da) from TMAB labels usually occurs. y′ ) y-59, b′′ ) b-118.
were found to contain very low levels of peptides relative to the aqueous extracts (data not shown). Furthermore, the reproducibility of most peptides extracted using the hot water/ cold acid method among replicates of animals is good (Figure 4), suggesting the efficient extraction of these peptides without the need for organic solvents. Recently, Parkin et al. reported that repeated sonication of the tissue in 8 mM SDS and 0.25% acetic acid was necessary for the efficient extraction of many neuropeptides from microwave-irradiated rat striatum.33 However, half of the reported “neuropeptides” were fragments of cytosolic proteins such as thymosin β-4, thymosin β-10, and 4672
Journal of Proteome Research • Vol. 6, No. 12, 2007
phosphatidylethanolamine-binding protein.33 Furthermore, many of the peptides reported to have a C-terminal amide group were not encoded by a precursor with a Gly in the appropriate position;33 amidation requires a C-terminal Gly which is converted to the amide by peptidyl-glycine R-amidating monooxygenase.45 Because of these discrepancies, it is difficult to interpret the results of Parkin et al. Using the optimal extraction protocol, the variability among three different groups of mice was fairly low; the average ratio of peptides was 0.95, which is very close to the theoretical 1.00 ratio, and the standard deviation was less than 0.20 for the
dynein light chain 2 ES1 heat shock protein 1 (chaperonin 10) gemoglobin R macrophage migration inhibitory factor macrophage migration inhibitory factor microtubule associated protein tau myelin basic protein myelin basic protein myelin basic protein myelin basic protein Na and Cl-dependent GABA transporter (GAT4) NADH dehydrogenase peptidyl-prolyl isomerase peptidyl-prolyl isomerase seryl tRNA synthetase
Cgi-38 protein, chain A cytochrome C oxidase subunit VIIb
actin (β or γ) aspartate transaminase ATP synthase, R subunit ATP synthase, subunit F calmodulin-1 calmodulin-1 calmodulin-3 calmodulin-3 cathepsin D
precursor
AKLKEIVTNFLAGFEP PKFEVIDKPQS GQVNYEEFVQMMTAK GDGQVNYEEFVQMMTAK Ac-ADQLTEEQIAEFKEAFSLFD Ac-ADQLTEEQIAEFKEAFSLFDKD YTVFDRDNNRVGFANAVV
C-terminal fragment
C-terminal fragment
C-terminal fragment C-terminal fragment N-terminal fragment N-terminal fragment internal fragment (near C-terminus) N-terminal fragment
Ac-ASQKRPSQRSKYLATA Ac-ASQKRPSQRSKYLATAS Ac-ASQKRPSQRSKYLATASTM Ac-ASQKRPSQRSKYLATASTMD GTISAITEKETHF PTKEPEPVVHYDI EDENFILKHTGPGILSM VNPTVFFDITADDEPLGRVSF VLDLDLFRVDKGGD
N-terminal fragment
N-terminal fragment
N-terminal fragment
C-terminal fragment
C-terminal fragment Internal fragment
N-terminal fragment
N-terminal fragment
PMFIVNTNVPRASVPEGFLSELTQQL
N-terminal fragment
N-terminal fragment
KFLASVSTVLTSKYR AQATGKPAQYIAVHVVPDQL
C-terminal fragment internal fragment
