Amine modification of digested peptide at C-terminal end during protein digestion by protease

Article abstract of DOI:10.1248/bpb.30.1838

We recently reported that C-terminal polyamine modification occurs when proteins are digested with trypsin in the presence of polyamine [Biochem. Biophys. Res. Commun., 356, 159-162 (2007)]. In the present study, the characteristics of this C-terminal modification in the presence of protease and amine were investigated. When hemoglobin (HB) was digested with trypsin in the presence of N-(2-pyridyl)-1,4-diaminobutane (Py4), formation of the modified peptide was dependent on time and on HB or Py4 concentration. When synthetic peptide was treated with trypsin in the presence of Py4, ca. 0.1% of the peptide was modified with Py4. When HB or cytochrome C was treated with a range of serine proteases in the presence of various amines (Py4, N-(2-pyridyl)-1,3- diaminopropane, tranexamic acid, isonicotinic acid hydrazide and ampicillin), the modified peptide was detected in all cases tested, thus suggesting that amine modification widely accompanies digestion by proteases.

Full text of DOI:10.1248/bpb.30.1838

1838
Biol. Pharm. Bull. 30(10) 1838—1843 (2007)
Vol. 30, No. 10
Amine Modification of Digested Peptide at C-Terminal End during
Protein Digestion by Protease
a
b
b
a
,a,b
*
Toshiyuki ITO, Yoshiaki SUGITA, Koichi TAKAO, Yoshihiko IKEGUCHI, and Akira SHIRAHATA
a Department of Biochemistry, Faculty of Pharmaceutical Sciences, Josai University; and b Department of Human Nutrition
and Clinical Dietetics, Faculty of Pharmaceutical Sciences, Josai University; 1–1 Keyakidai, Sakado, Saitama 350–0295,
Japan. Received May 22, 2007; accepted August 5, 2007; published online August 7, 2007
We recently reported that C-terminal polyamine modification occurs when proteins are digested with
trypsin in the presence of polyamine [Biochem. Biophys. Res. Commun., 356, 159—162 (2007)]. In the present
study, the characteristics of this C-terminal modification in the presence of protease and amine were investigated.
When hemoglobin (HB) was digested with trypsin in the presence of N-(2-pyridyl)-1,4-diaminobutane (Py4), for-
mation of the modified peptide was dependent on time and on HB or Py4 concentration. When synthetic peptide
was treated with trypsin in the presence of Py4, ca. 0.1% of the peptide was modified with Py4. When HB or cy-
tochrome C was treated with a range of serine proteases in the presence of various amines (Py4, N-(2-pyridyl)-
1,3-diaminopropane, tranexamic acid, isonicotinic acid hydrazide and ampicillin), the modified peptide was de-
tected in all cases tested, thus suggesting that amine modification widely accompanies digestion by proteases.
Key words amine; protease; modification; C-terminus; amino group; digestion
Transglutaminase (TGase) is a typical enzyme that cat- more, the possibility of in vitro production of peptides modi-
alyzes the post-translational cross-linking of proteins by acyl fied by a variety of alkylamines, including drugs, was evalu-
transfer in mammalian cells. TGases use a cysteine protease- ated.
like catalytic mechanism to release ammonia from protein-
bound glutamine by forming a g-glutamyl-enzyme thioester MATERIALS AND METHODS
intermediate, which subsequently transfers the g-glutamyl
moiety to another amine (transamidation), an aliphatic alco-
Materials Bovine HB, ampicillin (Amp), 1-octaneslu-
hol (esterification), or water (glutamine hydrolysis; deamida- fonic acid sodium salt and high performance liquid chro-
tion).1) In vitro, numerous amines, diamines, polyamines, and matography (HPLC) grade trifluoroacetic acid were obtained
alcohols are capable of interacting with the protein-acyl-en- from Wako Pure Chemical Industries (Osaka, Japan); isoni-
zyme intermediate.1) Therefore, all enzymatic reactions via cotinic acid hydrazide (INH), tranexamic acid (TA) and 4-
protein-acyl-enzyme intermediates have the potential for pro- bromobenzylamine (BrBA) hydrochloride were obtained
ducing amine-modified peptides or proteins in enzymatic from Tokyo Chemical Industry Co. (Tokyo, Japan); chy-
systems in the presence of amino compounds.
