Amidation/non-amidation top-down analysis of endogenous neuropeptide Y in brain tissue by nano flow liquid chromatography orbitrap Fourier transform mass spectrometry.

Neuropeptide Y (NPY) is a transmitter molecule in nerve system, and it was an over 4-kDa large peptide with the C-terminal end amidation. NPY is biosynthesized through many maturation processes from a large pre-pro-peptide with peptide-cleavages and amidation that is important to study the biosynthesis regulation. Previously, it was reported that cathepsin L participates in the production of NPY and that cathepsin L generates both of amidated and non-amidated NPYs. However, the non-amidated NPY (NPY-COOH) has not been reported in brain tissues until now. In this study, endogenous NPY-COOH in mouse brain tissue was detected and identified by using nano flow liquid chromatography (nanoLC) orbitrap Fourier transform mass spectrometry (FT-MS) after the effective purification and separation of NPY-COOH from NPY-amide and other peptides using two different gel-filtration chromatography. Amidated NPY was eluted earlier than non-amidated NPY-COOH in the C18 reversed phase nanoLC and the silica-based gel-filtration chromatogram with hydrophobic interaction. The amount of endogenous NPY-COOH was about 0.05% of the matured NPY-amide amount in adult mouse brain.

INTRODUCTION

Neuropeptides function as neurotransmitters or neuroendocrine hormones in the central nervous system and are stored in secretory granules. Neuropeptides are coded on their genes and are synthesized as a long precursor pre‐pro‐peptide or protein through transcription and translation. After that, the precursor peptides are digested, modified, for example, via amidation, and maturated as a bioactive peptide form. It is important to study these maturation processes via direct analysis of the various peptide forms and their modifications using mass spectrometry (MS).

Neuropeptide Y (NPY) is a linear polypeptide with 36 amino acid residues and is amidated at the C‐terminal as the active form (Figure 1), this form being linked to basic biological functions such as the regulation of feeding behavior, the control of blood pressure, and so on. , , , , NPY (NPY‐amide) is generated and transferred into secretory vesicles by proteolytic processing of the precursor pre‐pro‐NPY polypeptide that has 97 amino acid residues as shown in Figure 2. A peptidase recognizes a dibasic processing site (NPY‐Gly‐Lys‐Arg‐) in proNPY, and “NPY‐Gly” is generated proteolytically such as prohormone convertases and carboxypeptidase E. Then, the NPY‐Gly is amidated by peptidylglycine alpha‐hydroxylating monooxygenase (PAM) to NPY‐amide (Figure 2). However, another proteolytic processing of proNPY was reported. Funkelstein reported that cathepsin L participates in the production of NPY directly. ProNPY was cleaved by cathepsin L at the Lys‐Arg dibasic processing site, and two product peptides, NPY‐Gly and NPY‐COOH, were generated (Figure 2). The NPY‐Gly was amidated into NPY‐amide by PAM. Therefore, both NPY‐amide and NPY‐COOH peptides may be detected as final products in brain. However, it was paradoxical that the presence of NPY‐COOH in brain have not been reported. Here, we try to detect NPY‐COOH directly in mouse brain using a high sensitive system of nano liquid chromatography (nanoLC) mass spectrometry (MS).

FIGURE 1

Structure of neuropeptide Y

FIGURE 2

The assumed maturation processes of NPY. CPE, carboxypeptidase E; PAM, peptidylglycine‐α‐amidating monooxygenase; PC, prohormone convertase. The up arrows show the cleavage sites of peptidases

Normally, the presence of NPY are estimated with the trypsin‐digested fragments of NPY by LC–MS/MS with multiple reaction monitoring due to the sensitivity, which cannot distinguish between NPY‐NH2 and NPY‐COOH. We also analyzed trypsin‐digested fragments of NPY; however, the fragments including the C‐terminal ends were not detected (data no shown). The proteomics technique based on the digested short peptide search was useful for identifying the large variety proteins, , , but it was not suite for distinguishing among small different modifications in long peptides. The top‐down identification method was thought to be more advantageous than the bottom‐up method based on the digested peptide search. We focused our attention to the detection of NPY‐COOH, and it was essential to detect the whole peptides directly like a top‐down analysis to study the neuropeptide maturation processes. As reported, the amidation/non‐amidation analysis of long peptides over 4 kDa using LC–MS is challenging. The 36 amino acid sequences of NPY‐amide and NPY‐COOH are the same in Figure 1, and the structural difference is only at the C‐terminal of the carboxyamide (‐CONH2) and the carboxyl groups (‐COOH). A potential problem with MS measurement is that the mono‐isotopic ion of NPY‐COOH is severely overlapped by the second isotopic peak of NPY‐amide. Addition to that, the amount of NPY was trace with large amount of other contaminants in the mouse brain tissue sample, and there could be difficulties in sensitivity and separation. , Thus, it is essential to use high‐resolution MS and MS/MS measurements with high‐sensitive capillary nanoLC and to develop pre‐purification such as effective gel‐filtration chromatography (GFC) to detect the presence of NPY‐COOH in mouse brain tissue.

