Capillary zone electrophoresis of amino acid derivatives
Electrophoresis 1990,11,777-780
Karen C. Waldron Shaole Wu Colin W. Earle Heather R. Harke Norman J. Dovichi Department of Chemistry, University of Alberta, Edmonton, Alberta
777
Capillary zone electrophoresis separation and laser-based detection of both fluorescein thiohydantoin and dimethylaminoazobenzene thiohydantoin derivatives of amino acids Capillary zone electrophoresis is employed for the separation and analysis of both fluorescein thiohydantoin and dimethylaminoazobenzene thiohydantoin derivatives of amino acids. Detection of minute amounts of these amino acid derivatives is an important milestone in.the development of a high sensitivity protein se uencer. Current detection limits for the fluorescein derivative is on the order of 10-'1 moles whereas detection limits for the dimethylaminoazobenzene derivative is on the order of moles.
'
1 Introduction The amino acid sequence of a protein is of fundamental value in the study of the structure and function of the molecule. The Edman degradation procedure is commonly employed to determine the amino acid sequence of proteins 11, 21. I n this procedure, phenyl isothiocyanate reacts with the amine group on the terminal amino acid of the protein under basic conditions to produce a thiocarbamyl protein derivative. Under acidic conditions, the thiocarbamyl cyclizes with the carboxilic acid group to cleave the terminal amino acid from the protein and to produce a thiohydantoin amino acid derivative. The thiohydantoin derivative is collected and the degradation is repeated to cleave the next amino acid from the protein. Liquid chromatography, with ultraviolet absorbance detection, is usually used to identify the amino acid produced from ech step in the procedure. The power of the Edman degradation is demonstrated by its popularity; there are very few competing technologies for protein sequencing. However, the conventional approach to protein sequencing suffers from several limitations. First, the macroscopic size of the protein sequencing instruments and the use of conventional liquid chromatography for amino acid detection requires that relatively large amounts of protein or peptide be available for sequencing. Roughly a picomole of peptide is required for sequencing. Second, the use of ultraviolet absorbance detection results in a large background signal due to impurities in the derivatization reagent. The reagent blank ultimately determines the detection limits for an analysis 131. T o improve detection limits, modified Edman degradation schemes have been developed. In each case, the isothiocyanate functional group remains while the phenyl group is replaced with a different chromophore. Perhaps the best developed modified Edman degradation scheme is based on dimethylaminoazobenzene isothiocyanate (DABITC) 141. This molecule couples with the N-terminus of the protein under basic conditions to produce the thiocarbamyl (DABTC)
Correspondence: Dr. Norman J. Dovichi, Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2 Abbreviations: DABITC, dimethylaminoazobenzene isothiocyanate; DABTC, dimethylaminoazobenzene thiocarbamyl; DABTH, dimethylaminoazobenzene thiohydantoin; FITC, fluorescein isothiocyanate; FTC, fluorescein thiocarbamyl; FTH, fluorescein thiohydantoin
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
and cyclizes under acidic conditions to cleave the terminal amino acid as a thiohydantoin (DABTH). Dimethylaminoazobenzene derivatives have a strong absorbance in the blue portionofthespectrum,&=:40,000LmoI~'cm ',and areusually separated by use of liquid chromatography with absorbance detection at -440 nm. An alternative reagent for the modified Edman degradation scheme is fluorescein isothiocyanate(F1TC) 151. Again, under basic conditions, the molecule couples with the N-terminus amino acid to form the thiocarbamyl (FTC) and under acidic conditions the terminal amino acid is cleaved as the thiohydantoin (FTH). Fluorescein derivatives have high absorbance in the blue, E FZ 80.000 L mol-' cm ', and high fluorescent quantum yield, Q z 0.5 to 1.0. The few reports of protein sequencing using this reagent have employed liquid chromatography separation and fluorescence detection 161. These highly colored or fluorescent reagents produce improvements in the minimum concentration of amino acid and protein that can be analyzed. Additional improvements in the mass of protein required for analysis will follow from reduction in the size of sequencing instrumentation. This research group has developed capillary zone electrophoresis systems for separation and identification of minute amounts of thiohydantoin derivatives of amino acids. Capillary electrophoreais offers significant advantages in both mass sensitivity and speed over liquid chromatography and will play a key role in the development of an atto-scale protein sequencer.