motrypsin (sequencing grade) was obtained from Roche
Serine proteases are known to form protein-acyl-enzyme (Mannheim, Germany); modified trypsin (sequencing grade)
intermediates.2) These proteases hydrolyze proteins in two was obtained from Promega (Madison, WI, U.S.A.); 2-[4-(2-
steps. The first step is the acylation of the serine residue, hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES),
which results in the liberation of the C-terminal peptide. The elastase, horse cytochrome C (CytC), angiotensin II, bovine
second step is hydrolysis of the acyl-enzyme intermediate, insulin b chain and a-cyano-4-hydroxycinnamic acid were
which results in elimination of the N-terminal peptide. We obtained from SIGMA (St. Louis, MO, U.S.A.); synthetic
recently reported that C-terminal polyamine modification of peptides (SLIGKV-OH and SFLLRN-NH2) were obtained
digested peptides occurs when hemoglobin (HB) is digested from Bachem (Bubendorf, Switzerland); and HPLC grade
with trypsin in the presence of polyamine.3) This suggests acetonitrile was purchased from KANTO KAGAKU (Tokyo,
that removal of the C-terminal peptide in the hydrolysis reac- Japan).
tion produces sufficient space in the vicinity of the protein- N-(2-Pyridyl)-diaminoalkanes were synthesized in our lab-
acyl-trypsin intermediate to allow an alkylamine with an un- oratory.
shared electron pair, such as polyamine, to attack the protein-
For N-(2-pyridyl)-1,3-diaminopropane (Py3), a mixture of
acyl-enzyme intermediate instead of a hydroxyl ion. Such 2-bromopyridine (5.0 ml, 51.6 mmol) and 1,3-diamino-
modification could occur widely with other amines including propane (21 ml, 258 mmol) were stirred under a nitrogen
drugs having an amino group, as well as polyamines, during atmosphere at 130 °C for 19 h. Then, 4 mol/l ammonia solu-
protease digestion. If biological amine- or drug-modified tion (15 ml) was added to the mixture, and the product was
peptides were produced in the intestinal tract, this modifica- extracted with chloroform (6ꢀ30 ml). The organic layer was
tion may be related to unknown nutrient–nutrient interactions dried (MgSO4), and the solvent was evaporated. The oil
or drug–nutrient interactions.
residue was distilled under reduced pressure (160—165 °C,
In the present study, in order to investigate the characteris- 12 mmHg) to give Py3. After adding ethanol (10 ml) and
tics of amine modification, the production of modified pep- concentrated hydrochloric acid (6.2 ml), the solvent was
tides was examined using a model alkylamine, N-(2-pyridyl)- evaporated to give Py3 hydrochloride. The white crystals ob-
1,4-diaminobutane (Py4), with intestinal proteases (trypsin, tained were recrystallized with ethanol. White crystals, 47%
chymotrypsin and elastase) and synthetic peptides. Further- yield; 1H-NMR (D2O) d 1.90—1.97 (2H, m, H-2), 2.99 (2H,
∗ To whom correspondence should be addressed. e-mail: ashiraha@josai.ac.jp
© 2007 Pharmaceutical Society of Japan

October 2007
1839
t, Jꢁ7.8 Hz, H-3), 3.34 (2H, t, Jꢁ6.8 Hz, H-1), 6.89 (1H, d, (MALDI-TOF MS).
Jꢁ9.1 Hz, PyH-3), 6.76 (1H, ddd, Jꢁ7.0, 6.4, 0.9 Hz, PyH-
Analysis of Py4 in Py4-Modified Peptide Py4-modified
5), 7.65 (1H, dd, Jꢁ6.4, 1.5 Hz, PyH-6), 7.76 (1H, ddd, synthetic peptides were separated and collected by HPLC.
