Anal. Chem. 1995,67,4276-4282
Development of an Efficient Amino Acid Sequencing Method Using Fluorescent Edman Reagent 7-[(N,N-Dimethylamino)sulfonyl]-2,1,3benzoxadiazol-4-yllsothiocyanate Hirokazu Matsunaga, Tomofumi Santa, Kenichi Hagiwara, Hiroshi Homma, and Karuhiro Imai* Faculty of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 1 13, Japan Sonoko Uzu Branch Hospital Pharmacy, University of Tokyo, 3-28-6 Mejirodai, Bunkyo-ku, Tokyo 112, Japan Kenichiro Nakashima and Shuzo Akyama
-
School of Pharmaceutical Sciences, Nagasaki University, 1 14 Bunkyo-Machi, Nagasaki 852, Japan
In this paper, a new method is described for N-terminal isothiocyanate (FITC) ,5 4{[ (N-(butoxycarbonyl)aminolmethyl}phenyl isothiocyanate (BAMPITC),6 4(N,N-dimethylamino)-lamino acid sequencing of peptides using the fluorescent reagent 7-~(N~-dimethylamino)sulfonyll-2,l,3-benzoxa- naphthyl isothiocyanate @NTC) ,7 4[N-[l-(dimethylamino)naphdiazol-4-ylisothiocyanate (DBD-NCS). Sequence deterthalene-5sulfonyll amino]phenyl isothiocyanate @NSAPITC) ,a 4( [ (5(dimethylamino)-1-naphthyl)sulfonyl]amino) phenyl isothiomination is carried out by identifying thiazolinone 0 cyanate (dansylamino-PITC)? 3- and 4(Zphenanthra[9',10'-d]amino acids, which are generally unstable and W c u l t to detect The employed system can easily and quickly oxazoly1)phenyl isothiocyanate (3- and ~ P O P I C S )and , ~ ~4(2-1cyanoisoindoly1)phenyl isothiocyanate (CIPIC).ll Although the derive "2 amino acids using the Edman reaction with sensitivity for detecting fluorescent TH amino acids by HPLC is DBD-NCS; these amino acids are also stable enough to greater than that for PTH amino acids (e.g., < 10 fmol for FITC5 be efficiently detected by high-performance liquid chroversus 1pmol for PITC12), a sample of 1-10 pmol is still required matography. Resultant detection limits for DBD-"2 amino acids range from 50 fmol to a sub-picomolelevel (S/N = for analysis for the following reasons: (1) fluorescent reagents are less reactive to N-terminal amino acids, hence normally 3). This system successfullyanalyzed sequences of Leu5necessitating parallel coupling of the residual N-terminal amino enkephalin (25pmol) and angiotensinI(lO0 pmol) using acid with PITC, and (2) during the subsequent conversion fluorometric detection at 524 nm with excitation at 387 reaction, only about 50% of the produced TZ amino acids are nm. converted to TH amino acids. Therefore, a more desirable Amino acid sequence analysis of peptides and proteins is fluorescent reagent is needed, namely, one that is more reactive and which enables quantitative conversion of TZ to TH amino conventionally done using the Edman method; i.e, the N-terminal amino acids are derivatized by aryl isothiocyanate and then cleaved acids. Another solution is to directly detect TZ amino acids, and cyclized into thiazolinone 0amino acids by anhydrous acid, thereby making the conversion reaction unnecessary. However, after which they are recyclized into phenylthiohydantoin (PTH) our literature search found that the Edman method has never been used to identify anilinothiazolinone (ATZ) amino acids, probably amino acids and identified by high-performance liquid chromasince they are unstable and difficult to detect. tography (HPLC). The liquid phase manual Edman method, which usually uses phenyl isothiocyanate (PITC), requires 0.1-5 In our previous study, we developed a new fluorescent Edman nmol of peptides and proteins (15 cycles from 100 pmol of protein reagent, 7-[ (NJ-dimethylamino) sulfonyll-2,1,3-benzoxadiazol-4yl using PITC and manual sequencing'), whereas the vapor phase isothiocyanate @BD-NCS) , which derivatized the N-terminal amino acids of the Ala-Phe dipeptide such that it could be detected automated Edman method, where the reaction time is carefully controlled and byproducts are completely removed, requires only (5) Muramoto, IC; Nokihara, IC; Ueda, A: Kamiya, H. Biosci. Biotechnol. Biochem. 