Anal. Chem. 1990, 62,1573-1577
Figure 12. The slopes of the calibration lines are different for the three eluent systems of benzenesulfonate solution. Total Area of the Three Peaks. In all the cases calculated, the total area of the first system peak, the analyte peak, and the second system peak in a chromatogram was proportional to the volume of the sample injected. The proportional coefficient was independent of the analyte concentration. This seems to be reasonable according to mass balance and to be evidence that the calculations were carried out correctly. In a special case, where the first system peak has a constant intensity independent of the analyte concentration (a salt eluent and acid sample combination), the analyte peak and the second system peak have areas equal in absolute value but opposite in sign. For the purpose of detailed comparison between the actual ion chromatograms and the calculation results, it is necessary to increase the theoretical plate number to at least lo3. Further, the eluents in common use are of the bibasic type,
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such as phthalate solution. In the near future, the author will revise the simulation programs so as to simulate the chromatography of ions more generally by using a mainframe computer. Registry No. HBz, 65-85-0; NaBz, 532-32-1; HBs, 98-11-3; NaBs, 515-42-4; C1, 16887-00-6.
LITERATURE CITED (1) Brunt, K. I n Trace Analysis; Lawrence, J. F., Ed.; Academic Press: New York, 1981; Vol. 1, p 47. (2) Small, H.; Miller, T. E., Jr. Anal. Chem. 1982, 54, 462. (3) Okada, T.; Kuwamoto. T. Anal. Chem. 1884, 56, 2073. (4) Jackson, P. E.; Haddad, P. R. J. Chromatogr. 1885, 346, 125. (5) Sato, H. Anal. Chim. Acta 1988, 206, 281. (6) Sato, H. Bunseki Kagaku 1982, 37,97. (7) Fritz, J. S.;DuVal, D. L.; Barron, R. E. Anal. Chem. 1884, 56, 1177. (8) Gjerde, D. T.; Fritz, J. S. Anal. Chem. 1881, 53, 2324.
RECEIVEDfor review December 20, 1989. Accepted April 4, 1990.
Quantitative Amino Acid Analysis of Individual Snail Neurons by Open Tubular Liquid Chromatography Mary D. Oates, Bruce R. Cooper, and James W. Jorgenson*
Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290
A method Is descrlbed for the determlnatlon of amino aclds In lndlvlduai cells. The amino acids are derivatized with naphthalene2,3dkarboxaWyde and then analyzed by open tubular llquld chromatography wHh amperometrlc detectlon. The total v o h e present after derlvatlzatlon is approxlmately 25 nL. It was posslble to quantitatlvely determlne 17 amino aclds In three dlfferent neurons of the land snall Helix asp e m . Quantltatlon was accompHshed through the use of two Internal standards and a calibration curve. Alanlne was found to be the most abundant amlno acld by about a factor of 2 over glutamine In an three types of neurons. Thls method has the advantages of sendtlvky In the attomole range (5 X 10-o M) and selectlvlty for a specific class of compounds and is at least as reliable as other methods used for slngle-cell analysis.