Jꢁ9.1, 7.0, 1.5 Hz, PyH-4). FAB-MS m/z: 152 (Calcd for The collected fractions were lyophilized, and then dissolved
C8H14N3: 152). Anal. Calcd for C8H15N3Cl2: C, 42.87; H, in 100 ml of water. After the solution was diluted with the
6.75; N, 18.75. Found: C, 42.87; H, 6.82; N, 18.76.
same volume of conc. HCl (final 6 M HCl), the mixture was
For N-(2-pyridyl)-1,4-diaminobutane (Py4), a mixture of heated at 110 °C for 24 h under nitrogen. The reaction mix-
2-bromopyridine (20 ml, 0.21 mol) and 1,4-diaminobutane ture was lyophilized, and then dissolved in 100 ml of 30%
(106 ml, 1.06 mol) were stirred under a nitrogen atmosphere acetonitrile containing 0.1% trifluoroacetic acid.
at 120 °C for 10 h. Then, 4 mol/l ammonia solution (200 ml)
Py4 was analyzed by HPLC on a packed C18 column
was added to the mixture and the product was extracted (TOSOH ODS 120T, 4.6ꢀ250 mm). Flow rate was 1.0
with chloroform (4ꢀ600 ml). The organic layer was dried ml/min and column temperature was 40 °C. Fluorescence
(MgSO4), and the solvent was evaporated. The oil residue was monitored at Ex 310 nm and Em 390 nm. The sample
was purified on a chromatography column (silica gel) using a injection volume was 10 ml. Solvent for HPLC was 25%
mixture of chloroform–methanol (10 : 1) to give Py4. After acetonitrile containing 0.1% trifluoroacetic acid and 8 mM
adding ethanol (20 ml) and concentrated hydrochloric acid 1-octaneslufonic acid. Py4 was eluted at 9.3 min.
(26.7 ml), the solvent was evaporated to give Py4 hydrochlo-
Mass Spectrometry All mass spectra were obtained in
ride. The white crystals obtained were recrystallized with the reflectron mode using an AXIMA-CFR MALDI-TOF
ethanol. White crystals, 59% yield; 1H-NMR (D2O) d 1.56— (Shimadzu/Kratos, Manchester, U.K.) equipped with a
1.68 (4H, m, H-2, H-3), 2.90 (2H, t, Jꢁ7.3 Hz, H-4), 3.26 337 nm pulsed nitrogen laser. External mass calibration was
(2H, t, Jꢁ6.4 Hz, H-1), 6.71 (1H, ddd, Jꢁ7.0, 6.4, 0.9 Hz, performed with a mixture of peptide standards (angiotensin
PyH-5), 6.86 (1H, d, Jꢁ9.1 Hz, PyH-3), 7.61 (1H, dd, Jꢁ6.4, II, m/z 1046.54; insulin b chain, m/z 3495.65). The matrix,
1.5 Hz, PyH-6), 7.72 (1H, ddd, Jꢁ9.1, 7.0, 1.5 Hz, PyH-4). a-cyano-4-hydroxycinnamic acid, was prepared at a concen-
FAB-MS m/z: 166 (Calcd for C9H16N3: 166). Anal. Calcd for tration of 10 mg/ml in 2 : 3 acetonitrile/0.1% trifluoroacetic
C9H17N3Cl2: C, 45.39; H, 7.19; N, 17.64. Found: C, 45.23; H, acid. The sample (1.0 ml) was spotted onto the sample plate,
7.28; N, 17.54.
1.0 ml of matrix was added, and the spot was allowed to dry
Enzymatic Digestion of Proteins or Peptides HB or at room temperature. Peptide sequences were determined by
CytC digestion with protease (trypsin, chymotrypsin or elas- the post-source decay (PSD) method.
tase) was performed at a concentration of 1.0 mg/ml in
100 mM HEPES buffer (pH 7.5) in the presence or absence RESULTS
of 50 mM alkylamines. Digestion of synthetic peptides
(SLIGKV-OH or SFLLRN-NH2) was performed at a concen-
Detection of Py4-Modified Peptide during Protease Di-
tration of 12.5 mM. The mass ratio of HB, CytC, SLIGKV- gestion Although the polyamine modification of peptides
OH or SFLLRN-NH2 to proteases was 1 : 20, 1 : 20, 1 : 60 or during tryptic digestion was previously confirmed by mass
1 : 75, respectively. Reaction mixtures were incubated at spectrometry,3) the modification was not fully investigated
37 °C for 16 h. Some reactions were performed in a mixture because polyamine modified peptides were hard to be distin-
of 100 mM HEPES buffer (pH 7.5) and methanol.