SPQLATLADEVSASLAKQGL
GIGAMVKNVLELTGK Ac-AGQAFRKFLPLFDRV
C-terminal fragment N-terminal fragment
C-terminal fragment
Ac-SDRKAVIKNADMSEDMQQDAVD
SHQKRAPSFHDKYGNAILA
N-terminal fragment (after removal of mitochondrial targeting peptide) N-terminal fragment
Ac-AASTDIAGLEESFRKFAIHGD
ALPHAILRL SSWWTHVEMGPPD
peptide sequence
internal fragment N-terminal fragment
peptide name
Table 2. Peptides Derived from Cytosolic Proteinsa
1560.85
2338.07
1522.72 1900.00
1432.68
2267.15
2152.12
1920.03
1832.97
1998.04
2886.52
1699.00 2105.11
1528.89 1806.00
2507.04
2139.11
2276.18
1773.82 1945.86 2372.09 2615.18 2056.03
1286.65
1776.01
1002.63 1527.66
obsd mass
1560.82
2338.15
1522.77 1899.95
1432.72
2267.14
2152.12
1920.03
1833.00
1998.07
2886.50
1698.97 2105.13
1528.86 1805.98
2507.13
2139.11
2276.12
1773.81 1945.86 2372.11 2615.23 2056.03
1286.69
1775.98
1002.64 1527.65
theor. mass
58 69
-37 25
21
71
79
60
-27
27
51
51 4
0
48
54
-16 0
73
84
65 64
-17
9
17 -10
78 67
57
-37 20 11
52
78
0
28
73 73 89 84 64
56
-36 2 -2 -7 -18 0
83
61 66
-10 12 17
elute (min)
diff (ppm)
3
3
3 2
3
2
3 3
3
4
4
3,4
3,4
2,3
3
4 3
3 3
3
4–6
3
3 2,3 2,3 2,3 3
z
2
1
2 2
2
2
2
2
2
2
1
3 2
3 1
2
2,3
1
2 2 1 2 1
2,3
3
1 1
#T
1.89
1.04
2.63 0.88
2.17
2.86
n/d
n/d
2.13
>5
1.15
1.85 1.14
2.78 1.28
2.70
n/d
1.96
10 >10 1.39 >20 0.96
5.88
3.57
n/d 3.85
hot acid:water
n/d
1.52 ( 0.63 (3)
n/d n/d
n/d
0.97 (1)
1.05 (1)
0.89 (1)
0.93 (1)
n/d
1.90 ( 0.88 (2)
n/d 1.00 ( 0.77 (3)
n/d n/d
2.04 ( 0.27 (3)
1.08 ( 0.03 (2)
n/d
n/d 1.32 ( 0.23 (2) n/d n/d 0.86 ( 0.05 (3)
1.01 (1)
n/d
1.32 (1) n/d
WT:WT ( sd(n)
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1.12 (1) 0.88 2
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Abbreviations are as defined in Table 1, except that the observed and theoretical masses of thymosin β-4 are average; all others are monoisotopic. a
61 1396.70 AGGHKLGLGLEFQA
1396.75
-36
3
n/d 1.89 1 2,3 63 1 1781.91 Ac-ASASGAMAKHEQILVLD
N-terminal fragment from upstream ATG C-terminal fragment
1781.91
n/d 2.13 1 60 Ac-AKHEQILVLD N-terminal fragment
1206.64
1206.67
-27
2
1.12 (1) n/d 1 4 53 1692.91 1692.93
12
0.92 ( 0.45 (3) 1.85 2 4 55 -19 1688.76
MREIVHIQAGQC+ Glutathione EVRPQVHPNYRVTV
tubulin β (isoforms 2-6) tubulin β (isoforms 2-6) + glutathione vacuolar ATP synthase subunit 2 VAP-33 (VAMP associated protein) VAP-33 (VAMP associated protein) voltage-dependent anion channel protein 1 (VDAC-1)
N-terminal fragment + glutathione on Cys C-terminal fragment
1688.73
1.88 ( 0.93 (3) 4.55 1 55 1280.63
1280.67
-31
3
1.43 ( 0.01 (2) 0.92 ( 0.30 (2) 0.88 0.90 3 9 3 9 65 61 30 -20 1603.88 4963.54
VKLIESKEAFQEAL Ac-SDKPDMAEIEKFDKSKLKKTET QEKNPLPSKETIEQEKQAGES MREIVHIQAGQ N-terminal fragment Entire protein (without N-terminal Met) N-terminal fragment
1603.93 4963.44
n/d 2.94 2 2 79 15 1257.70 PLADLNIKDFL
1257.72
Che et al.