2 Materials and methods 2.1 DABTH-amino acid derivatives 2.1.1 Preparation Twenty standard DABTH-amino acid derivatives were prepared using a method similar to Chang's [4].Amino acid (Fluka) stock solutions, 5 x ~ O - * M ,were prepared in a solution made up of 50 mL acetone, 10 mL water, 5 mL 0.2 M acetic acid, 0.5 mL triethylamine, pH 10.6, and stored at 4 "C. Where necessary, the pH was adjusted by addition of 1 M NaOH. A 4 x M DABITC (Sigma) stock solution was prepared in acetone and stored at -10 OC. To prepare the DABTH-amino acid derivatives, 100 yL of each amino acid solution was mixed with 50 yL DABITC stock solution in a 1.5 m L disposable centrifuge vial, placed in a 52 & 2 OC water 0173-0835/00/0909-0777 $3.50+.25/0
778
Eleclrophoresis 1990,1I, 777-780
K. C. Waldron ef al.
bath for 1 h, and then dried under vacuum at 3OOC. The buffer. Stock 1.3 x lo-' M FITC (Sigma) was prepared in orange-yellow DABTC-amino acid derivatives were acidified acetone. One mL of each amino acid solution was mixed with with 40 pL water and 80 pL of a 1 : 2 mixture of 6 M hydro- 0.1 mL of the FITC solution and allowed to react for 4 h at chloric acid/glacial acetic acid. After heating for 50 min room temperature in the dark, to produce the thiocarbamyl and drying under vacuum, the red DABTKamino acids were derivative. Next, 0.5 mL of each thiocarbamyl solution was dissolved in 500 pL of a 10 mM phosphate buffer, pH 2.5, to mixed with 0.5 mL of trifluoroacetic acid and allowed to react yield a 4 x 1 0 - 4solution. ~ A sample made up of 30 pL of each for 15 h in the dark. The F T H amino acids were diluted at least of 20 DABTH-amino acids, final concentrationof 2 x 1 0 - 5 ~ , 4000-fold in 0.0 1 Mphosphate buffer, ph 7, before injection. was prepared in a disposable centrifuge tube.
2.2.2 Capillary electrophoresis 2.1.2 Capillary electrophoresis Capillary electrophoresis of the DABTH-amino acids was performed with a 1.1 m long, 50 pm inner diameter fused silica capillary, Polymicro. A mixed 10 mM sodium dodecyl sulfate, 40 % acetonitrile, 60 % aqueous 10 mM pH 2.5 phosphate buffer was used for the separation. The sample was injected electrokinetically at 20 kVfor 5 s, manually timed. Thermo-optical detection was performed 5.5 cm from the ground end of the capillary. The detector has been described in detail elsewhere [7, 81. In this experiment, a 15 mW helium-cadmium laser, h= 442 nm, provided the pump beam and a 2 mW helium-neon laser, h = 632.8 nm, provided the probe beam. The pump laser was focused with a 7 x objective whereas the probe beam was focused with a 10 x objective. A chopping frequency of 84 Hz was employed and the lock-in time constant was 1 s. The analog output of the lock-in amplifier was directed to a strip chart recorder.