guished by HPLC with a UV detector. Therefore, it was nec-
Fractionation of Enzyme Digestion Products Protease essary to select a model alkylamine for high sensitivity de-
digestion products were fractionated by HPLC on a packed tection of modified peptides by HPLC analysis. In the pres-
C18 column (TOSOH ODS 120T, 4.6ꢀ250 mm). Flow rate ent study, Py4 (Fig. 1) was synthesized and used as a model
was 1.0 ml/min and column temperature was 35 °C. UV ab- alkylamine because of its resemblance to polyamine and its
sorption was monitored at 220 nm. Fluorescence was moni- high fluorescence, which allows detection without any signif-
tored at Ex 310 nm and Em 390 nm.
icant effect on sensitivity in MALDI-TOF MS analysis (data
For fractionation of digested HB or CytC, solvents for not shown). The detection limit of Py4 on the HPLC with
HPLC were: A, 5% acetonitrile containing 0.1% trifluoro- fluorescence detection was about 0.5 pmol (S/Nꢁ3).
acetic acid; B, 80% acetonitrile containing 0.1% trifluoro-
acetic acid. The column was equilibrated with 100% A. The
HPLC protocol consisted of 100% A for 10 min, followed by
a gradient of 0—100% B over 90 min.
For fractionation of digested synthetic peptide (SLIGKV-
OH- or SFLLRN-NH2), solvents for HPLC were: A, 5% ace-
tonitrile containing 0.1% trifluoroacetic acid; B, 25% ace-
tonitrile containing 0.1% trifluoroacetic acid. The column
was equilibrated with 100% A. The HPLC protocol consisted
of 100% A for 20 min, a gradient of 0—20% B over 5 min,
followed by a gradient of 20—100% B over 60 min.
Sample injection volume was 10 ml. Protein digests
fractions were collected every 30 s. Each fraction was
lyophilized, and then dissolved in 20 ml of 2 : 3 acetoni-
trile/0.1% trifluoroacetic acid for analysis of matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry Fig. 1. Structure of Tested Alkylamines

1840
Vol. 30, No. 10
In order to confirm the modification of peptide with Py4, merous peaks were detected for those in the presence of Py4
HB was used as a model protein, and was treated with (Fig. 2D).
trypsin in the presence of 50 mM Py4. The products were sep-
When each fraction was analyzed by MALDI-TOF MS
arated by HPLC and collected every 30 s. On the chro- and PSD, it was found that eight peptides were modified with
matogram, digested peptides were eluted between 30 and Py4 at the C-terminus, as shown by the arrows in Fig. 2D and
55 min, while intact a and b chains of HB were eluted at 68 Table 1. The PSD spectra of a modified peptide eluted at
and 72 min, respectively, as shown in Figs. 2A and B. Al- 37 min (c in Fig. 2D) is shown in Fig. 3. Based on the parent
though the exact mechanisms are unclear, trypsin digestion ion and the fragment ions, the sequence of the 1677 Da pep-
was slightly hindered by the added Py4. Some fluorescence tide was confirmed to be VGGHAAEYGAEALER-Py4
peaks were detected on the chromatograms for samples (17—31 of HB a chain), which corresponds to a portion of
treated with trypsin in the absence of Py4 (Fig. 2C), but nu- the HB sequence (Swiss-Prot Accession No. P01966 and
P02070). The modified peptides were not always detected by
MS (mass spectrometry) in every fluorescence peaks fraction