C-terminal fragment
synaptosomal-associated protein (Snap91) thioredoxin 1 thymosin β-4
precursor
Table 2. Continued
peptide name
peptide sequence
obsd mass
theor. mass
diff (ppm)
elute (min)
z
#T
hot acid:water
WT:WT ( sd(n)
research articles
majority of the peptides. Still, some peptides showed large variability among the groups. The reason for the variability is not known but may involve naturally occurring differences in peptide levels among mice, or in the dissection, and/or analysis. It is interesting that many of the peptides with the largest variability among replicates of wild type samples are derived from proopiomelanocortin (Table 1). Because the proopiomelanocortin system is known to be regulated by stress,46,47 it is possible that the variability in these peptides reflects differences in the animals. While efforts were made to reduce the levels of stress in the mice, the mice used in this study were group housed, and it is stressful to mice when their cage-mates are removed.48 Thus, the stress on the mice sacrificed first is less than those sacrificed last, and no effort was made to control for this. Other possible causes of variation such as gender and age were controlled among the groups. Still, with the exception of a small number of peptides, the technique is fairly reproducible for neuropeptides and other peptides derived from secretory pathway proteins. In contrast to the neuropeptides and other secretory pathway peptides, the variability among replicates for cytosolic protein degradation fragments is considerably larger (Table 2), presumably because of the transient nature of these fragments. Whereas neuropeptides are produced and stored within secretory vesicles prior to release,49 cytosolic proteins are typically degraded by the proteasome into peptides which are then either selected for antigen presentation or further degraded by intracellular peptidases.44 Thus, small fluctuations in either the initial proteolysis of proteins or in subsequent cleavages by peptidases could have a large impact on the cellular levels of these fragments. In addition, some of the observed protein degradation fragments may still result from postmortem breakdown at labile bonds during the microwave irradiation or in the subsequent extraction with cold acid. Nine of the secretory pathway peptides identified in the present study have not been previously reported in the literature, to the best of our knowledge. These novel peptides include cerebellin 1-15, chromogranin B 438-453, prodynorphin 177-199, prodynorphin 183-199, proenkephalin 114-133, little SAAS 5-16, big SAAS 1-24, prothyrotropin releasing hormone 160-169, secretogranin II 287-297, and VGF 489-507. Some of these peptides are flanked by pairs of basic residues, and therefore it was predicted that they would exist in the brain. Neuropeptide precursors are expressed together with enzymes that cleave at pairs of basic residues: first prohormone convertases cleave to the C-terminal side of the basic residues, and then carboxypeptidases E or D remove these basic residues.2–6 Most of the previously identified peptides found in the present study are also produced by this processing pathway. However, some of the previously identified peptides and a few of the novel peptides appear to result from a different set of processing enzymes. Examples of peptides that require cleavage at nonbasic sites include cerebellin, cerebellin 1-15, chromogranin B 438-453, procholecystokinin 46-62, and several novel forms of SAAS. Further studies are required to identify the enzymes that are responsible for the nonbasic mediated cleavages found in the present study, as well as in previous studies of rodent brain peptides.33,18,14,10,27,28,31
Conclusions The purpose of this study was to optimize the extraction of neuropeptides and related secretory pathway peptides from a mouse brain, while minimizing postmortem degradation frag-
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Peptidomics of the Mouse Hypothalamus
Figure 3. Summary of the results of experiments 1 (left) and 2 (right) examining the relative levels of hypothalamic peptides in hot acid versus hot water extracts (experiment 1) or in cold water versus hot water extracts (experiment 2). The results for the cytosolic protein fragments are subdivided into peptides that result from cleavage C-terminal to an Asp residue (D-X) and all other cytosolic protein fragments. Sec Path Peptides refers to peptides derived from secretory pathway proteins, which include neuropeptides as well as putative neuropeptides and related molecules. For experiment 1, the results for the secretory pathway peptides are expanded in the middle panel. The figure was generated using the SigmaPlot 2001 program with data extracted from Tables 1 and 2.
extraction technique will be useful for searches for novel brain neuropeptides.
Acknowledgment. This work was supported primarily by National Institutes of Health grants DA-004494 (L.D.F.). Mass spectrometry was performed within the Laboratory for Macromolecular Analysis of the Albert Einstein College of Medicine (Dr. Ruth Angeletti, director). Special thanks to Ben Segall for creating a program for de novo sequencing of isotopic tagged peptides, which was used to determine the sequence of several of the peptides described in this paper. Supporting Information Available: MS/MS spectra that represent the peptides in Table 1, arranged in order of ascending mass. For some of the peptides, both Mascot results and the manual interpretation are shown. This material is available free of charge via the Internet at http://pubs.acs.org. Figure 4. Summary of the results of experiment 3. The figure was generated using the SigmaPlot 2001 program with data extracted from Tables 1 and 2. Sec Path Peptides, neuropeptides and other peptides from secretory pathway proteins; Cytosol Prot Fragments, peptides resulting from degradation of cytosolic proteins.
ments of proteins. Microwave irradiation of the mouse heads following decapitation is an important step that improves peptide levels and decreases protein degradation fragments, but also of importance is the avoidance of hot acidic extraction conditions. Extraction of microwave-irradiated brain regions in hot water followed by the addition of ice-cold acid provides for the reproducible recovery of many neuropeptides while greatly reducing the levels of cytosolic protein fragments. This
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