Capillary electrophoresis separation of the FTH-amino acids was performed in a 1.O mlong, 50 pminner diameter capillary. A 10 mM phosphate buffer, p H 7.0, was used for the separation. The sample was injected electrokinetically at 0.5 kV for 10 s, manuallytimed, and separation proceeded at 30 kV. The fluorescence detector was identical to that described before [9-111. The detection end of the capillary was placed within the flow chamber of a sheath flow cuvette. A 25 mW beam from an argon ion laser, h = 488 nm, was focused into the cuvette. Fluorescence was collected at right angles with a 32 x 0.65 numerical aperture microscope objective. The collected light was passed through a bandpass filter to reject scattered laser light. The fluorescencewas imaged onto apinhole matched in size to the illuminated sample stream and detected with a high quantum yield photomultiplier tube. A 0.3 s resistorcapacitor (RC) filter was used to smooth the photomultiplier output before display on a strip chart recorder.
-
0
2.2 FTH-amino acid derivative 2.2.1 Preparation FTH-derivatives of amino acids were prepared using a method similar to that reported by Kawauchi [51. Stock amino acid solutions, 5 x 1 0 - 3 ~werepreparedinapH , 9.1 carbonate
Figirrc 1. Structure of the neutral form of DABTH-glycine.
9
a 7
6
5 . -
z
c m
g4 3
2
1
0 20
30
40
50 Time I Min
60
70
80
Figure 2. Analysis of DABTH-amino acids. Peak (1) is Cys, (2) His. (3) Lys. (4) Arg, ( 5 ) Lys, (6) CYS,(7) Asn, (8) G ~ Y(9) , Thr, (10) Asp, (1 I ) Ala,(12) Gln,(l3)Glu, (14) Pro, (15) Val, (16) Tyr, (17) Met, ( I 8) Phe, (19) Trp. Injected concentration is 2 x 10 'M.
Eiecirophoresis 1990, 1I , 777-780
Capillary zone electrophoresis of amino acid derivatives
0.06-
7 79
13
12
D.'D5. 16
0.134-
. 2
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17
0.03.
0
i7j
0.02-
0.oi.
(1.00-
x_
I
I
9
8
I
9
I
10 Time I Min
I
I
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11
12
13
3 Results and discussion Separation of DABTH-amino acid derivatives by electrophoresis is not simple. Figure 1 presents the structure of a DABTH-amino acid. The reaction scheme removes both the primary amine and carboxylic acid groups from the amino acid. At neutral pH, the molecules are not charged and therefore have zero electrophoretic mobility. However, under acidic conditions, the secondary amine group does protonate, with a pK, between 3 and 4. These DABTH-cations may be separated using capillary zone electrophoresis (Fig. 2). Careful adjustment of the separation buffer is required to achieve separation of the amino acids. After investigating a number of separation buffers, a 40 % acetonitrile, 10 mM sodium dodecyl sulfate/lO mM phosphate buffer, pH 2.5, was used to obtain the separation of 15 DABTH-amino acids. As in separation of dimethylaminoazobenzene sulfonyl chloride (DABSYL) derivatives of amino acids by capillary electrophoresis, the acetonitrile is added to reduce the zeta potential of the capillary walls, decreasing electroosmosis, increasing the separation time, and increasing the resolution of analyte. The sodium dodecyl sulfate interacts with the amino acids, perhaps through an ion pairing mechanism, to increase the plate count of the separation. However, separation at low pH is not ideal in fused silica capillaries; relatively low plate counts are produced, 1 to 4 x lo5, presumably due to interaction of the positively charged amino acid derivatives with the negatively charged capillary walls. Detection limits for the separation are on the order of 2 x 10 M,similar to reported capillary electrophoresis separation and thermo-optic detection of the dimethylaminoazobenzene sulfonyl chloride-amino acid derivatives [7, 81.