INTRODUCTION The ability to accurately determine the contents of individual cells is important, particularly in the field of neurochemistry. Nervous tissues consist of large populations of cells, and each cell may have a unique biochemistry and function ( I ) . The amino acids present in a neuron can provide clues to its role in the nervous system, and several techniques have been developed for their determination in one or more cells. For example, Osborne and co-workers (2,3) pooled 4-8 neurons from the land snail Helix pomatia, reacted the contents with (*4C)dansylchloride, and separated the derivatives with micro-thin-layer chromatography (TLC). This technique was not sensitive enough to analyze individual neurons, and quantitation was cumbersome. Absolute amounts of amino acids could not be determined, and results were given as a
percentage of the total amount of measured substances. Gas chromatography/mass spectrometry (GC/MS) has also been used to determine amino acids in individual neurons ( 4 , 5 ) . The sensitivity needed for single-cell analysis required that the selected ion mode be used, however, limiting the analysis to a few previously selected compounds. Recently in our laboratory a method was developed that overcame several of the drawbacks of these earlier techniques. Open tubular liquid chromatography (OTLC) with voltammetric detection was used for the quantitative determination of several compounds in individual neurons of the snail Helix aspersa (6, 7). The number of moles of tyrosine, tryptophan, dopamine, and serotonin were measured in three different types of neurons. Only the amino acids tyrosine and tryptophan could be determined because the other amino acids are not intrinsically electroactive. The advantages of the OTLC method, including single-cell sensitivity and accurate, absolute quantitation, can be combined with the ability to analyze many more substances, in particular those that are not intrinsically electroactive, by combining OTLC with a derivatization scheme. An entire class of compounds can then be determined by reacting them with a reagent that provides a common detectable group. Our lab has recently been investigating the use of naphthalene2,3-dicarboxaldehyde (NDA) for the derivatization of primary amines (8-10). NDA reacts with amines in the presence of the cyanide ion to produce N-substituted l-cyanobenz[flisoindole derivatives, which are both fluorescent and electroactive. This report describes the use of NDA for the derivatization of amino acids present in individual snail neurons. The final volume present after derivatization was approximately 25 nL. The tagged amino acids were separated by using OTLC with gradient elution and detected amperometrically. Quantitative
0003-2700/90/0362-1573$02.50/00 1990 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990
measurements were obtained for 17 amino acids from three different types of neurons. The data present here have appeared in a review of microcolumn separation methods used for the analysis of single cells (11). However, no experimental detail was given in the review article. T h e purpose of this report is to present the data along with the vital information necessary to reproduce the work and make it a usable technique for others.
EXPERIMENTAL SECTION Apparatus. The chromatographic system used in this work has been described previously (10, 12). Briefly, a Waters 600E multisolvent delivery system provided mobile phase at a flow rate of 1 mL/min. The flow was split by using two fused silica capillaries, resulting in the delivery of approximately 60 nL/min of mobile phase to the analytical column. This column had an inner diameter of 15 pm, was 150 cm long, and had octadecylsilane bound to its superficially porous glass walls (13). The electrochemical detector used has also been described earlier (12, 14). A carbon fiber, 1 mm long and 9 pm in diameter, served as the working electrode. It was inserted directly into the outlet end of the capillary column. All chromatograms were obtained in the amperometric mode. The peak potential of the NDA derivatives is approximately 0.8 V vs Ag/AgCl, so 0.9 V, on the plateau of the wave, was chosen as the working potential. Data were acquired at 1point/s with 16-bit accuracy through the use of a microcomputer. A Model 427 current amplifier (Keithley Instruments, Inc., Cleveland, OH) with a 500-ms rise time and a Model 3341 low-pass filter (Krohn-Hite Corp., Avon. MA), set a t 10 Hz, were also used. Sample Preparation. Three giant neurons from the land snail Helix aspersa, labeled F1, E4, and D2 according to the map of Kerkut et al. (15),were analyzed for their amino acid content. The cells were isolated as described previously (16). After isolation the cells were transferred with approximately 5 nL of Ringer solution to a 200-nL microvial. Then 1 nL of lo-* M normetanephrine was added as an internal standard. All additions to the microvial were made by using a pneumatic micropipet made of borosilicate glass. The pipet was calibrated for solutions made with water as the solvent by measuring the size of water droplets in mineral oil, as described previously (17). For reagents made in solvents