MATERIALS AND METHODS

Materials

Adult mouse brain tissue (snap frozen; Rockland Immunochemicals, Inc., PA, USA) was purchased from Cosmo Bio Co., LTD., Tokyo, Japan. Acetonitrile (HPLC grade), 99% acetic acid (guaranteed regents), and trifluoroacetic acid (TFA) were purchased from Nacalai Tesque Inc., Kyoto, Japan. Milli‐Q water (Merck Millipore, Burlington, MA, USA) was used. The chemically synthesized NPY‐amide and NPY‐COOH were purchased from Peptide Institute, Inc., Osaka, Japan.

Protocol of neuropeptide extraction from mouse brain

The experimental protocol is outlined in Figure 3. Three pieces of frozen adult mouse brain were smashed into small fragments. The frozen fragments were then boiled in 25‐ml water at 100°C for 10 min in an eggplant flask (Figure 3). After cooling on ice, 99% acetic acid was added to the flask resulting in the 1‐M final concentration. The samples were homogenized in a TissueLyser II (QIAGEN N. V., Hilden, Germany) for 1 min at 28 frequency using zirconia beads in microtubes. The peptide mixtures were obtained from the supernatants following centrifugation at 150,000 rpm for 10 min (TOMY MX‐107 centrifuge, TOMY SEIKO, Co., Ltd., Tokyo, Japan). A C18 Sep‐Pak solid phase extraction (tC18 cartridges, Vac 12 cc, 2 g) (Waters Corporation, Milford, MA, USA) was pre‐washed by acetonitrile and then treated with 5% acetonitrile 0.1% TFA aqueous solution. A centrifuged supernatant sample was loaded onto the Sep‐Pak cartridge, which was then washed with 12 ml of 5% acetonitrile 0.1%TFA aqueous solution (four times of the cartridge volume). Then, the peptides were fractionated by 40% acetonitrile 0.1% TFA aqueous solution, and the total extraction was 12 ml. The NPY‐COOH rich fraction was used for the identification of NPY‐COOH/NPY‐amide. The ratio of NPY‐COOH/NPY‐amide was estimated by the ultra‐high resolution MS analysis of the total solid extraction sample. The total lyophilized sample (3.67 mg) was obtained in the solid extraction step.

FIGURE 3

Protocol of neuropeptide analysis using LC–MS

Large‐scale GFC chromatography

The GFC column of TSKgel G2000SW (7.5 × 600 mm; 10 μm) with a guard column (TOSOH Corporation, Tokyo, Japan) was used as the first column. The eluent was an aqueous solution of 40% acetonitrile/0.1%TFA, and a flow rate of 1 ml/min was used. The sample fractions were collected at intervals of 1 min. The lyophilized peptide extract (2.9 mg) was dissolved in 100 μl of 40% acetonitrile/0.1%TFA aqueous solution, and all of them were loaded to the GFC column. The eluted fractions were taken to each tube systematically for 1 min. The NPYs were eluted at the 18 min fraction as shown in Figure 4, which was dried up by a centrifugal evaporator system (EYELA Tokyo Rikakikai Co. Ltd., Tokyo, Japan), and it was dissolved in 100 μl of 40% acetonitrile/0.1%TFA aqueous solution for the subsequent triple analytical GFC.