'
Unlike DABTH derivatives of amino acids, FTH amino acids are anions under basic conditions. These derivatives possess
Figure 3. Analysis of FTH amino acids. Peak (1) is Arg, (2) His, (3) Leu, (4) Ile, (5) Tyr, (6) Met and Thr, (7) Val, (8) Hyp, (9) Pro, (10) Thr (Ser), (1 1) Ala, ( I 2) Gly, (1 3) Cys, Ser, (14) FITC, (15) Gln, (16) Glu, (1 7) Aso. Iniected concentration was 1.5 x . < 10.'~ for serine and 1 x 1 0 - 9 ~ for the others. A small serine peak coelutes with threonine, peak (10).
low interaction with the capillary walls at pH > 7 and may be separated with high effeciency in capillary zone electrophoresis. Figure 3 presents the analysis of a mixture of 17 FTHamino acids in an aqueous, low ionic strength buffer. The separationproduces -1 x lo6plates for these analytes atthis pH. A total of 17 peaks, including one reagent peak, are obtained and 15 amino acids may be identified unambiguously. Rather surprisingly, the retention time of some of the neutral amino acids depends strongly upon pH. Presumably, the electron withdrawing properties of the particular side chain of the amino acid can influence the ionization constant of the carboxylic acid group of fluorescein. Reproducible buffer pH is required to obtain reproducible separations. Also, several of the FTH-amino acids show more than one peak, presumably due to degratation of the molecule under the derivatization conditions.
4 Concluding remarks Capillary zone electrophoresis separation of FTH-amino acids and DABTH-amino acids is an important step in the generation of protein sequence information on subfemtomole samples. If funding becomes available, it will be possible to construct a highly miniaturized protein sequencer with sensitivity that is three to six orders of magnitude superior to the present state of the art in amino acid sequencing.
This work wasfunded by the National Science andEngineering Research Fund through both the operating and equipment grant programs. Ruedi Aebersold of the University of British Columbia kindly provided samples of DABITC-amino acids that were used in our preliminary work. Received December 4, 1989
780
Eleclrophuresis 1990, / I . 780- 783
I . Foret el nl.
5 References I I 1 Iidman, P., Acfn Chem. Scand. 1950,4,277-283. 121 Edman, P. and Begg. G.. Eur. J . Biochern. 1967. 1. 80-9 I . 131 Smith. L.,Anul. Chern. 1988.60.381-390. 141
Electrophoresis 1990,11,777-780
Karen C. Waldron Shaole Wu Colin W. Earle Heather R. Harke Norman J. Dovichi Department of Chemistry, University of Alberta, Edmonton, Alberta
777
Capillary zone electrophoresis separation and laser-based detection of both fluorescein thiohydantoin and dimethylaminoazobenzene thiohydantoin derivatives of amino acids Capillary zone electrophoresis is employed for the separation and analysis of both fluorescein thiohydantoin and dimethylaminoazobenzene thiohydantoin derivatives of amino acids. Detection of minute amounts of these amino acid derivatives is an important milestone in.the development of a high sensitivity protein se uencer. Current detection limits for the fluorescein derivative is on the order of 10-'1 moles whereas detection limits for the dimethylaminoazobenzene derivative is on the order of moles.