1994,58, 300-304. io-100 pm01.2-4 (6) Palacz, 2.; Salnikow. J.; Jin, S.-W.; Wittmann-Liebold, B. FEES Leb. 1984, Various fluorescent Edman reagents have recently been 176, 365-370. introduced to increase detection sensitivity of the final product (7) Miyano, H.; Nakajima, T.; Imai, IC Biomed. Chromatogr, 1987,2,139144. aryl thiohydantoin (TH) amino acids. These include fluorescein (8) Jin, S.-W.; Chen, G.-X.; Palacz, Z.; Wittmann-Liebold, B. FEBS Lett. 1986, (1) Haniu, M.; Shively. J. Anal. Biochem. 1988,173, 196-306. (2) Bidlingmeyer, B. A.; Cohen, S. A: Tarvin. T. L. J. Chromatogr. 1984,336, 93-104. (3) Matsudaira, P.J. Bid. Chem. 1987,262,10035-10038. (4) Hewick, R. M.; Hankapiller, M. W.; Hood, L. E.; Dreyer, W. J. J. Biol. Chem. 1981,256, 7990-7997.
4276 Analytical Chemistry, Vol. 67, No. 23, December 1 , 1995
198,150-154. (9) Hirano, H.: Wittmann-Liebold, B. Biol. Chem. 1986,367, 1259-1265. (10) Imakyure, 0.;Kai, M.; Mitsui, T.; Nohta, H.; Ohkura, Y. Anal. Sci. 1993, 9, 647-652. (11) Imakyure, 0.;Kai, M.; Ohkura, Y. Anal. Chim. Acta. 1994,291, 197-204. (12) Cohen, S. A; Bidlingmeyer, B. A.; Tarvin, T. L. Nature 1986,320,769770. 0003-2700/95/0367-4276$9.00/0 0 1995 American Chemical Society
at a subpicomole 1e~el.l~ We hypothesize that DBD-NCS is more reactive than other fluorescent Edman reagents because the benzofurazan moiety is electron deficient and the electron density of the carbon atom of the isothiocyanate group is very low. This work led to the present study, in which we use the liquid phase manual Edman method with DBD-NCS to identify Leu5enkephalin and angiotensin I sequences. Specifically, we report on their formation, characteristics, and HPLC results. In addition, we demonstrate that the employed system can determine the Nterminal amino acid sequence of a peptide sample ranging from 25 to 100 pmol. EXPERIMENTAL SECTION Materials. DBD-NCS was synthesized from C[(N,"ethylamino)sulfonyl]-7-fluoro-2,1,3-benzoxadiazoleas previously described.13 We used the following materials: insulin chain B (oxidized) and dipeptides (Ala-Gly,AspAla,Arg-Phe, Cys-Gly, GluAla, His-Phe, Ile-Asn, Lys-Ala, Met-Ala, Phe-Gly, Pro-Leu, Ser-Phe, Thr-Leu,TrpAla, Tyr-Val, Val-Ala) (Sigma Chemical Co., St. Louis, MO) ; Led-enkephalin Vyr-Ala-Gly-Phe-Leu), [Asn1,Va15,Asngl angiotensin I (salmon, Asn-Arg-Valcryr-Val-His-Pr~Phe-Asn-~u), Leu-Gly, and Gly-Leu (Peptide Institute Inc., Osaka, Japan); phenylthiohydantoin 0 - L e u , HPLC-grade acetonitrile, and sequencer-grade trifluoroacetic acid VA) mako Pure Chemicals, Tokyo, Japan); and sequencer-grade pyridine (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan). Water was purified on a Milli-Q system (Millipore, Bedford, MA). All other reagents were analytical reagent grade and were used without further purification. HPLC. HPLC was carried out using an intelligent pump (L 6200;Hitachi, Tokyo, Japan) equipped with a Rheodyne injector (Model 7125,injection loop, 20 pb Cotati, CA). DBD-TZ amino acids were separated using two columns in tandem, i.e., an ODS column (YMC J'shere ODS H-80,250x 4.6 mm i.d., spherical, 5 pm, 80 A; YMC Co., Ltd., Kyoto, Japan) and