FIGURE 4

Gel‐filtration chromatograms of large‐scale GFC of the crude peptide extracts (A) and the subsequent triple‐analytical GFC (B)

Triple analytical GFC chromatography

Three GFC columns, TSKgel Super SW3000, SW2000, and SW2000 (4.6 × 300 mm; 4 μm) with a guard column (TOSOH Corporation, Tokyo, Japan), were connected in series as a triple‐analytical GFC column. The eluent was an aqueous solution of 40% acetonitrile/0.1%TFA, and a flow rate of 0.15 ml/min was used. An aliquot of 100‐ml sample of large‐scale GFC was loaded to the triple‐analytical GFC column. The eluted fractions were taken to each tube systematically for two mins. NPY samples were eluted in 54–66 min for six fractions of Fr.1–Fr.6 as shown in Figure 4. These fractions were dried‐up with a centrifugal evaporator system (EYELA Tokyo Rikakikai Co. Ltd., Tokyo, Japan), and then, they were resolved in 100 μl of 40% acetonitrile/0.1%TFA aqueous solution for nanoLC‐orbitrap MS analysis.

NanoLC‐orbitrap MS

All the MS and MS/MS data were acquired with an FT Orbitrap Elite MS instrument (Thermofisher Scientific, MA, USA). The EASY‐nLC 1000 system (Thermofisher Scientific, MA, USA) was used as the front end of the nanoLC–MS instrument. A reversed phase ODS column, Acclaim PepMap RSLC (C18 50 μm i.d. × 150 mm; 2 μm, 100 A) (Thermofisher Scientific, MA, USA), was used for peptide separation. The eluent consisted of an 0.1% aqueous solution of formic acid (solution A) and acetonitrile/0.1% formic acid (solution B), and the flow rate was 300 nl/min. The solvent ratios for solution B in the gradient program were increased gradually from 0% to 40% over 70 min (0–70 min), then quickly from 40% to 100% over 70–72 min; thereafter, solution B was kept at 100% for 72–80 min for column washing. NanoLC‐orbitrap MS data were measured with full scan FT‐MS at 240,000 mass resolution. NanoLC‐orbitrap MS/MS data were measured with full scan FT‐MS at 120,000 resolution and with data dependent ion‐trap MS/MS. The solvent ratios for solution B in the gradient program were increased gradually from 0% to 40% over 50 min (0–50 min), then quickly from 40% to 100% over 50–52 min; thereafter, solution B was kept at 100% for 52–60 min for column washing.

RESULTS AND DISCUSSION

Neuropeptides extraction protocol from mouse brain

In generally, neuropeptides and the precursor peptides are stored in secretary vesicles rather than cytoplasm in cell. It is in homogenize process that there is possibility that the artificially digested peptides could generate with cytoplasm proteases. To inactivate all peptidases and proteases, frozen mouse brain tissues were boiled at 100°C for over 10 min before homogenizing them as shown in Figure 1. After extraction of solid phase, the peptide extraction was applied to the gel‐filtration chromatography (GFC). The GFC process was essential for the detection of neuropeptides with the subsequent nanoLC–MS because the abundant contaminating proteins and small molecules such as lipids.

In this study, the solid phase peptide extraction was applied to the large‐scale GFC separation for a rough fractionation of NPYs, and the NPY fraction was eluted at 18 min as shown in Figure 4A. Moreover, the concentrated NPY fraction was applied to the triple analytical GFC column, and NPYs were separated into six fractions Fr.1–Fr.6 for the elution times 54 to 66 min as shown in Figure 4B.

Separation and detection of NPY‐amide and NPY‐COOH with nanoLC‐orbitrap‐MS

The molecular weight of NPY‐amide is 1 Da smaller than that of NPY‐COOH. The mono‐isotopic ion of NPY‐amide is easily identified. The mono‐isotopic ion of NPY‐COOH is severely overlapped to the second isotopic ion of NPY‐amide in their mass spectra (Figure 5). It is difficult to identify NPY‐COOH with the mono‐isotopic mass signal because the m/z value differences of their isotope ions are 0.003 in the [M + 6H]6+ peaks. Therefore, it is essential for the identification of NPY‐COOH to separate between NPY‐amide and NPY‐COOH with their LC chromatogram even though the high‐resolution MS was used.

FIGURE 5

NanoLC‐orbitrap‐MS raw spectra of (A) NPY‐amide and (C) NPY‐COOH and their calculated spectra (B and D)

The NPY‐amide standard was eluted earlier for about one min than NPY‐COOH in the capillary nanoLC‐orbitrap MS with a C18‐reversed phase column as shown in Figure 6A,B. Although their structure differences are just at the C‐terminal ends of NPY‐amide and NPY‐COOH, NPY‐amide and NPY‐COOH were separated because the capillary nanoLC separation was effective and the C‐terminal amidation in peptides gives them high hydrophilicity more than we expected.