'
1 Introduction The amino acid sequence of a protein is of fundamental value in the study of the structure and function of the molecule. The Edman degradation procedure is commonly employed to determine the amino acid sequence of proteins 11, 21. I n this procedure, phenyl isothiocyanate reacts with the amine group on the terminal amino acid of the protein under basic conditions to produce a thiocarbamyl protein derivative. Under acidic conditions, the thiocarbamyl cyclizes with the carboxilic acid group to cleave the terminal amino acid from the protein and to produce a thiohydantoin amino acid derivative. The thiohydantoin derivative is collected and the degradation is repeated to cleave the next amino acid from the protein. Liquid chromatography, with ultraviolet absorbance detection, is usually used to identify the amino acid produced from ech step in the procedure. The power of the Edman degradation is demonstrated by its popularity; there are very few competing technologies for protein sequencing. However, the conventional approach to protein sequencing suffers from several limitations. First, the macroscopic size of the protein sequencing instruments and the use of conventional liquid chromatography for amino acid detection requires that relatively large amounts of protein or peptide be available for sequencing. Roughly a picomole of peptide is required for sequencing. Second, the use of ultraviolet absorbance detection results in a large background signal due to impurities in the derivatization reagent. The reagent blank ultimately determines the detection limits for an analysis 131. T o improve detection limits, modified Edman degradation schemes have been developed. In each case, the isothiocyanate functional group remains while the phenyl group is replaced with a different chromophore. Perhaps the best developed modified Edman degradation scheme is based on dimethylaminoazobenzene isothiocyanate (DABITC) 141. This molecule couples with the N-terminus of the protein under basic conditions to produce the thiocarbamyl (DABTC)
Correspondence: Dr. Norman J. Dovichi, Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2 Abbreviations: DABITC, dimethylaminoazobenzene isothiocyanate; DABTC, dimethylaminoazobenzene thiocarbamyl; DABTH, dimethylaminoazobenzene thiohydantoin; FITC, fluorescein isothiocyanate; FTC, fluorescein thiocarbamyl; FTH, fluorescein thiohydantoin
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
and cyclizes under acidic conditions to cleave the terminal amino acid as a thiohydantoin (DABTH). Dimethylaminoazobenzene derivatives have a strong absorbance in the blue portionofthespectrum,&=:40,000LmoI~'cm ',and areusually separated by use of liquid chromatography with absorbance detection at -440 nm. An alternative reagent for the modified Edman degradation scheme is fluorescein isothiocyanate(F1TC) 151. Again, under basic conditions, the molecule couples with the N-terminus amino acid to form the thiocarbamyl (FTC) and under acidic conditions the terminal amino acid is cleaved as the thiohydantoin (FTH). Fluorescein derivatives have high absorbance in the blue, E FZ 80.000 L mol-' cm ', and high fluorescent quantum yield, Q z 0.5 to 1.0. The few reports of protein sequencing using this reagent have employed liquid chromatography separation and fluorescence detection 161. These highly colored or fluorescent reagents produce improvements in the minimum concentration of amino acid and protein that can be analyzed. Additional improvements in the mass of protein required for analysis will follow from reduction in the size of sequencing instrumentation. This research group has developed capillary zone electrophoresis systems for separation and identification of minute amounts of thiohydantoin derivatives of amino acids. Capillary electrophoreais offers significant advantages in both mass sensitivity and speed over liquid chromatography and will play a key role in the development of an atto-scale protein sequencer.
2 Materials and methods 2.1 DABTH-amino acid derivatives 2.1.1 Preparation Twenty standard DABTH-amino acid derivatives were prepared using a method similar to Chang's [4].Amino acid (Fluka) stock solutions, 5 x ~ O - * M ,were prepared in a solution made up of 50 mL acetone, 10 mL water, 5 mL 0.2 M acetic acid, 0.5 mL triethylamine, pH 10.6, and stored at 4 "C. Where necessary, the pH was adjusted by addition of 1 M NaOH. A 4 x M DABITC (Sigma) stock solution was prepared in acetone and stored at -10 OC. To prepare the DABTH-amino acid derivatives, 100 yL of each amino acid solution was mixed with 50 yL DABITC stock solution in a 1.5 m L disposable centrifuge vial, placed in a 52 & 2 OC water 0173-0835/00/0909-0777 $3.50+.25/0
778
Eleclrophoresis 1990,1I, 777-780
K. C. Waldron ef al.
bath for 1 h, and then dried under vacuum at 3OOC. The buffer. Stock 1.3 x lo-' M FITC (Sigma) was prepared in orange-yellow DABTC-amino acid derivatives were acidified acetone. One mL of each amino acid solution was mixed with with 40 pL water and 80 pL of a 1 : 2 mixture of 6 M hydro- 0.1 mL of the FITC solution and allowed to react for 4 h at chloric acid/glacial acetic acid. After heating for 50 min room temperature in the dark, to produce the thiocarbamyl and drying under vacuum, the red DABTKamino acids were derivative. Next, 0.5 mL of each thiocarbamyl solution was dissolved in 500 pL of a 10 mM phosphate buffer, pH 2.5, to mixed with 0.5 mL of trifluoroacetic acid and allowed to react yield a 4 x 1 0 - 4solution. ~ A sample made up of 30 pL of each for 15 h in the dark. The F T H amino acids were diluted at least of 20 DABTH-amino acids, final concentrationof 2 x 1 0 - 5 ~ , 4000-fold in 0.0 1 Mphosphate buffer, ph 7, before injection. was prepared in a disposable centrifuge tube.
