Abstract
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.
Keywords: amidation, brain, nanoLC, neuropeptide, orbitrap‐MS
1. 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), 1 this form being linked to basic biological functions such as the regulation of feeding behavior, the control of blood pressure, and so on. 1 , 2 , 3 , 4 , 5 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. 6 Then, the NPY‐Gly is amidated by peptidylglycine alpha‐hydroxylating monooxygenase (PAM) to NPY‐amide (Figure 2). 7 However, another proteolytic processing of proNPY was reported. Funkelstein reported that cathepsin L participates in the production of NPY directly. 8 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). 8 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, 9 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, 10 , 11 , 12 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. 13 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. 14 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. 15 , 16 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.
2. MATERIALS AND METHODS
2.1. 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.
2.2. 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
2.3. 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)
2.4. 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.
2.5. 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.
3. RESULTS AND DISCUSSION
3.1. 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. 17
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.
3.2. 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. 18
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.
3.3. 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)
3.4. 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.
3.5. 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. 19 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.
3.6. 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. 8
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. 8 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.
4. 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.
ACKNOWLEDGEMENTS
We thank for useful technical advises of purification of neuropeptides to Prof. Naoto Minamino and Prof. Kenji Kangawa in National Cerebral and Cardiovascular Center, Osaka, Japan.
Yamagaki T, Kimura Y, Yamazaki T. Amidation/non‐amidation top‐down analysis of endogenous neuropeptide Y in brain tissue by nano flow liquid chromatography orbitrap Fourier transform mass spectrometry. J Mass Spectrom. 2021;56:e4716. 10.1002/jms.4716
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