FIGURE 6

Total ion chromatogram of NPY‐amide (A), NPY‐COOH standard (B), and the NPY fraction Fr.6 (C), and the mass chromatogram of NPY‐amide (D) and NPY‐COOH (E) in the nanoLC‐orbitrap MS of Fr. 6 after purified by large‐scale GFC and triple‐analytical GFC chromatography

The nanoLC separation between NPY‐amide and NPY‐COOH was enough to identify them; however, it was difficult to detect a small amount of NPY‐COOH in the predominant NPY‐amide mixtures in the nanoLC–MS because the signal of NPY‐COOH was close and it was covered with the peak tail of NPY‐amide. Actually, NPY‐COOH was not detected in each NPY fraction of the large‐scale GFC and triple analytical GFC separately. To reduce the contaminating peptides, we tried to combine these two different GFCs purification of the NPY peptides as described above (Figure 4). We did not expect the separation between NPY‐amide and NPY‐COOH with the GFC purification steps, however, the double GFC purification processes separate them effectively as described later.

Identification of endogenous NPY‐COOH in mouse brain tissue

Six NPY fractions Fr.1 to Fr.6 in the triple‐analytical GFC were analyzed by nanoLC–MS (Figures 6 and 7). The extracted mass chromatogram peak of the most abundant ions at m/z 713.0191 of NPY‐COOH was detected at 50.4 min with the fourth isotope ion of NPY‐amide at 49.5 min because of their overlapped mass signals in the extracting mass window at m/z 713.01 to 713.03 as shown in Figures 5 and 6. The NPY‐COOH and NPY‐amide of the brain peptide extracts were eluted earlier for one minute than those of the standard each (Figure 6). It was because the targets of NPY‐COOH and NPY‐amide could not absorb well to the column due to the large amount of other peptides and proteins and because the absorb capacity of the capillary column could be too small. The mass spectra of the NPY‐amide at 49.5 min and NPY‐COOH at 50.4 min in nanoLC‐orbitrap MS were consistent with the calculated spectra within 2.0 ppm error as shown in Figure 5. The MS/MS data of NPY‐COOH also proved the sequence structure of NPY‐COOH (Figure 8) as describe later. Therefore, it was first proved that the endogenous NPY‐COOH exists in mouse brain with NPY‐amide.

FIGURE 7

The table of the peak area of NPY‐amide and NPY‐COOH in their mass chromatograms of Figure 6 (A) and the logarithmic graph of NPY‐amide and NPY in each fraction Fr.1–Fr.6 of the triple‐analytical GFC by nanoLC‐orbitrap MS (B)

FIGURE 8

MS/MS spectra of NPY‐amide (A) and NPY‐COOH (B)

NPY‐COOH/NPY‐amide ratio in the triple analytical GFC fractions

The mass chromatogram of NPY‐amide was extracted with the mass window from m/z 712.85 to 712.87 according to the most abundant ion at m/z 712.8551. The mass chromatogram of NPY‐COOH was extracted with the mass window from m/z 713.01 to 713.03 according to the most abundant ion at m/z 713.0191 (Figures 6 and 7). The NPY‐COOH was detected in Fr. 4–6, and the amount of NPY‐COOH was estimated from the ion signals and summarized in Figure 7. NPY‐COOH existed in 0.05% amount of NPY‐amide in mouse brain tissue.

The assignments of the fragment ions were summarized in Tables 1 and 2. The red and blue colored ions were corresponded to the colored mass numbers in tables.