a phenyl function bonded porous silica gel column (YMC-Pack Ph, 250 x 4.6 mm i.d., spherical, 5 pm, 120 A; YMC Co., Ltd.). An isocratic elution of DBD-TZ amino acids was employed with acetonitrile-water (64 v/v) containing 10 mM formic acid at a flow rate of 0.5 mL/ min. An intelligent spectrometer (820-FPJASCO, Tokyo, Japan) detected fluorescence at 524 nm with excitation at 387 nm, and the resultant data were analyzed using a chromatointegrator @2500;Hitachi). For identification of DBD derivatives, the isocratic HPLC system was also used. Elution was carried out at a flow rate of 1.0 mL/min using acetonitrile-water (55:45 v/v) containing 10 mM formic acid. Fluorometric detection was the same as described above. Absorbance was detected at 269 nm (L4200 W-vis detector; Hitachi).
liquid Chromatography/Mass Spectrometry (LC/MS). To perform LC/MS, an M-1200H LC/APCI-MS system was connected to the intelligent pump. The mass spectrometer was operated in the APCI mode, with the drift, focus, and multiplier voltages respectively set at 40,120,and 1800V. The temperatures of the vaporizer and desolvation regions were set at 220 and 399 "C,respectively. The separation of DBD-TZ amino acids was performed as for HPLC. Elution was carried out at a flow rate of 1.0 mL/min in the isocratic mode using acetonitrile-water (55: 45 v/v) containing 10 mM formic acid. (13) Imai, IC;Uzu, S.; Nakashima, IC;Akiyama, S. Biomed. Chromutogr, 1992, 7, 56-57.
Preparationof Standard DBD-TZAmino Acids. Solutions of various dipeptides dissolved in 50% (vlv) pyridine in water (10 pL) and 20 mM DBD-NCS in 50% (vh) pyridine (10pL) were vortex mixed and heated at 50 "C for 15 min. After the coupling reaction, the excess reagent and byproducts were removed by washing three times with 100 pL of n-heptane containing 10% (v/ v) dichloromethane. The aqueous phase was dried by the centrifugal evaporator (SPE200, Shimadzu, Kyoto, Japan) at 50 "C for 15 min, and TFA (30pL) was added to the resultant residue, which was heated at 50 "C for 10 min. Since the cleavage reaction with TFA is strongly affected by moisture, which also degrades DBD-TZ amino acids, the reaction was performed under NZ purging, and the reaction products were dried under a stream of Nz. The obtained residue was finally dissolved in acetonitrile. Dipeptide with N-terminal Asn or Gln could not be obtained commercially, so Asn-DBD-TZ was made with [Asn1,Va15,Asngl angiotensin I. Identification of DBD-TZ-, DBD-TC-, and DBD-TH-Leu. DBD-TZLeu was prepared from dipeptide Leu-Gly as described above. The fluorescence peak corresponding to DBD-TZLeu was collected by HPLC. Ethanethiol was added to the fraction (about 0.1% v/v) in order to prevent oxidation of the thiocarbamoyl group, and the fraction was dried under a stream of NZ gas. For conversion to DBD-TH-Leu, 30 pL of 50% (v/v) TFA in water containing about 0.1% ethanethiol was added to the residue, which was then heated at 50 "C for 10 min and dried under Nz. The resultant residue was dissolved in the mobile phase (acetonitrilewater (5545v/v) containing 10 mM formic acid), and a portion of the solution was subjected to HPLC and LC/MS as described above. DBD-TC, DBD-TZ, and DBD-TH amino acids were separated and collected by HPLC, and the fluorescence intensity and UV absorbance of their DBD derivatives were measured respectively with a fluorescence spectrophotometer (F.2010; Hitachi) and a UV-vis spectrophotometer (Ubest 50;JASCO). Effects of pH and Sohrent Qpe on Fluorescence Intensity of DBD-TZ Amino