other than water, such as the NDA and borate solutions, the size of droplets of these solvents were measured in mineral oil. After addition of the internal standard, the cell was homogenized by using a small glass rod. The microvial containing the cell contents was then centrifuged for 30 min at 3000 g, after which the supernatant was transferred to a clean microvial. Approximately 1-2 nL of liquid was available for derivatization after transferring the supernatant to the new microvial. No attempt was made to remove proteins from the solution prior to derivatization. The order of addition of the tagging reagents is critical and began with the addition of 4 nL of 0.5 M sodium cyanide to the cellular fluid in the microvial. This was followed by 9 nL of 0.1 M boric acid, pH 9.5, which contained 10% acetonitrile to improve the solubility of the hydrophobic NDA deM rivatives. The boric acid solution also contained 1.8 X norleucine, a second internal standard. Finally, 2 nL of 0.25 M NDA was added. The NDA was dissolved in an 80/20 mixture of acetonitrile and 1-propanol. When NDA is dissolved only in acetonitrile, the solution creeps out the end of the micropipet used to add it to the microvial. This results in the immediate evaporation of the acetonitrile, causing the pipet to plug with solid NDA. The addition of 1-propanol allowed the NDA solution to remain inside the pipet and did not adversely effect the derivatization reaction or the chromatographic results. The mixture was allowed to react for 5 min after the NDA was added. Due to the small volumes involved, evaporation of the contents of the microvial was a problem during both the addition of reagents to the vial and the 5-min reaction time. Therefore, the microvial was attached to an aluminum stand with doublesided tape, and the stand was placed in a Pyrex dish containing ice. This sufficiently cooled the microvial so that no detectable evaporation occurred. A small piece of parafilm was placed over the open end of the microvial during the reaction period to further reduce the evaporation rate.
After 5 min, 8 nL of 0.05 M phosphate buffer (pH 7.0) was added to the microvial. This dilution was necessary to reduce the concentration of organic solvents in the mixture, which adversely affected the chromatography. Approximately 25% of the total volume, or 4-5 nL, was withdrawn into a clean micropipet and injected onto the column. All of the sample preparation and injections were done with the aid of a Wolfe Selectra I1 stereomicroscope, a Brinkman micromanipulator, and an Oriel micropositioner. Reagents. HPLC grade acetonitrile (Fisher Scientific Co., Fair Lawn, NJ) was used as received. Amino acids were obtained from Sigma (St. Louis, MO) while reagent grade sodium cyanide was purchased from Aldrich (Milwaukee, WI). All water used was purified by a Barnstead water purification system. NDA was obtained from two sources, Molecular Probes (Eugene, OR) and Polysciences, Inc. (Warrington, PA). When the Molecular Probes NDA was used as received, many large crystals formed upon adding the NDA to the reaction vial. This was true for standards as well as the cellular fluid. It is not known what these crystals were. However, the electrochemical signal obtained for standards derivatized with this NDA was far below that expected on the basis of experiments done with larger sample volumes. We have used this NDA extensively for derivatizations on the milliliter scale and have not encountered this problem. Polysciences NDA was then purified according to a procedure provided by Stobaugh (18) and based on vacuum LC (19). The commercial NDA was crystallized from ethyl acetate and then dissolved in chloroform. This solution was applied uniformly to the top of a vacuum LC column made with TLC grade silica gel. Elution was accomplished using a mixture of hexane and methylene chloride. The amount of methylene chloride was gradually increased from 0 to 40% over the course of the separation. Fractions were collected and the solid residue from each fraction was recovered by evaporation of the liquid. Pure NDA consists of white crystals, while the impurities present are bright yellow and orange. TLC was used to confirm the identity of the white crystals as pure NDA. Upon addition of the purified NDA to the sample, many very small particles were formed. However, the signal from the resulting derivatives was much closer to the level expected. Purified NDA was thus used in all experiments. Solutions. Buffers. Borate buffer (pH 9.5, 0.1 M) was prepared by dissolving boric acid in water and adding sodium hydroxide until the desired pH was reached. Phosphate buffer (pH 7.0,0.05 M) was made by diluting phosphoric acid in water and adjusting the pH with sodium hydroxide. The mobile phase was prepared by adding 1 mM EDTA to the pH 7 phosphate buffer and filtering through a 0.22-wm nylon filter (Fisher). Stock Solutions. Amino acid stock solutions M) were dissolved in water and kept in the refrigerator for up to 1week. NDA solutions were prepared fresh daily, while the cyanide and borate solutions were used for up to 1month. Purified NDA was kept in the dark in a desiccator and used for up to 1 year after purification.