in HPLC. Ionization of the modified peptide in MS may be
repressed because of co-presence of easily-ionized non-mod-
Table 1. Detection of C-Terminal Py4-Modified Peptide by MALDI-TOF
MS Analysis and PSD
Monoisotopic
Retention
time
[min]
MHꢂ at m/z
Fragment
Sequence
(Calculated
monoisotopic mass)
34.5
36.0
37.0
38.5
39.0
39.5
43.0
44.0
b 19—29
b 95—103
a 17—31
b 132—143
a 91—99
b 8—18
VDEVGGEALGR-Py4
LHVDPENFK-Py4
VGGHAAEYGAEALER-Py4
VVAGVANALAHR-Py4
LRVDPVNFK-Py4
1248.6 (1248.8)
1245.5 (1244.8)
1676.4 (1676.9)
1324.5 (1324.9)
1234.3 (1234.8)
1324.3 (1324.9)
2093.7 (2094.2)
1569.9 (1569.9)
Fig. 2. HPLC Chromatograms of Tryptic Digestion Products of HB in the
Presence or Absence of Py4
Tryptic digestion products of HB in the absence (A, C) or in the presence (B, D) of
Py4 were analyzed by HPLC with UV absorbance (A, B) and fluorescence detection
(C, D) on a packed C18 column (TOSOH ODS 120T, 4.6ꢀ250 mm). UV absorption
was monitored at 220 nm. Fluorescence detection was monitored at Ex 310 nm and Em
390 nm. Eluates were collected every 30 s for mass analysis. White and gray arrows
(A, B) indicate the retention time of the a and b chains of HB, respectively. Black
arrow of a, b, c, d, e, f, g or h indicates the retention time of modified peptide of b19—
29, b95—103, a17—31, b132—143, a91—99, b8—18, b65—81 or b120—131, re-
spectively, identified by MS as shown in Table 1.
AAVTAFWGKVK-Py4
b 65—81 KVLDSFSNGMKHLDDLK-Py4
EFTPVLQADFQK-Py4
b 120—131
HB digestion with trypsin was performed in the presence of 50 mM Py4. Tryptic di-
gestion products were separated and fractionated every 30 s by HPLC, and fractions in-
cluding fluorescence peaks on HPLC chromatograms (Fig. 2D) were analyzed by
MALDI-TOF MS and PSD.
Fig. 3. PSD Spectrum of Protonated C-Terminus of Py4-Modified Peptide (1677 Da)
When the HB a chain Py4-modified peptide spanning residues 17 to 31 (VGGHAAEYGAEALER-Py4, 1677 Da) was used as a parent ion for PSD, sequential fragment b ions
(4—12, 14) and y ions (1—11, 14, 15) were observed. Calculated mass (Da) of b ions are 100.1 (b1), 157.2 (b2), 214.2 (b3), 351.4 (b4), 422.5 (b5), 493.5 (b6), 622.7 (b7), 785.8
(b8), 842.9 (b9), 914.0 (b10), 1043.0 (b11), 1114.1 (b12), 1227.3 (b13), 1356.4 (b14) and 1512.6 (b15). Calculated mass (Da) of y ions are 166.2 (y1), 322.4 (y2), 451.5 (y3),
564.7 (y4), 635.8 (y5), 764.9 (y6), 836.0 (y7), 893.0 (y8), 1056.2 (y9), 1185.3 (y10), 1256.4 (y11), 1327.5 (y12), 1464.6 (y13), 1521.7 (y14) and 1578.7 (y15). “ꢀ200” or “ꢀ50”
indicates the magnification of ion intensity.

October 2007
1841
ified peptide.
cation was enzymatic.
Alkylamination in Mixed Methanol–Water Solvent
Py4-modified peptides were also identified among the di-
gestion products of HB and CytC (Swiss-Prot Accession No. The effects of methanol on formation of Py4-modified pep-
P01966, P02070, P00004) treated with trypsin, chymotrypsin tide were examined. When HB was treated with trypsin in
or elastase in the presence of 50 mM Py4, as shown in Table 17, 34 or 50% methanol in the presence of Py4, the peak area
2. All modified peptides were modified at the C-terminus of the modified peptide (17—31 of HB a chain) on the chro-
(data not shown).