TABLE 1

MS/MS fragment ion list of NPY‐amide in Figure 8

#1	b+	b2+	b3+	b4+	b5+	Seq.	y+	y2+	y3+	y4+	y5+	#2
1	164.07061	82.53894	55.36172	41.77311	33.61994	Y						36
2	261.12337	131.06532	87.71264	66.03630	53.03050	P	4107.02535	2054.01632	1369.67997	1027.51180	822.21089	35
3	348.15540	174.58134	116.72332	87.79431	70.43690	S	4009.97259	2005.48993	1337.32905	1003.24861	802.80034	34
4	476.25036	238.62882	159.42164	119.81805	96.05589	K	3922.94056	1961.97392	1308.31837	981.49060	785.39393	33
5	573.30312	287.15520	191.77256	144.08124	115.46645	P	3794.84560	1897.92644	1265.62005	949.46686	759.77494	32
6	688.33007	344.66867	230.11487	172.83797	138.47183	D	3697.79284	1849.40006	1233.26913	925.20367	740.36439	31
7	802.37299	401.69014	268.12918	201.34871	161.28042	N	3582.76589	1791.88658	1194.92682	896.44693	717.35900	30
8	899.42576	450.21652	300.48010	225.61190	180.69097	P	3468.72296	1734.86512	1156.91251	867.93620	694.55041	29
9	956.44722	478.72725	319.48726	239.86726	192.09527	G	3371.67020	1686.33874	1124.56158	843.67301	675.13986	28
10	1085.48981	543.24855	362.50146	272.12791	217.90378	E	3314.64874	1657.82801	1105.55443	829.41764	663.73557	27
11	1200.51676	600.76202	400.84377	300.88465	240.90917	D	3185.60614	1593.30671	1062.54023	797.15699	637.92705	26
12	1271.55387	636.28057	424.52281	318.64393	255.11660	A	3070.57920	1535.79324	1024.19792	768.40026	614.92166	25
13	1368.60664	684.80696	456.87373	342.90712	274.52715	P	2999.54209	1500.27468	1000.51888	750.64098	600.71424	24
14	1439.64375	720.32551	480.55277	360.66639	288.73457	A	2902.48932	1451.74830	968.16796	726.37779	581.30369	23
15	1568.68634	784.84681	523.56697	392.92704	314.54309	E	2831.45221	1416.22974	944.48892	708.61851	567.09626	22
16	1683.71329	842.36028	561.90928	421.68378	337.54848	D	2702.40962	1351.70845	901.47472	676.35786	541.28774	21
17	1814.75377	907.88052	605.58944	454.44390	363.75658	M	2587.38267	1294.19498	863.13241	647.60113	518.28236	20
18	1885.79088	943.39908	629.26848	472.20318	377.96400	A	2456.34219	1228.67473	819.45225	614.84100	492.07426	19
19	2041.89199	1021.44964	681.30218	511.22846	409.18422	R	2385.30508	1193.15618	795.77321	597.08173	477.86684	18
20	2204.95532	1102.98130	735.65663	551.99429	441.79689	Y	2229.20396	1115.10562	743.73951	558.05645	446.64661	17
21	2368.01865	1184.51296	790.01107	592.76012	474.40955	Y	2066.14064	1033.57396	689.38506	517.29062	414.03395	16
22	2455.05068	1228.02898	819.02174	614.51813	491.81596	S	1903.07731	952.04229	635.03062	476.52478	381.42128	15
23	2526.08779	1263.54754	842.70078	632.27741	506.02338	A	1816.04528	908.52628	606.01994	454.76678	364.01488	14
24	2639.17186	1320.08957	880.39547	660.54842	528.64019	L	1745.00817	873.00772	582.34091	437.00750	349.80745	13
25	2795.27297	1398.14012	932.42917	699.57370	559.86041	R	1631.92410	816.46569	544.64622	408.73648	327.19064	12
26	2932.33188	1466.66958	978.11548	733.83843	587.27220	H	1475.82299	738.41513	492.61251	369.71121	295.97042	11
27	3095.39521	1548.20124	1032.46992	774.60426	619.88486	Y	1338.76408	669.88568	446.92621	335.44648	268.55864	10
28	3208.47927	1604.74327	1070.16461	802.87528	642.50168	I	1175.70075	588.35401	392.57177	294.68065	235.94597	9
29	3322.52220	1661.76474	1108.17892	831.38601	665.31026	N	1062.61669	531.81198	354.87708	266.40963	213.32916	8
30	3435.60626	1718.30677	1145.87361	859.65702	687.92707	L	948.57376	474.79052	316.86277	237.89890	190.52057	7
31	3548.69033	1774.84880	1183.56829	887.92804	710.54389	I	835.48970	418.24849	279.16808	209.62788	167.90376	6
32	3649.73801	1825.37264	1217.25085	913.18996	730.75342	T	722.40563	361.70645	241.47339	181.35687	145.28695	5
33	3805.83912	1903.42320	1269.28456	952.21524	761.97364	R	621.35795	311.18261	207.79084	156.09495	125.07741	4
34	3933.89769	1967.45249	1311.97075	984.22988	787.58536	Q	465.25684	233.13206	155.75713	117.06967	93.85719	3
35	4089.99881	2045.50304	1364.00445	1023.25516	818.80558	R	337.19826	169.10277	113.07094	85.05502	68.24547	2
36						Y‐Amidated	181.09715	91.05222	61.03724	46.02975	37.02525	1

Note: The numbers of the list are the ideal fragment ion masses, and the colored numbers by red and blue were experimentally detected in the MS/MS spectra and they were corresponded to the detected fragment ions in Figure 8.