RESULTS AND DISCUSSION The amino acid contents of five E4 cells, one F1, and one D2 cell were determined. A typical chromatogram is shown in Figure 1. It was possible to analyze 17 amino acids. In addition, many unknown peaks, presumably other primary amines, are also evident in the chromatogram. A gradient was necessary to separate all the amino acids in a reasonable time. Mobile phase A was 3% tetrahydrofuran in 0.05 M phosphate buffer and mobile phase B was pure acetonitrile. A linear gradient from 100% A to 89% A/11% B in 38 min and then to 47% A/53% B in 80 min was used. I t was not possible, with this method, to determine lysine, dopamine, or serotonin. They are lost during sample preparation, perhaps due to adsorption to the glassware (micropipet or microvial) or due to precipitation from solution. Quantitation. Quantitation was accomplished through the use of a calibration curve and two internal standards. The first internal standard, normetanephrine, was added prior to homogenizing the cell and should account for losses of cellular
ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990
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Table I. Data from a Typical Calibration Curve
amino acid
0.15 pmol
ratio to norleucine 0.55 pmol
2.15 pmol
slope
int
corr
ASP
0.524 0.280 0.299 0.286 0.483 0.190 0.314 0.114 0.328 0.232 0.384 0.231 0.139 0.138 0.169 0.128 0.198
1.00 0.692 0.938 0.726 1.04 0.858 0.933 0.411 0.941 0.750 1.14 0.629 0.541 0.345 0.461 0.414 0.483
4.00 2.85 3.40 2.94 3.74 4.04 3.40 1.82 3.14 2.51 4.20 2.72 2.12 1.45 1.77 1.85 2.09
0.0018 0.0013 0.0015 0.0013 0.0016 0.0019 0.0015 0.0009 0.0014 0.0011 0.0019 0.0013 0.0010 0.0007 0.0008 0.0009 0.0010
0.1530 0.0359 0.0759 0.0413 0.1912 0.1501 0.0835 0.0362 0.1426 0.0925 0.0942 0.0059 0.0067 0.0117 0.0347 0.0305 0.0091
0.9980 0.9992 1.000 0.9994 0.9996 0.9996 1.000 0.9996 0.9998 0.9996 1.000 0.9992 1.OOO 0.9991 0.9998 0.9994 0.9988
Glu
Am
Ser Gln His
GlY Thr Ala Arg
TYr Val
Met TrP Ile Phe Leu
9
I
19
BZ
0.04 nA
I
5 2
I
I
B1 1
4
L
1
A
I
I
1
3 1
11
0.6 nA
5
6
J
Ud"
53 TIME (min)
Figure 1. Chromatogram of NDA-tagged amino acids from a single E4 neuron from Helix a s p e m . Peak numbers 1-17 correspond to those listed in Table I. Peaks 15-1 7 are the three peaks immediately following 82. Peak 14 is obscured in this run by 82. 8 1 and 82 are present in blank runs, while peaks 18 and 19 correspond to the internal standards norleucine and normetanephrine, respectively. All unlabeled peaks are unknowns. material that occur during sample preparation. A calibration curve was constructed for normetanephrine so that the number of moles of normetanephrine detected in each snail run could be determined. This was divided by the number of moles of normetanephrine added (a constant amount was added to each snail sample prior to cell homogenization) to indicate what fraction of the cell contents were recovered. This ratio ranged from 0.2 to 0.5. The second internal standard, norleucine, was added to account for variability in the tagging reaction. The same quantity of norleucine was added to all standard runs and to all snail runs. The calibration curve was constructed by derivatizing three sets of standard amino acids that contained 0.15 (6.0 X lo4 M), 0.55 (2.2 X M), and 2.15 pmol (8.6 X 10" MI of each amino acid. They also each contained 1.26 pmol (5.0 x M) of norleucine. Figure 2 shows the chromatogram for a set of standards containing 2.15 pmol of each amino acid. The standards were derivatized in the microvial exactly as described earlier for the cellular fluid. The ratio of the peak area for each amino acid to the area of the norleucine peak in that run was found, and these values were plotted vs the number of moles of amino acid in the three runs to obtain the
