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
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990
mineral oil, as described previously (22). The gas-phase hydrolysis procedure used was adapted from one described in the literature (29). The microvial containing only the protein residue (no water) was placed in a 0.5-mL test tube. The test tube was put into a 25-mL vial (Pierce, Rockford, IL) containing 0.5 mL of constant boiling 6 N HCl (Pierce). An argon stream was bubbled through the HCl for 30 s and then directed into the test tube for 15 s. The vial was capped and placed in a 165 “C oven for 40 min. The microvials were removed from the larger vial immediately after the hydrolysis to prevent HCl from condensing inside them. (Caution: 25-mL hydrolysis vials other than the ones recommended here have been known to explode while in the oven). Derivatization Procedure. The proteins and standards were derivatized in an identical manner following the hydrolysis procedure. The residue in the microvial was redissolved in 5 nL of water, followed by the addition of 4 nL of sodium cyanide solution. Next, 7 nL of 0.1 M boric acid, pH 9.5, was added. Finally, 2 nL of NDA, dissolved in an 80/20 mixture of acetonitrile and 1propanol, was added to the microvial. The concentration of the cyanide and NDA used in the derivatizations depended on the amount of protein hydrolyzed. For 1ng of protein, the cyanide concentration was 0.5 M, and the level of NDA was 0.1 M. When these reagent concentrations were used with the 0.1-ng hydrolysis products, however, large amounts of precipitate were formed. The identity of the precipitate is not known; however, it resulted in electrochemicalsignals for the NDA derivatives that were lower than expected. Therefore, for the case of 0.1 ng of protein hydrolysate, the concentrations of cyanide and NDA were lowered to 0.1 and 0.01 M, respectively. The mixture was allowed to react for 5 min after the NDA was added. The microvial was cooled during both the addition of reagents and the reaction time to prevent evaporation. A small piece of Parafilm was placed over the open end of the microvial to further reduce the evaporation rate. After 5 min, 4 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 4-5 nL was then 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.
RESULTS AND DISCUSSION The amino acid composition of bovine chymotrypsinogen A was determined for 2 different amounts of protein, namely, 1 and 0.1 ng. Also, the amino acid content of a protein whose identity was unknown to the researcher was determined in order to better evaluate the usefulness of the method in a more realistic situation. Quantitation. Quantitation was accomplished through the use of calibration curves and an internal standard. Norleucine was chosen as the internal standard because it is well resolved from the amino acid peaks chromatographically and is available in high purity. The method of quantitation used for the two amounts of protein was identical; only the levels of amino acids used in the standard mixtures differed. For 1 ng of protein, the standards used contained 0.17,0.85, and 1.7 pmol of each amino acid, with 0.85 pmol of norleucine present in each. Norleucine (0.85 pmol) was also added to the protein prior to the hydrolysis. For 0.1 ng of bovine chymotrypsinogen, the levels of the amino acids as well as the internal standard were decreased by a factor of 10. A calibration curve was constructed each day that proteins were analyzed. The standards and five protein samples were “hydrolyzed” a t the same time in the same 25-mL vial. It was found that “hydrolyzing” the standards produced more accurate results than if the standards were simply derivatized without subjecting them to hydrolysis conditions. This was particularly true for the amino acids threonine and serine, presumably because they are partially destroyed by acid hydrolysis. Taking the standards through the same procedure
Table I. Data from a Typical Calibration Curve amino acid ASP