Acids. DBD-=Leu was prepared from dipeptide Leu-Gly as described above. The fluorescence peak corresponding to DBD-TZLeu was collected by HPLC and diluted with buffers having various pH values or with various organic solvents; i.e., buffer solutions were 0.1 M phosphate buffer (PH 2.03-8.03),while the organic solvents were acetonitrile, methanol, hexane, ethyl acetate, benzene, or dichloromethane. Solutions were quickly measured after preparation. Effects of pH and Solvent Qpe on Stability of DBD-'IZ Amino Acids. The DBD-TZLeu fraction collected by HPLC was similarly diluted with buffer solutions or organic solvents (methanol or acetonitrile) as described above and kept at 25 "C. Aliquots were withdrawn after appropriate time intervals and quickly subjected to HPLC. Fluorescence intensity of DBD-TZ-Leu showed pseudo-st-order kinetics, and the observed rate constant (kobs) for the disappearance of DBD-TZLeu was calculated using In [AI = -kobst c, where [AI is the residual content of DBDTZ-Leu, while t and c are respectively the time and intercept values. Sequencing Coupling Reaction. The yield of the coupling reaction was measured with DBD-TZ amino acids generated after the cleavage reaction with TFA of DBD-TC dipeptides, since DBDTC dipeptides have weak fluorescence to quantify. A solution of various dipeptides (10pM) dissolved in 50% (v/v) pyridine in water (100pL) and 20 mM DBD-NCS in 50% (v/v) pyridine (100pL)
+
Analytical Chemistry, Vol. 67, No. 23, December 1, 7995
4277
was vortex mixed and heated at 50 "C. A 20 pL aliquot of the reaction mixture was withdrawn after appropriate time intervals, and the excess reagent and byproducts were removed by washing three times with 100 pL of n-heptane containing 10% (v/v) dichloromethane. The aqueous phase was dried by the centrifugal evaporator at 50 "C for 15 min, and TFA (30 pL) was added to the resultant residue. After 10 min incubation at 50 "C, TFA was removed under a stream of N2. The residue obtained was dissolved in acetonitrile and subjected to HPLC. Sequencing Cleavage Reaction. In several sample tubes, a solution of dipeptides (10 pM) dissolved in 50% (v/v) pyridine in water (10 pL) and 20 mM DBD-NCS in 50% (v/v) pyridine (10 pL) was vortex mixed and then heated at 50 "C for 15 min. The excess reagent and byproducts were removed by washing three times with 100 pL of n-heptane containing 10% (v/v) dichloromethane. The aqueous phase was dried by the centrifugal evaporator at 50 "C for 15 min. TFA (30 pL) was added to the residue in each tube and heated at 50 "C for 2.5-20 min. After an appropriate time interval, each sample tube was withdrawn in sequence, and TFA was removed under a stream of Nz. The residue obtained was dissolved in acetonitrile and subjected to HPLC for measurement of the yield of DBD-TZ amino acids. Determination of the Optimal Dichloromethane Concentration for SolventWashing of DBD-CoupledPeptides. DBDNCS was reacted with several peptides (Leu5-enkephalin, [Asnl,Valj,Asnglangiotensin I, and insulin chain B), and after the coupling reaction, the reaction mixture was washed using various dichloromethane concentrations (0-50% v/v) in n-heptane in order to determine the optimal dichloromethane concentration. To do this, the aqueous phase was withdrawn, dried, and processed for the cleavage reaction as described above. Following the first cleavage reaction, the produced DBD-TZ amino acids were extracted by 50% (v/v) dichloromethane in n-heptane and subjected to HPLC. llquid Phase Sequencing with DBD-NCS. A peptide solution (10 pL) dissolved in 50%(v/v) pyridine in water and 20 mM DBD-NCS in 50%pyridine (10 pL) were vortex mixed and heated at 50 OC for 15 min. After the coupling reaction, the mixture was washed with 3 x 200 p L of n-heptane containing dichloromethane (10-50% v/v). We vaned the ratio of dichloromethane in n-heptane because the optimal content of dichloromethane in n-heptane was different according to the length of the peptide. The aqueous phase was dried by the centrifugal evaporator at 50 "C for 15 min, TFA (30 pL) was added to the residue, and the mixture was again heated at 50 "C for 10 min and dried under a stream of N2. The resultant residue was mixed with distilled water (20 pL) and extraction solvent (100 pL) and centrifuged at lOOOg for 5 min. The extraction solvent for DBDTZ amino acid was n-heptane containing a 10% (v/v) higher dichloromethane concentration than the washing solvent in order to maximally remove excess reagent and byproducts. The extraction was repeated three times, and the combined organic phase including DBD-TZ amino acids was dried under a Nz stream and dissolved in acetonitrile for the subsequent HPLC analysis. Standards derivatives used for identification were prepared with the corresponding dipeptides. The aqueous phase containing the residual peptide was dried in a centrifugal evaporator and subjected to the next cycle. 4278 Analytical Chemistry, Vol. 67, No. 23, December 1 , 1995
a-2 x
.%
DBD-TZ-Leu
a c,
-
B
b-1
DBD-TC-Leu
1.DBD-ru I
DBD-TH-Leu
_1
Figure 1. Chromatograms of DBD derivatives. (a) DBD-TZ-Leuand (b) after conversion of the DBD-TZ-Leu fraction. Fluorescence intensity was measured in chromatograms a-1 and b-1, while UV absorption was measured in chromatograms a-2 and b-2. DBD-TZLeu was prepared as described in the Experimental Section.
RESULTS AND DISCUSSION
Identification of DBD-TZ, DBD-TC-, and DBD-TH-Leu. HPLC analysis of DBD-TZ-Leu, which was prepared as described in the Experimental Section, showed a strong fluorescence peak (Figure 1,chromatogram a-1, t~ = 22.7 min, A, = 394 nm, Aem = 531 nm) and a weak W absorption (Figure 1, chromatogram a-2, at 269 nm). This peak was considered to be DBD-'IZLeu on the basis of the proposed Edman reaction method (Scheme 1). LC/ MS of the peak fraction indicated a mass number of 398 (M H)+,which supports that the peak is DBD-TZ-Leu (MW 397). Since the final Edman product, DBD-TH-Leu, also has a MW of 397, we subjected the fraction of the fluorescence peak after conversion reaction and analyzed it by HPLC to confirm that the peak fraction is DBD-TZ-Leu (Figure 1,chromatogram b-1). The intensity of the fluorescence peak was decreased, and two new UV absorption peaks were detected (Figure 1,chromatogram b 2 , t~ = 7.1 and 9.1 min, at 269 nm), one of which corresponded to DBD-TC-Leu ( t =~ 7.1 min, (M + H)+, 416), and the other ( t =~ 9.1 min, (M + H)+,398) was assigned as DBD-TH-Leu on the basis of the LC/MS measurement. Figure 2 shows UV spectra of the fractions obtained. One of the fractions ( t = ~ 9.1 min) showed a strong absorption at 265 nm (Figure 2c), while the fraction of the fluorescence peak ( t =~ 22.7 min) did not (Figure 2a). FTH-Leu standard indicated a maximal W absorption at 266 nm which has been assigned to FTH ring.I4 Thus, the fluorescence peak due to the cleavage reaction and two UV absorption
+
0.75
I
1
t
0,5 1
0.751
0.25
275
200
Wavelength (nm)
Wavelength (nm)
350
425
Wavelength (nm)
Figure 2. UV absorption curves of DBD-TZ-Leu (a), DBD-TC-Leu (b), and DBD-TH-Leu (c) in HPLC elution.