TIME(min)
Figure 2. Chromatogram of NDA-tagged amino ackl standards. Peaks 1-17 correspond to those listed in Table I, while 18 and 19 are the internal standards norleucine and normetanephrine, respectively. calibration curve for each amino acid. The calibration data varied from day to day. The relative standard deviation of the ratio of the peak area of each amino acid to the area of norleucine in that run was determined for each concentration by examining three calibration curves done during a 2-week period. The average of the values for all the amino acids was 23.6% at 0.15 pmol, 23.7% at 0.55 pmol, and 19.6% a t 2.15 pmol. Due to this lack of precision at the individual analyte concentrations, a calibration curve was constructed each day that cell analyses were performed to get the most reliable results. Table I gives the slope, intercept, and correlation coefficient found for each amino acid from a typical set of calibration curves obtained on one day. These values were determined by using the equation for a straight line. The correlation coefficients range from 0.9980 for aspartic acid to 1.000 for asparagine, glycine, tyrosine, and methionine. The electrode was electrochemically cleaned between each chromatographic run by using a method developed in this lab (20). A triangular potential wave from 0 to 1.8 V at 1 V/s was applied to the electrode for 30 s. This was necessary because NDA derivatives appear to foul the electrode (IO). The number of moles of each amino acid present in the snail cell were obtained by calculating the ratio of the peak area of each amino acid to the peak area of norleucine in that run. Using the calibration curve, this yielded a value for each amino
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990
Table 11. Amino Acid Profiles of Single Snail Neurons
measd amt, fmol F1 D2
peak
amino acid
E4
1
ASP Glu Asn Ser Gln His
500 f 100 1300 f 440 300 f 130 950 f 370 1900 f 1100 320 f 55 870 f 300 290 f 24 4200 f 2400 39 f 14 260 f 100 200 f 63 120 f 57 69 f 28 170 f 46 380 f 160 250 f 150
2
3 4
5
-
6 I
8
9 10 11 12
13 14 15 16 1:
G~Y
Thr Ala Arg TYr Val Met TrP Tile Phe Leu
300 430 500
570 930 110 510 140 2100 73 80
150 48 19 110 290 160
560 6000 940 2300 14000 1000 2400 920 25000 56 500 950 210 130 860 1300 870
method (7) yielded relative standard deviations (RSD) ranging from 30.6% for dopamine to 77.3% for tyrosine. Also, the RSD of serotonin as measured in leech neurons by GC/MS was 60% ( 4 ) ,while an RSD of 17.8% was obtained for dopamine by using HPLC with electrochemical detection for a different leech neuron (22). The RSDs found in Table I1 range from 8% for Thr to 59% for Leu. The source of the variations may be due to experimental error or biological variation. One analytical error is possible contamination of the analyzed cells by other, smaller cells that adhere to it during the dissection process. Also, the use of a single internal standard to account for all sample losses may lead to erroneous results. The internal standard should be as similar as possible to the analytes of interest to accurately reflect the behavior of the analytes. For a sample as diverse as one that contains may of the amino acids, this would require many internal standards. Biological factors that could produce variance include differences in the neurons a t the time of dissection and differences in the sizes of the cells analyzed.