Glu Ser His
Thr GlY
Ala TYr Arg Val Met Ile Phe Leu
ratio to norleucine fmol 170 fmol slope
17 fmol 85 0.389 0.294 0.357 0.510 0.243 0.622 0.448 0.382 0.243 0.336 0.372 0.210 0.199 0.216
1.92 1.73 1.74 2.51 1.36 2.08 1.78 1.89 0.803 1.64 1.36 1.02 0.728 1.04
3.62 3.37 3.48 5.30 3.11 4.53 4.14 3.79 1.97 3.27 2.97 1.90 1.63 2.04
0.0211 0.0201 0.0204 0.0314 0.0188 0.0257 0.0243 0.0223 0.0114 0.0192 0.0171 0.0110 0.0094 0.0119
int
corr
0.0658 -0.0218 0.0081 -0.0708 -0.1348 0.0826 0.0787 0.0008 -0.0271 0.0100 0.0205 0.0444 -0.0003 0.0184
0.9994 0.9997 1.000 0.9995 0.9980 0.9967 0.9955 1.000 0.9907 1.000 0.9973 0.9992 0.9964 1.000
should a t least partially compensate for any losses that occur to these protein residues. Following uhydrolysis”the standards were derivatized in the microvial exactly as described earlier for the protein hydrolysate. 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 each amino acid in the three calibration runs. This produced a 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 1-month period. The average of the values for all the amino acids was 10.8% a t 17 fmol, 17.1% a t 85 fmol, and 13.1% a t 170 fmol. Similar results were obtained for the calibration curves done with 10 times more amino acid. Due to this lack of precision a t the individual analyte concentrations, a calibration curve was constructed each day that proteins were analyzed to get the most reliable results. Table I gives the slope, intercept, and correlation coefficient found for each amino acid from one set of calibration curves. These values were determined by using the equation for a straight line. The correlation coefficients range from 0.9907 for arginine to 1.000 for serine, tyrosine, valine, and leucine. The electrode was electrochemically cleaned between each chromatographic run by using a method developed in this laboratory. A triangular potential wave from 0 to 1.8 V vs Ag/AgCl was applied to the electrode for 30 s. This was necessary because NDA derivatives appear to foul the electrode ( 2 1 ) . Chromatograms obtained from the NDA-tagged hydrolysis products of 1 and 0.1 ng of bovine chymotrypsinogen are shown in Figure 1. Note the good S / N obtained from 0.1 ng of protein, indicating that still smaller quantities of protein could be quantitatively analyzed. A gradient was used to separate all of the amino acids in a reasonable time. Mobile phase A was 100% 0.05 M phosphate buffer, p H 7.0, and mobile phase B was pure acetonitrile. A linear gradient from 100% A t o 50% A/50% B in 50 min was used. Due to the acid hydrolysis, asparagine and glutamine are converted to aspartic acid and glutamic acid, respectively, and tryptophan is totally destroyed. Lysine is lost during the derivatization that follows the hydrolysis procedure. Lysine is tagged twice by NDA, and may be lost due to adsorption to the glassware (micropipet or microvial) or through precipitation from solution. Table I1 gives the quantitative results from five separate hydrolyses for both 1 and 0.1 ng of chymotrypsinogen. The ratio of the area of each amino acid peak to the area of the norleucine peak in that run was used to determine the number of moles of each amino acid present. T o determine the
qALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990
6 6
a
TIME[min]
1579
7 1
TIME[min] 6
15
Figure 2. Chromatogram of the NDA-tagged hydrolysis products of 1 ng of the "unknown" protein (lysozyme). The peak numbers correspond to the amino acids listed in Table 11. Peak 15 is the internal standard norleucine.
Table 111. Amino Acid Composition of "Unknown" Protein (Lysozyme)
I/
no. of actual
no. of found
amino acid
residues
residues"
1 2
Ask
21
Glx
3
Ser His Thr
5 10
22.3 f 3.25 4.70 f 0.721 9.00 f 1.85
peak
4
5 6 7
8 9 10 TIME[rnin]
11
Figure 1. Chromatograms of the NDA-tagged hydrolysis products of (a) 1 ng of bovine chymotrypsinogen and (b) 0.1 ng of bovine chymotrypsinogen. The peak numbers correspond to the amino acids listed in Table I . Peak 15 is the internal standard norleucine.