Scheme 1. Reaction Scheme of the Edman Method
,
I
,
I
1.6-
E
.P
=8
1.21.2
8 E
Coupling
5
0.80.8
NH
'
I
R
0.4'b
I
1
4
,
6
1
8
PH
Cleavage 0
CH-
7
C
I
I
N
y
4
S
I
+
R2
I
+H,N
-CH -
Table 1. Effect of Solvent Type on Fluorescence Spectrometric Properties of DBD-TZAeu
NH
I
aryl
Aryl-TZ Conversion
7
CH-
NH
I
I
o//c'
NC
I
Figure 3. Effect of pH on fluorescence intensity of DBD-TZ-Leu. Fluorescence intensity was measured at 531 nm with excitation at 394 nm.
+S
aryl
Aryl-TH
peaks ( t =~ 7.1 and 9.1 min) due to the conversion reaction were identified as the DBD-TZ, TC, and TH-Leu signals, respectively. DBD-TC-Leu and -TH-Leu revealed only weak fluorescence (Figure 1, chromatogram b-1). Maximal ratio of fluorescence/ absorption was obtained in DBD-TZLeu among these DBDderivatives, supporting that a DBD-TZ amino acid is suitable and sensitive for the sequencing analysis.
solvent
lex (nm)
A,, (nm)
re1 fluor intenso
water acetonitrile methanol hexane ethyl acetate benzene dichloromethane
391 387 392 385 387 389 388
551 509 517 468 491 485 491
1 21 14 8 13 21 23
-
The fluorescence intensity of DBD-TZ-Leu in water was arbitrarily taken as 1.
Effects of pH and Solvent Qpe on the Fluorescence Intensity of DBD-72 Amino Acids. As shown in Figure 3, the highest fluorescence intensity of DBD-'ELeu was obtained at pH 7, about 3.5 times higher than that at pH 2. The slight decrease in fluorescence intensity at pH 8 may be due to degradation of the derivative. Note that the correspondingfluorescence intensity in acetonitrile, benzene, and dichloromethane was about 20 times higher than that in water (Table 1). Effects of pH and Solvent m e on the Stability of DBDTZ Amino Acids. Figure 4 shows the observed rate constant (kobs) for the disappearance of DBD-TZ-Leu at 25 "C versus pH where this compound is obviously most stable, from pH 4 to 5, though even where most stable, its corresponding tllz is only 1.3 h. In an aprotic solvent like acetonitrile, DBD-TZ-Leu was Analytical Chemistry, Vol. 67, No. 23, December 1, 1995
4279
i'
2 !c
i OL 2
,
3
'
4
'
PH
5
'
6
,
j
'
4
I
Figure 4. Observed rate constant (kbs) versus pH for the degradation of DBD-TZ-Leu in an aqueous solution at 25 "C. Table 2. Effect of Solvent Type on the Stabllity of DBD-TZ-Leu
solvent water methanol acetonitrile
'
-5
1.5
kobs
(h-')
0.99 0.19 0.011
t09"
I
0
3
E
01)
0.11 0.56 10.0
Time at which the residual amount of the derivative is 90%of the initial amount.
3 I
0
I 1
I!
I 1 1 , I1 l l l l l l I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I I I I I I I I
5
10
15
20
25
30
35
40
45
I
50
time (min)
Figure 6. Chromatogram of DBD-TZ amino acids. Each peak of DBD-TZ amino acids corresponds to 100 (peak 7, Asn), 50 (8, His; 15, Lys; 5, Tyr), 20 (2, Arg), 10 (4, Gly; 6, Ala; 10, Asp; 11, Glu; 16, He; 12, Met; 1, Ser; 3, Thr; 13, Val; 14, Phe; 17, Leu), and 0.5 pmol (9, Pro).