CONCLUSIONS Table 111. Comparison of Amino Acid Values Obtained from Individual H e l i x aspersa Neurons by Two OTLC MethodsD
cell
method
D2
original with deriv original with deriv original with deriv
E4 F1
measd amt, fmol TY r TrP 340 f 98 500 550 f 420 260 f 100 490 f 140 80
160 f 20 130 59 f 22 69 f 28 89 f 24 19
"All values from the original method are the average of five runs with the standard deviation given to the side. Only the values for cell E4 from the derivatization method are the average of five runs. The values for D2 and F1 are from single runs. acid that was uncorrected for sample losses. This uncorrected value was then divided by the ratio of normetanephrine detected to normetanephrine added. Without the use of normetanephrine as an internal standard, considerably lower values for the amino acids would have been obtained due to the substantial amount of sample loss that apparently occurs. Table I1 gives the amino acid profiles obtained for seven cells, five of cell E4 and one each of D2 and F1. For all three types of cells, alanine is present at the highest level. This is not surprising since pyruvate, when not needed by the cell for energy production via the tricarboxylic acid cycle, can be stored in the form of alanine (21). The high levels of amino acids in cell D2 as compared to the other cells were due to this particular specimen's large size. The accuracy of these results is difficult to assess because very little quantitative work has been done with amino acids at the single cell level. However, Table I11 compares the results obtained from the original method (7) developed in this lab (OTLC with no derivatization) to those obtained with the method described here. Note the close agreement for both tyrosine and tryptophan between the two sets of E4 measurements. The comparison is not as valid for D2 and F1 due to the lack of repetitive measurements for these cells. Relative amounts of amino acids can be compared to the results obtained by Osborne using the micro-TLC method on similar neurons in Helix pomatia (3). The micro-TLC technique did not permit absolute quantitation. Osborne found that alanine was present at the highest level by approximately a factor of 2 over glycine, followed by glutamic acid. The standard deviations seen in Table I1 for the five E4 cells are comparable to what has been reported for other methods used for single-cell analysis. The original OTLC
There are several problems with this technique. The most disturbing is the loss of lysine, dopamine, and serotonin during sample preparation. Another drawback is that the derivatizing reagent (NDA) must be purified prior to use. The purification procedure is necessary simply because NDA is not commercially available in a pure enough form for the single-cell work. In spite of these limitations, the method presented in this report is a valuable new technique for the quantitative analysis of amino acids in single cells. The total volume present after derivatization and dilution is approximately 25 nL, the smallest volume we are aware of for any derivatization scheme. The fact that only about 25% of the final volume was injected onto the column suggests that cells much smaller than the giant snail neurons can be analyzed. This method has the advantages of sensitivity, with detection limits in the attomole M) (IO),and selectivity for a particular range (about 5 x class of compounds due to the derivatization process. It has also been shown to be a t least as reliable as other methods developed for single-cell analysis.
ACKNOWLEDGMENT We thank Waters Associates (Milford, MA) for the gift of the 600E Multisolvent Delivery system. Registry No. Asp, 56-84-8; Glu, 56-86-0; Asn, 70-47-3; Ser, 56-45-1; Gln, 56-85-9; His, 71-00-1; Gly, 56-40-6; Thr, 72-19-5; Ala, 56-41-7;Arg, 74-79-3;Tyr, 60-18-4; Val, 72-18-4; Met, 63-68-3; Trp, 73-22-3; Ile, 73-32-5; Phe, 63-91-2; Leu, 61-90-5.