matogram was about 3.2ꢃ0.9, 14.0ꢃ0.9 or 18.6ꢃ1.1 times
Dose and Time Dependency of Alkylamination The greater than that of the control sample (0% methanol), re-
dependency of reaction time and dose for HB or Py4 was ex- spectively (nꢁ3). These results suggest that methanol en-
amined by monitoring the formation of Py4-modified pep-
tides (17—31 of HB a chain), which could be detected from
the early reaction stages. When degradation of HB was mon-
itored by detecting the HB a chain peak, the increase in for-
mation of Py4-modified peptide was accompanied by the
degradation of HB (Fig. 4). The formation of modified pep-
tide was dependent on HB or Py4 concentration and was sat-
urated at 25 mM Py4 (Fig. 5), thus suggesting that the modifi-
Fig. 5. Effects of HB or Py4 Concentration on Py4 Modification in
Trypsin Digestion
Fig. 4. Time Course of Py4 Modification Reaction in Trypsin Digestion
and Degradation of HB
Tryptic digestion products of HB in the presence of Py4 were analyzed by HPLC.
Formation of Py4-modified peptide (17—31 of HB a chain, VGGHAAEYGAEALER-
Py4) in the presence of 1 mg/ml HB (A) or 50 mM Py4 (B) was monitored in order to
determine the effects Py4-concentration (A) and HB-concentration (B), respectively
(nꢁ3).
Tryptic digestion products of HB in the presence of Py4 were analyzed by HPLC.
Relative fluorescence intensity of the peak area of Py4-modified peptide (17—31 of HB
a chain, VGGHAAEYGAEALER-Py4) and absorbance unit of peak area of HB a
chain are shown (nꢁ3).
Table 2. Detection of C-Terminal Py4-Modified Peptide by MALDI-TOF MS Analysis and PSD
Monoisotopic MHꢂ at m/z
(Calculated monoisotopic mass)
Protease
Trypsin
Protein
CytC
Fragment
Sequence
39—53
40—53
KTGQAPGFTYTDANK-Py4
TGQAPGFTYTDANK-Py4
1618.2 (1617.9)
1746.7 (1746.0)
Chymotrypsin
HB
a 1—24
a 3—14
VLSAADKGNVKAAWGKVGGHAAEY-Py4
SAADKGNVKAAW-Py4
GKVGGHAAEY-Py4
SFPTTKTY-Py4
2545.0 (2545.4)
1364.6 (1364.8)
1135.6 (1135.7)
1091.6 (1091.7)
944.5 (944.6)
a 15—24
a 35—42
a 67—73
a 92—98
a 110—117
b 15—27
b 32—36
b 96—102
b 122—129
1—10
TKAVEHL-Py4
RVDPVNF-Py4
ASHLPSDF-Py4
GKVKVDEVGGEAL-Py4
VVYPW-Py4
HVDPENF-Py4
TPVLQADF-Py4
Ac-GDVEKGKKIF-Py4
993.5 (993.6)
1020.5 (1020.6)
1448.0 (1447.9)
810.4 (810.5)
1004.4 (1004.6)
1037.8 (1037.7)
1310.1 (1309.9)
CytC
HB
Elastase
a 35—48
b 31—47
b 126—137
4—15
SFPTTKTYFPHFDL-Py4
LVVYPWTQRFFESFGDL-Py4
QADFQKVVAGVA-Py4
EKGKKIFVQKCA-Py4
KKIFVQKCA-Py4
1847.9 (1848.1)
2251.1 (2251.3)
1379.3 (1379.9)
1525.9 (1526.0)
1211.8 (1211.8)
1534.8 (1535.8)
925.3 (925.6)
CytC
7—15
12—24
35—41
QKCAQCHTVEKGG-Py4
LFGRKTG-Py4
85—98
IKKKTEREDLIAYL-Py4
1867.2 (1867.2)
HB or CytC digestion with trypsin, chymotrypsin or elastase was performed in the presence of 50 mM Py4. Proteolytic digests were separated and fractionated every 30 s by
HPLC, and fractions including fluorescence peaks on HPLC chromatograms were analyzed by MALDI-TOF MS and PSD.