d
extremely stable in comparison with a protic solvent like water or methanol Vable 2); hence, acetonitrile is the best of the solvents considered for use in a stock solution or HPLC eluent. Figure 5 shows the relationship between the acetonitrile concentration in a stock solution and the rate constant of DBD-TZ-Leu's disappearance at 25 and 5 "C, where the rate constant decreases with increasing acetonitrile concentration, being approximately constant at > 30%. Based on these results, when the acetonitrile concentration is greater than 30%, DBD-TZ amino acids can be adequately identilied by HPLC. Separation of DBDrlZ Amino Acids. Figure 6 shows HPLC separation of a mixture of 17 DBD-TZ amino acids. To achieve a good separation, two analytical columns were used in tandem, i.e., an ODS column and a phenyl bonded silica gel column. The benzofurazan moiety in the derivatives shows a remarkable retention on HPLC due to a r - ninteraction with an aromatic moiety of the stationary phase.lj In this HPLC system, a good separation of 17 DBD-TZ amino acids could be accomplished with a simple isocratic mode. DBD-TZ-Trp and -Cys could not be 4280 Analytical Chemistry, Vol. 67, No. 23, December 1, 7995
O
4 10 20 30 Time (min)
Figure 7. Time course for the coupling reaction of dipeptides with DBD-NCS at 50 "C. The yield of the coupling reaction at 50 "C for 30 min was taken as 100%. The reaction conditions of dipeptides with DBD-NCS are described in the text.
detected. Association of two fluorophores, i.e., DBD-TZ and an aminoindole moiety of Trp,might result in the quenching of DBDTZ-Trp. The reason why DBD-Z-Cys was not detected remains to be known. DBD-TZ-Lys was detected with two peaks ( t = ~ 32.74 and 44.34 min), presumably since Lys possesses two amino groups. It is supposed that one of the peaks (tR = 32.74 min) (14) Edman, P. Acta Chem. Scand. 1956, 10, 761-768. (15) Fukushima, T.:, Kato. M.: Santa, T.; Imai, K Biomed. Chromatogr. 1995, 9,10-17.
O
*
' 100-
-
80-
60-
40 20
-
while the other derivatives were detected from 0.2 to the s u b picomole level. Sequencing Coupling Reaction. Several dipeptides were reacted with DBD-NCS to determine the optimal coupling reaction conditions. After the reagent and dipeptides were dissolved in 50% (v/v) pyridine in water at 50 "C, the reaction was nearly completed in about 10 min Figure 7), with the exception of Asp, whose ionized sidechain carboxyl group may have interfered with the coupling reaction (the same applies to Glu). To determine the optimal DBD-NCS concentration, 100pmol of Leu-Gly or GlyLeu was reacted with various concentrations of DBD-NCS (25400 nmol). The resultant yield was approximately constant at a concentration 2000 times greater than that of the peptide. SequencingCleavage Reaction. The cleavage reaction with TFA at 50 "C for producing DBD-TZ amino acids was nearly completed within 2.5 min (Figure 8), although an amino acid with an aromatic group on the side chain (''and IF a secondary ) amino acid (Pro) required 10 min. Optimal Content of Dichloromethane in the Washout Solvent of DBD-Coupled Peptides. The residual DBD-NCS and byproducts must be removed after the coupling reaction with a minimal loss of the peptide coupled to DBD-NCS. When the n-heptane and dichloromethane mixture was used as the washout solvent (Figure 9), DBD-TZ amino acids recovery after the cleavage reaction was dependent on both the dichloromethane concentration and the number of the amino acids of the peptide; Le., the dichloromethane concentration should be varied on the basis of the number of the amino acids. Microsequencing of Peptides with DBD-NCS. Figure 10 shows the results of sequencing 25 pmol of Leu5-enkephalin, where four cycles sequentially yielded DBD-TZT~T(cycle l), DBD-TZGly (cycle 2), DBD-TZ-Gly (cycle 3), and DBD-TZ-Phe (cycle 4), i.e., yielding results that correspond to a sequence of peptide Tyr-Gly-Gly-Phe-Leu. Figure 11 shows the obtained chromatograms for nine sequence analysis cycles of 100 pmol of [Asn1,Va15,Asnglangiotensin I, where the nine amino acid sequences were obtained, although Leu could not be detected in the last cycle. The residual Germinal amino acid, Leu, possibly afforded a lesser amount of DBD-TZ-Leu since DBD-TH-Leu was also produced by the strong acid.16 In addition, the loss of DBDTC amino acids might be caused by the washing step. The fluorescent impurities detected in HPLC were probably due to
1
00
10
20
Time (min) Figure 8. Time course for the cleavage reaction of DBD dipeptides. The yield of the cleavage reaction at 50 "C for 10 min was taken as 100%. The reaction conditions of dipeptides with DBD-NCS are described in the text. I1
II
0
'
I
I
I
10
,
I
'
I
,
20
'
I
1
,
1
3
I
,
40
30
1 1
II
50
Dichloromethane (%) Figure 9. Optimal dichloromethane concentration in n-heptane washing solvent of DBD-coupled peptides. Recovery of DBD-TZ amino acids from each peptide after washing with rt-heptane (0% dichloromethane) was taken as 100%.