LITERATURE CITED (1) Osborne, N. N. I n Biochemistry of Characterized Neurons; Osborne, N. N., Ed.; Pergamon: New York, 1978. (2) Briel, G.; Neuhoff, V.; Osborne, N. N. Int. J. Neurosci. 1971, 2 , 129-136 . -. . - -. (3) Osborne, N. N.; Szczepaniak, A. C.; Neuhoff, V. Int. J. Neurosci. 1973. 5 . 125-131. (4) McAdoo, D. J. I n Biochemistry of Characterized Neurons; Osborne, N. N., Ed.; Pergamon: New York, 1978. (5) Abramson, F. P.; McCaman, M. W.; McCaman, R. E. Anal. Biochem. 1974, 5 7 , 482-499. (6) Kennedy, R. T.; St. Claire, R. L.; White, J. G.; Jorgenson, J. W. Mikrochim. Acta 1987, 2 , 37-46. (7) Kennedy, R. T.; Jorgenson, J. W. Anal. Chem. 1980, 61, 436-441. (8) de Montigny, F.; Stobaugh, J. F.; Givens, R . S.; Carlson. R. G.; Srinivasachar, K.: Sternson, L. A.; Higuchi, T. Anal. Chem. 1987, 59, 1096-1101. (9) Roach, M. C.; Harmony, M. D. Anal. Chem. 1987, 59, 411-415. (10) Oates, M. D.; Jorgenson, J. W. Anal. Cbem. 1989, 67, 432-435. (11 ) Kennedy, R. T.; Oates. M. D.;Cooper, B.R.; Nickerson, B.; Jorgenson, J. W. Science, 1989, 246, 57-63. (12) Knecht, L. A.; Guthrie, E. J.; Jorgenson, J. W. Anal. Chem. 1984. 56. 479-482. (13) St. Claire, R . L. Ph.D. Thesis, University of North Carolina at Chapel Hill, 1986. (14) White, J. G.; St. Claire, R . L.;Jorgenson, J. W. Anal. Chem. 1988, 58 293-298 .., -. . -. -. (15) Kerkut, G. A.; Lambert, J. D. C.; Gayton, D.; Walker, R. J. Comp. Biochem. Physiol. A 1975, 50A, 1-25.
Anal. Chem. 1990, 62, 1577-1580 Walker, R. J. Exp. Physiol. Biochem. 1974, 7 , 331-345. Kennedy, R. T.; Jorgenson, J. W. Anal. Chem. 1988, 60, 1521-1524. Stobaugh, J. F. Personal communlcatlon, University of Kansas, 1988. Pelletier, S. W.; Chokshl, H. P.; Desal, H. K. J . Nat. Prod. 1986, 4 9 , 892-900. (20) St. Claire, R. L.; Jorgenson, J. W. J . Chromatogr. Sci. 1985, 23, 186-191. (21) Gardiner, M. S. I n Mc&aw-Hi// Series in Organismic B / ~ /Fuller, ~ ~ ; M. S.,Llcht, P., Eds.; McGraw-Hill: New York, 1972.
(16) (17) (18) (19)
1577
(22) Lent, C.M.; Meuller, R. L.; Haycock, D. A. J . Neurochem. 1973, 4 7 , 48 1-490.
RECEIVEDfor review December 27,1989. Accepted April 30, W3l Support for this work was provided by the National Institute of Health under Grant No. GM39515.
Quantitative Amino Acid Analysis of Subnanogram Levels of Protein by Open Tubular Liquid Chromatography Mary D. Oates and James W. Jorgenson*
Chemistry Department, University of North Carolina, Chapel Hill,North Carolina 27599-3290
A method Is descrlbed for the hydrolysis and quantltatlve amlno acid analysis of as llttle as 0.1 ng (4 fmol) of proteln. Hydrolysis Is performed by uslng a gas-phase method. The resultlng amlno aclds are derlvatlzed with naphthalene-2,3dlcarboxaldehyde and then analyzed by open tubular llquld chromatography wHh amperometrlc detection. The total volume present afler derlvatlzatlon Is approxknately 25 nL. To our knowledge this Is currently the lowest level of proteln to be quantltatlvely analyzed, as well as the smallest volume In which derlvatlzatlon has been accompllshed. I t was possible to quantitatively determine 14 amlno aclds with an error of 5.0% for a known protein and 8.5% for a protein whose ldentlty was unknown to the researcher.