1842
Vol. 30, No. 10
hances the reactivity of alkylamine to the protein-acyl-en- of Py4-modified peptide, synthetic peptide (SLIGKV-OH or
zyme intermediate. SFLLRN-NH2) was treated with trypsin in the presence of
Evaluation of Formation Rate of Py4-Modified Peptide Py4. The products (SLIGK-OH, SLIGK-Py4, SFLLR-OH or
in Protein Digestion In order to analyze the formation rate SFLLR-Py4) were separated and collected by HPLC and
identified by PSD. After hydrolysis of the collected SLIGK-
Table 3. Detection of Alkylamine-Modified Peptide by MALDI-TOF MS
Analysis and PSD
Py4 or SFLLR-Py4 fraction with 6 M HCl, the recovered Py4
was analyzed by HPLC to calculate the amount of modified
peptide, and the formation rate was calculated by dividing
the amount of modified peptide by the initial amount of syn-
thetic peptide used. The formation rates of SLIGK-Py4 and
SFLLR-Py4 were 0.07ꢃ0.01% and 0.09ꢃ0.01%, respec-
tively (nꢁ3).
Modification with Various Alkylamines Peptide modi-
fication was examined with a range of alkylamines. HB was
treated with trypsin in the presence of 50 mM of Py3, BrBA,
INH, TA or Amp (Fig. 1) for 16 h. In all cases, alkylamine-
modified peptides were detected by MALDI-TOF MS analy-
sis (Table 3) and identified by PSD. In case of Amp, HB was
treated with trypsin in 25% methanol in order to increase the
formation of the modified peptide. A PSD spectrum from a
sample digested in the presence of Amp is shown in Fig. 6.
Average MHꢂ
at m/z
Alkylamine
Py3
Fragment
Sequence
(Calculated average
mass)
a 12—16
a 17—31
b 76—81
b 1—7
b 17—29
a 91—99
a 17—31
a 41—56
b 132—143
b 30—39
a 8—16
a 91—99
a 128—139
a 17—31
a 1—16
b 82—94
b 66—81
a 1—7
AAWGK-Py3
666.4 (665.8)
1664.1 (1663.8)
874.8 (874.0)
VGGHAAEYGAEALER-Py3
HLDDLK-Py3
MLTAEEK-Py3
954.0 (955.2)
VKVDEVGGEALGR-Py3
LRVDPVNFK-BrBA
1462.7 (1462.7)
1256.7 (1255.6)
1698.8 (1697.7)
2003.5 (2001.9)
1346.8 (1345.7)
1443.9 (1442.7)
1049.3 (1050.2)
1207.1 (1207.4)
1398.8 (1399.6)
1649.1 (1649.8)
1735.8 (1735.0)
1511.8 (1511.7)
1939.4 (1939.2)
842.9 (843.0)
BrBA
INH
VGGHAAEYGAEALER-BrBA
TYFPHFDLSHGSAQVK-BrBA
VVAGVANALAHR-BrBA
LLVVYPWTQR-BrBA
GNVKAAWGK-INH
LRVDPVNFK-INH
FLANVSTVLTSK-INH
VGGHAAEYGAEALER-INH
VLSAADKGNVKAAWGK-INH
GTFAALSELHCDK-INH
VLDSFSNGMKHLDDLK-INH
VLSAADK-TA
DISCUSSION
Our results clearly demonstrated that the amine modifica-
tion occurring in the presence of protease and alkylamine is
enzymatic, and that a variety of alkylamines, including Amp,
are able to form alkylamide bonds at the C-terminus of ser-
ine protease-digested peptides. In addition to intestinal serine
protease, other serine or cysteine proteases involved in pro-
tein degradation systems, such as plasmin4) or calpain,5) may
also produce modified peptides in the presence of biogenic
amines. Furthermore, lipases, serine hydrolases, catalyze the
hydrolysis of esters by the same double-displacement mecha-
nism via an acyl-enzyme intermediate, as observed with ser-
ine proteases.6) Therefore, the present results suggest that
TA
a 93—99
a 32—40
a 17—31
a 41—56
b 17—29
b 1—16
VDPVNFK-TA
958.0 (958.1)
MFLSFPTTK-TA
1212.4 (1211.5)
1669.5 (1669.8)
1973.2 (1974.2)
1467.9 (1468.7)
1893.1 (1893.3)
1862.5 (1862.0)
2969.6 (2969.4)
VGGHAAEYGAEALER-TA
TYFPHFDLSHGSAQVK-TA
VKVDEVGGEALGR-TA
MLTAEEKAAVTAFWGK-TA
VGGHAAEYGAEALER-Amp
AVEHLDDLPGALSELSDLHAHKLR-Amp
Amp
a 17—31
a 69—92
HB digestion with trypsin was performed in the presence of 50 mM of Py3, BrBA,
INH, TA or Amp. The proteolytic digests were separated and fractionated every 30 s by
HPLC, and every fraction was analyzed by MALDI-TOF MS and PSD.