corresponds to DBD-TZ-Lys and the other ( t = ~ 44.34 min) was the derivative, the side chain of which was further reacted with DBD-NCS. Note that DBD-TZPro was the most sensitively detected (50 fmol, S/N = 3) among the 17 DBD-TZ amino acids,
Cycle 1
Cycle 2
I
Cycle 3
n
Cycle4
n
Phe
Figure 10. Chromatograms obtained by manual liquid phase microsequencing of 25 pmol of Leu5-enkephalin.The reaction conditions and HPLC conditions are described in the text. Analytical Chemistty, Vol. 67, No. 23, December 1, 1995
4281
Cycle 1 R
Cycle 6
c-Pro
I ,E
Asn
I
1
Figure 11. Chromatograms obtained by manual liquid phase microsequencing of 100 pmol of [Asn' ,Va15,Asng]angiotensinI. The reaction conditions and HPLC conditions are described in the text.
byproducts produced during the coupling or cleavage reaction. For the sequencing of smaller peptides, these impurities were incompletely removed in the washing step, and they became significant with the progress of sequence cycles. For the sequencing of 25 pmol of Leu5-enkephalin, 20% dichloromethane in n-heptane was used for washing, and for 100 pmol of [Asn1,Va15,Asng]angiotensin I, 40%dichloromethane in n-heptane was used. In the sequencing of lAsn1,Va15&nglangiotensin I (Figure l l ) , the impurities were better eliminated than in that of Leu5-enkephalin (Figure 10). However, in the latter part of the sequencing of [Asn1,Va15,Asng]angiotensin I, loss of the peptide became significant due to the higher content of dichloromethane in the washing solvent. FITC is not suitable for the sequencing of smaller peptides since it is hydrophilic, and a polar solvent is required to remove excess FITC. The polar solvent also brings about loss of peptide, especially smaller ones. Muramoto et al. explained that loss of peptide will be inevitable during sequencing with FITC," whereas the system described herein required only (16) Inman. J. IC; Appella. E. Methods Enzymol. 1977,47, 374-385. (17) Muramoto. K., Kawauchi, H., Tuzimura, K. Agric. Bid. Chem. 1978,42, 1559-1563.
4282 Analytical Chemistry, Vol. 67, No. 23, December I , 1995
25-100 pmol of peptide sample in spite of the peptides used being smaller. The relative yield, as calculated by the fluorescence intensity of Asn' and h n g , was (0.81): We developed a simple yet sensitive amino acid sequencing method using DBD-NCS and compared its results to those obtained using manual liquid phase sequencing with PITC or FITC. The ability to determine TZ amino acids is very advantageous in that it can be carried out without using the conversion step to obtain TH amino acids; hence, it is a simple, rapid, and effective method. In addition, the byproducts formed in the conversion step are not included in the sample and do not appear on chromatograms. It is expected that the presented sequence analysis method can be substantially improved using automated sequencing, i.e., making it faster and more sensitive. Received for review April 3, 1995. Accepted September 6,
1995.B AC9503280 @Abstractpublished in Advance ACS Abstracts, November 1, 1995.