2.2.2 Capillary electrophoresis 2.1.2 Capillary electrophoresis Capillary electrophoresis of the DABTH-amino acids was performed with a 1.1 m long, 50 pm inner diameter fused silica capillary, Polymicro. A mixed 10 mM sodium dodecyl sulfate, 40 % acetonitrile, 60 % aqueous 10 mM pH 2.5 phosphate buffer was used for the separation. The sample was injected electrokinetically at 20 kVfor 5 s, manually timed. Thermo-optical detection was performed 5.5 cm from the ground end of the capillary. The detector has been described in detail elsewhere [7, 81. In this experiment, a 15 mW helium-cadmium laser, h= 442 nm, provided the pump beam and a 2 mW helium-neon laser, h = 632.8 nm, provided the probe beam. The pump laser was focused with a 7 x objective whereas the probe beam was focused with a 10 x objective. A chopping frequency of 84 Hz was employed and the lock-in time constant was 1 s. The analog output of the lock-in amplifier was directed to a strip chart recorder.
Capillary electrophoresis separation of the FTH-amino acids was performed in a 1.O mlong, 50 pminner diameter capillary. A 10 mM phosphate buffer, p H 7.0, was used for the separation. The sample was injected electrokinetically at 0.5 kV for 10 s, manuallytimed, and separation proceeded at 30 kV. The fluorescence detector was identical to that described before [9-111. The detection end of the capillary was placed within the flow chamber of a sheath flow cuvette. A 25 mW beam from an argon ion laser, h = 488 nm, was focused into the cuvette. Fluorescence was collected at right angles with a 32 x 0.65 numerical aperture microscope objective. The collected light was passed through a bandpass filter to reject scattered laser light. The fluorescencewas imaged onto apinhole matched in size to the illuminated sample stream and detected with a high quantum yield photomultiplier tube. A 0.3 s resistorcapacitor (RC) filter was used to smooth the photomultiplier output before display on a strip chart recorder.
-
0
2.2 FTH-amino acid derivative 2.2.1 Preparation FTH-derivatives of amino acids were prepared using a method similar to that reported by Kawauchi [51. Stock amino acid solutions, 5 x 1 0 - 3 ~werepreparedinapH , 9.1 carbonate
Figirrc 1. Structure of the neutral form of DABTH-glycine.
9
a 7
6
5 . -
z
c m
g4 3
2
1
0 20
30
40
50 Time I Min
60
70
80
Figure 2. Analysis of DABTH-amino acids. Peak (1) is Cys, (2) His. (3) Lys. (4) Arg, ( 5 ) Lys, (6) CYS,(7) Asn, (8) G ~ Y(9) , Thr, (10) Asp, (1 I ) Ala,(12) Gln,(l3)Glu, (14) Pro, (15) Val, (16) Tyr, (17) Met, ( I 8) Phe, (19) Trp. Injected concentration is 2 x 10 'M.
Eiecirophoresis 1990, 1I , 777-780
Capillary zone electrophoresis of amino acid derivatives
0.06-
7 79
13
12
D.'D5. 16
0.134-
. 2
>
17
0.03.
0
i7j
0.02-
0.oi.