INTRODUCTION The ability to determine the amino acid composition of ever smaller quantities of protein has been an important trend in recent years. It is now possible to routinely obtain quantitative results at the low picomole level (1-3), but a need to work with still smaller quantities of protein is evident. Several factors have led to this decrease in the amount of protein required for amino acid analysis. The development of more sensitive derivatizing reagents for amino acids has played a large role. These include dansyl chloride (4),fluorescamine (5),phenyl isothiocyanate (6), o-phthalaldehyde (7-9), and naphthalene-2,3-dicarboxaldehyde(IO, 1I). The development of smaller scale systems for sample handling and analysis has also been vital. Microcolumn liquid chromatography, for example, is ideally suited for the determination of amino acids from small quantities of protein due to its small volume requirements. Typical injection volumes for microcolumns are 5-10 nL. A microinjector capable of dispensing a few nanoliters directly onto an open tubular column was recently developed in this laboratory (12, 13). The use of this microinjector with open tubular liquid chromatography (OTLC) has allowed for the development of a quantitative method for the hydrolysis and analysis of as little as 0.1 ng (4 fmol) of protein. This report describes a gas-phase method for the acid hydrolysis of 0.1 and 1 ng (4 and 40 fmol) of bovine chymotrypsinogen. The hydrolysis products are derivatized with naphthalene-2,3-dicarboxaldehyde(NDA) in a total volume of approximaely 25 nL and analyzed by using OTLC with electrochemical detection. To our knowledge this is currently
the lowest level of protein to be quantitatively analyzed, as well as the smallest volume in which derivatization has been accomplished. While there are reports in the literature of 5-15 ng of protein being qualitatively analyzed with microcolumn methods (14, 15), the hydrolysis was performed in 1mL of HC1, and the totalsample volume following derivatization WBS a few microliters. Dissolving a nanograms in a few nanoliters means that while the amount of protein used in the analysis is very small, the concentration of amino acids in the injection volume is quite high. For the same amount of protein hydrolysate dissolved in 1p L , the concentration of amino acids would be 2-3 orders of magnitude less. Also, gas-phase hydrolysis has been shown to have several advantages over the more common liquid-phase method, including easier sample handling and less chance of contamination by the HC1 ( I ) . With this method it was possible to determine the number of residues for 14 amino acids in both 1and 0.1 ng of bovine chymotrypsinogen, a known protein, as well as determine the amino acid composition of a protein whose identity was unknown to the researcher.
EXPERIMENTAL SECTION Apparatus. The chromatographic system and electrochemical detector used were identical with those described in the article immediately preceding this one. The analytical column used had an inner diameter of 15 pm, was 100 cm long, and had octadecylsilane bound to its superficially porous glass walls. Reagents. HPLC grade acetonitrile (Fisher Scientific Co., Fair Lawn, NJ) was used as received. Amino acids and proteins were obtained from Sigma (St. Louis, MO) while reagent grade sodium cyanide was purchased from Aldrich (Milwaukee, WI). All water used was purified by a Barnstead water purification system. NDA was obtained from Polysciences, Inc. (Warrington, PA), and was purified as described in the first article. Buffers and stock solutions were also prepared as described earlier. Procedures. Protein Hydrolysis. One milligram of protein was accurately weighed and dissolved in 1 mL of water. The protein solution was then diluted with water to the desired concentration. An internal standard, norleucine, was added at this point to compensate for any loss of the amino acids that occurred during or after the hydrolysis. The same quantity of internal standard was also added to all standard mixtures used to make the calibration curves. All standards were taken through the following hydrolysis procedures in a manner identical with the protein. Five nanoliters of the protein or standard solution was dispensed into a 200-nL microvial, which was then placed on top of a warm oven to allow for evaporation of the water. All additions to the microvial were made using a pneumatic microsyringe. The syringe was made of borosilicate glass and was calibrated by measuring the size of water droplets dispensed into
0003-2700/90/0362-1577$02.50/0 0 1990 American Chemical Society