TABLE 2

MS/MS fragment ion list of NPY‐COOH in Figure 8

#1	b+	b2+	b3+	b4+	b5+	Seq.	y+	y2+	y3+	y4+	y5+	#2
1	164.07061	82.53894	55.36172	41.77311	33.61994	Y						36
2	261.12337	131.06532	87.71264	66.03630	53.03050	P	4108.00937	2054.50832	1370.00797	1027.75780	822.40770	35
3	348.15540	174.58134	116.72332	87.79431	70.43690	S	4010.95661	2005.98194	1337.65705	1003.49461	802.99714	34
4	476.25036	238.62882	159.42164	119.81805	96.05589	K	3923.92458	1962.46593	1308.64638	981.73660	785.59074	33
5	573.30312	287.15520	191.77256	144.08124	115.46645	P	3795.82962	1898.41845	1265.94806	949.71286	759.97174	32
6	688.33007	344.66867	230.11487	172.83797	138.47183	D	3698.77685	1849.89206	1233.59713	925.44967	740.56119	31
7	802.37299	401.69014	268.12918	201.34871	161.28042	N	3583.74991	1792.37859	1195.25482	896.69293	717.55580	30
8	899.42576	450.21652	300.48010	225.61190	180.69097	P	3469.70698	1735.35713	1157.24051	868.18220	694.74722	29
9	956.44722	478.72725	319.48726	239.86726	192.09527	G	3372.65422	1686.83075	1124.88959	843.91901	675.33666	28
10	1085.48981	543.24855	362.50146	272.12791	217.90378	E	3315.63275	1658.32002	1105.88244	829.66365	663.93237	27
11	1200.51676	600.76202	400.84377	300.88465	240.90917	D	3186.59016	1593.79872	1062.86824	797.40300	638.12385	26
12	1271.55387	636.28057	424.52281	318.64393	255.11660	A	3071.56322	1536.28525	1024.52592	768.64626	615.11846	25
13	1368.60664	684.80696	456.87373	342.90712	274.52715	P	3000.52610	1500.76669	1000.84689	750.88698	600.91104	24
14	1439.64375	720.32551	480.55277	360.66639	288.73457	A	2903.47334	1452.24031	968.49596	726.62379	581.50049	23
15	1568.68634	784.84681	523.56697	392.92704	314.54309	E	2832.43623	1416.72175	944.81693	708.86451	567.29307	22
16	1683.71329	842.36028	561.90928	421.68378	337.54848	D	2703.39363	1352.20045	901.80273	676.60387	541.48455	21
17	1814.75377	907.88052	605.58944	454.44390	363.75658	M	2588.36669	1294.68698	863.46041	647.84713	518.47916	20
18	1885.79088	943.39908	629.26848	472.20318	377.96400	A	2457.32621	1229.16674	819.78025	615.08701	492.27106	19
19	2041.89199	1021.44964	681.30218	511.22846	409.18422	R	2386.28909	1193.64818	796.10122	597.32773	478.06364	18
20	2204.95532	1102.98130	735.65663	551.99429	441.79689	Y	2230.18798	1115.59763	744.06751	558.30245	446.84342	17
21	2368.01865	1184.51296	790.01107	592.76012	474.40955	Y	2067.12465	1034.06596	689.71307	517.53662	414.23075	16
22	2455.05068	1228.02898	819.02174	614.51813	491.81596	S	1904.06132	952.53430	635.35863	476.77079	381.61809	15
23	2526.08779	1263.54754	842.70078	632.27741	506.02338	A	1817.02930	909.01829	606.34795	455.01278	364.21168	14
24	2639.17186	1320.08957	880.39547	660.54842	528.64019	L	1745.99218	873.49973	582.66891	437.25350	350.00426	13
25	2795.27297	1398.14012	932.42917	699.57370	559.86041	R	1632.90812	816.95770	544.97422	408.98249	327.38744	12
26	2932.33188	1466.66958	978.11548	733.83843	587.27220	H	1476.80701	738.90714	492.94052	369.95721	296.16722	11
27	3095.39521	1548.20124	1032.46992	774.60426	619.88486	Y	1339.74810	670.37769	447.25422	335.69248	268.75544	10
28	3208.47927	1604.74327	1070.16461	802.87528	642.50168	I	1176.68477	588.84602	392.89977	294.92665	236.14277	9
29	3322.52220	1661.76474	1108.17892	831.38601	665.31026	N	1063.60070	532.30399	355.20509	266.65563	213.52596	8
30	3435.60626	1718.30677	1145.87361	859.65702	687.92707	L	949.55778	475.28253	317.19078	238.14490	190.71738	7
31	3548.69033	1774.84880	1183.56829	887.92804	710.54389	I	836.47371	418.74049	279.49609	209.87389	168.10056	6
32	3649.73801	1825.37264	1217.25085	913.18996	730.75342	T	723.38965	362.19846	241.80140	181.60287	145.48375	5
33	3805.83912	1903.42320	1269.28456	952.21524	761.97364	R	622.34197	311.67462	208.11884	156.34095	125.27422	4
34	3933.89769	1967.45249	1311.97075	984.22988	787.58536	Q	466.24086	233.62407	156.08514	117.31567	94.05399	3
35	4089.99881	2045.50304	1364.00445	1023.25516	818.80558	R	338.18228	169.59478	113.39894	85.30103	68.44228	2
36						Y	182.08117	91.54422	61.36524	46.27575	37.22206	1