(1.00-
x_
I
I
9
8
I
9
I
10 Time I Min
I
I
1
11
12
13
3 Results and discussion Separation of DABTH-amino acid derivatives by electrophoresis is not simple. Figure 1 presents the structure of a DABTH-amino acid. The reaction scheme removes both the primary amine and carboxylic acid groups from the amino acid. At neutral pH, the molecules are not charged and therefore have zero electrophoretic mobility. However, under acidic conditions, the secondary amine group does protonate, with a pK, between 3 and 4. These DABTH-cations may be separated using capillary zone electrophoresis (Fig. 2). Careful adjustment of the separation buffer is required to achieve separation of the amino acids. After investigating a number of separation buffers, a 40 % acetonitrile, 10 mM sodium dodecyl sulfate/lO mM phosphate buffer, pH 2.5, was used to obtain the separation of 15 DABTH-amino acids. As in separation of dimethylaminoazobenzene sulfonyl chloride (DABSYL) derivatives of amino acids by capillary electrophoresis, the acetonitrile is added to reduce the zeta potential of the capillary walls, decreasing electroosmosis, increasing the separation time, and increasing the resolution of analyte. The sodium dodecyl sulfate interacts with the amino acids, perhaps through an ion pairing mechanism, to increase the plate count of the separation. However, separation at low pH is not ideal in fused silica capillaries; relatively low plate counts are produced, 1 to 4 x lo5, presumably due to interaction of the positively charged amino acid derivatives with the negatively charged capillary walls. Detection limits for the separation are on the order of 2 x 10 M,similar to reported capillary electrophoresis separation and thermo-optic detection of the dimethylaminoazobenzene sulfonyl chloride-amino acid derivatives [7, 81.
'
Unlike DABTH derivatives of amino acids, FTH amino acids are anions under basic conditions. These derivatives possess
Figure 3. Analysis of FTH amino acids. Peak (1) is Arg, (2) His, (3) Leu, (4) Ile, (5) Tyr, (6) Met and Thr, (7) Val, (8) Hyp, (9) Pro, (10) Thr (Ser), (1 1) Ala, ( I 2) Gly, (1 3) Cys, Ser, (14) FITC, (15) Gln, (16) Glu, (1 7) Aso. Iniected concentration was 1.5 x . < 10.'~ for serine and 1 x 1 0 - 9 ~ for the others. A small serine peak coelutes with threonine, peak (10).
low interaction with the capillary walls at pH > 7 and may be separated with high effeciency in capillary zone electrophoresis. Figure 3 presents the analysis of a mixture of 17 FTHamino acids in an aqueous, low ionic strength buffer. The separationproduces -1 x lo6plates for these analytes atthis pH. A total of 17 peaks, including one reagent peak, are obtained and 15 amino acids may be identified unambiguously. Rather surprisingly, the retention time of some of the neutral amino acids depends strongly upon pH. Presumably, the electron withdrawing properties of the particular side chain of the amino acid can influence the ionization constant of the carboxylic acid group of fluorescein. Reproducible buffer pH is required to obtain reproducible separations. Also, several of the FTH-amino acids show more than one peak, presumably due to degratation of the molecule under the derivatization conditions.
4 Concluding remarks Capillary zone electrophoresis separation of FTH-amino acids and DABTH-amino acids is an important step in the generation of protein sequence information on subfemtomole samples. If funding becomes available, it will be possible to construct a highly miniaturized protein sequencer with sensitivity that is three to six orders of magnitude superior to the present state of the art in amino acid sequencing.
This work wasfunded by the National Science andEngineering Research Fund through both the operating and equipment grant programs. Ruedi Aebersold of the University of British Columbia kindly provided samples of DABITC-amino acids that were used in our preliminary work. Received December 4, 1989
780
Eleclrophuresis 1990, / I . 780- 783
I . Foret el nl.
5 References I I 1 Iidman, P., Acfn Chem. Scand. 1950,4,277-283. 121 Edman, P. and Begg. G.. Eur. J . Biochern. 1967. 1. 80-9 I . 131 Smith. L.,Anul. Chern. 1988.60.381-390. 141