Fig. 6. PSD Spectrum of Protonated C-Terminus of Amp-Modified Peptide (1862 Da)
When the HB a chain Amp-modified peptide spanning residues 17 to 31 (VGGHAAEYGAEALER-Amp, 1862 Da) was used as a parent ion for PSD, sequential fragment b ions
(4—14) and y ions (5, 8, 9, 15) were observed. Calculated mass (Da) of b ions are 100.1 (b1), 157.2 (b2), 214.2 (b3), 351.4 (b4), 422.5 (b5), 493.5 (b6), 622.7 (b7), 785.8 (b8),
842.9 (b9), 914.0 (b10), 1043.1 (b11), 1114.1 (b12), 1227.3 (b13), 1356.4 (b14) and 1512.6 (b15). Calculated mass (Da) of y ions are 351.4 (y1), 507.6 (y2), 636.7 (y3), 749.9
(y4), 821.0 (y5), 950.1 (y6), 1021.2 (y7), 1078.2 (y8), 1241.4 (y9), 1370.5 (y10), 1441.6 (y11), 1512.7 (y12), 1649.8 (y13), 1706.8 (y14) and 1763.9 (y15). “ꢀ5” indicates the
magnification of ion intensity.

October 2007
1843
these modifications might occur during the hydrolysis of form modified peptides at high levels due to the higher drug
amides or ester bonds in the presence of amines in a variety concentrations in the intestine. Drug-modified peptides may
of biological systems.
thus be related to unknown and/or undesired pharmacologi-
The formation rate of modified products from synthetic cal effects.
peptides in the presence of 50 mM Py4 with trypsin was
below ca. 0.1%. The rate was not substantial in vitro, but
Acknowledgements The authors are grateful to Profes-
might be significant for protein digestion in the gastrointesti- sor S. Matsufuji (Jikei University, Tokyo, Japan) for access to
nal tract. As shown in Fig. 5A, the formation of modified the AXIMA-CFR MALDI-TOF. The authors would also like
peptides from HB in the presence of 1 mM Py4 was one tenth to thank Ms. M. Wada, Ms. M. Yashiki, Mr. S. Miyatake, Mr.
that of 50 mM Py4, thus suggesting that the formation rate is M. Kusakabe, Mr. K. Yamazaki, Mr. Y. Tezuka and Ms. I.
significant even in the presence of 1 mM of alkylamine. Con- Ishii for helpful discussions. This work was supported by a
sidering that the daily protein intake for an adult is about Grant-in-Aid for Scientific Research (C) (2) (No. 16590033)
60 g, or about 0.5 mol of amino acid (0.8 g/kg body weight),7) from the Japan Society for the Promotion of Science.
the total modified peptide or amino acid formed in the
gastrointestinal tract may reach the mmol/d level. As poly-
amine concentrations in the intestine reach almost mM levels
shortly after a meal,8) polyamine modification of peptides
would occur at significant levels every day. Therefore, the
polyamine-modified peptides formed would have several
novel effects on the body and such amine modification could
be involved in the effects of certain foods on disease state.
Bulky alkylamines, such as Amp, were also able to react
with acyl-enzyme intermediates during protein digestion.
Therefore, drugs having primary amino groups that are ad-
ministered at high doses may become a substrate for pro-
tease-mediated alkylamination. Drugs administered as en-
teric-coated preparations would be particularly expected to
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