Note: The numbers of the list are the ideal fragment ion masses, and the colored numbers by red and blue were experimentally detected in the MS/MS spectra and they were corresponded to the detected fragment ions in Figure 8.

MS/MS analyses of NPY‐COOH

Figure 8 showed the MS/MS spectra of NPY‐amide and NPY‐COOH from the [M + 5H]5+ at m/z 854.8258 and 855.0231, respectively. The observed fragment ions were summarized in Tables 1 and 2. These MS/MS data indicated that the peaks at 49.5 and 50.4 min in nanoLC‐orbitrap MS in Figure 6 were identified to NPY‐amide and NPY‐COOH.

In the MS/MS spectra of amidated small peptides within 1 kDa, the indicating fragment of NH2 loss was observed. However, the indicating fragments were not observed in the MS/MS spectra of NPY‐COOH because the structural difference between NPY‐amide and NPY‐COOH was too small in their whole molecules to progress the specific fragmentation.

The presence of NPY‐COOH in brain

Non‐amidated NPY‐COOH were identified using nanoLC orbitrap‐MS, MS/MS spectra, indicating that endogenous NPY‐COOH is surely produced in brain tissue. The presence of NPY‐COOH in brain suggested that cathepsin L concerned with the NPY maturation process.

NPY‐amide predominated in mouse brain, and the amount of NPY‐COOH was 0.05% of NPY‐amide. A peptidase of cathepsin L was reported to produce about equally NPY‐COOH and NPY‐Gly of the precursor substitute of NPY‐amide. NPY‐COOH and NPY‐amide could be generated equally in mouse brain; however, the amount of NPY‐COOH was very low. One of hypothesis is that another peptidase was expressed and digested NPY‐COOH without the C‐terminal amidation. Consequently, these mechanisms of NPY maturation processes should be revealed to elucidate the regulation of NPY activities and their functions.

CONCLUSION

Many neuropeptides are modified at C‐terminal amidation such as NPY. Interestingly, non‐amidated NPY‐COOH also exists in mouse brain. It was the first report that endogenous non‐amidated NPY‐COOH existence in brain was directly proved by the detection of the molecule with high resolution nanoLC‐orbitrap MS. It was essential to separate between NPY‐amide and NPY‐COOH. The C‐terminal amidation affects in the retention times of a reversed phase column LC and silica based gel‐filtration chromatography. In this study, silica based gel‐filtration chromatography was very useful to separate amide/non‐amide NPYs. This idea can be applied to the identification of the other neuropeptides with or without C‐terminal amidation, and the point of view in amidation/non‐amidation of neuropeptides was focused on in the LC‐MS system. Addition to that, the high‐resolution MS analyses were essential to distinguish and identify amide/non‐amidated peptides.