13 14
12
Table 11. Amino Acid Composition of 1 and 0.1 ng of Chymotrypsinogen actual no, of
peak
amino acid
residues
1 2
Asx
3 4 5 6 7 8 9 10
Ser His Thr
23 15 28
11 12
Met Ile Phe
13 14
Glx
GlY Ala Tyr Arg Val
Leu
no. of found residues" (fsd) 1 ng 0.1 ng
23.2 f 1.35 16.2 f 1.12 23.7 k 3.13 2.10 f 0.532 20.4 f 3.92 23.0 f 1.14
23.6 f 1.90 12.4 f 0.762 28.5 f 3.47 2.80 f 0.395 24.5 f 1.05 22.7 f 2.01
22
"22"
"22"
4 4 23
4.40 f 0.444 4.10 f 0.498 23.5 f 2.22 1.90 f 0.259 10.6 f 0.826 5.90 f 0.643 19.4 f 0.730
4.48 f 0.402 3.56 f 1.36 21.7 f 1.59 1.30 k 0.424 10.2 f 1.57 5.76 f 0.261 18.1 f 2.73
2
23 23
2 10
6 19
" Based on the assumption of
22 alanine residues; n = 5.
number of residues of each amino acid in the protein, the experimentally determined number of moles of alanine was divided by the actual number of alanine residues present. The other amino acids were divided by this number to yield the
GlY
Ala TYr
1 7 12 12
3
Arg
11
Val Met Ile Phe
6
Leu
2
6 3 8
"1"
7.30 f 1.17 10.8 f 1.25 11.7 f 1.84 2.40 f 0.850 14.3 f 3.96 6.00 f 0.207 1.70 f 0.290 5.30 f 1.11 2.60 f 0.402 7.20 f 1.02
" Based on the assumDtion of 1 histidine residue: n
= 5.
number of residues of each amino acid found. The number used as the ratio was the average value from five separate hydrolyses. At both the 1- and 0.1-ng level, the error from the known amino acid composition was found to be 5.0%. This number was calculated by summing the integer values by which the experimentally determined ratios for each type of amino acid were off from the correct values and dividing this by the total number of residues actually present. The error appears to be random, based on the improvement obtained when the average of five runs is used to determine the ratio rather than a single run. In biological analyses the identity of the protein of interest is often not known. To more effectively evaluate the usefulness of this method for amino acid analysis, the researcher (Oates) was given an unknown protein to analyze. The determination was carried out exactly as described earlier for 1 ng of chymotrypsinogen. Figure 2 shows a chromatogram of the NDA-tagged hydrolysis products of the "unknown" protein. Table I11 gives the quantitative results from five separate hydrolyses of this protein based on a 170-1700 fmol calibration curve done on the same day. Histidine was found to be present in the lowest amount, so it was assumed that there was one histidine residue per protein molecule. The number of moles for each of the other amino acids was then
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Anal. Chem. 1990. 62. 1580-1585
divided by the number of moles of histidine found in that run, yielding the number of residues for the other amino acids. On the basis of the number of residues found (106), a molecular weight of 11660 was estimated for the protein. This was calculated by using the rough assumption that each residue contributes, on average, 110 to the molecular weight (22). The identity of the protein was then disclosed to be lysozyme, with an actual molecular weight of 13 930. The weight calculated for the unknown protein is low due to the amino acids that cannot be measured with this method, i.e., tryptophan, cysteine, lysine, and proline. The amino acid composition of lysozyme is given in Table I11 and compared to what was experimentally found. The error for the average of five runs is 8.5%.
CONCLUSIONS The method presented in this report has been shown to be useful for the hydrolysis and analysis of proteins a t the low femtomole level. The key to this endeavor was the development of methods that can easily handle and analyze a few nanoliters of sample. There are a few problems with the technique, however. The most disturbing is the loss of lysine during the derivatization process (23). 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 work on the nanoliter scale. In spite of these limitations, the method was able to determine the amino acid composition of 0.1 ng (4 fmol) of protein with an error of only 5.0%. Further improvements in sample handling methods may decrease the amount of protein required for amino acid analysis to still lower levels. The least amount of protein that can be analyzed with this method may ultimately depend on the detection limit of the electrochemical detector used, which is approximately 1 amol, or M, at the present time.
Registry No. Asp, 56-84-8; Glu, 56-86-0; Ser, 56-45-1; His, 71-00-1;Thr, 72-19-5;Gly, 56-40-6;Ala, 56-41-7;Tyr, 60-18-4;Arg, 74-79-3;Val, 72-18-4; Met, 63-68-3;Ile, 73-32-5;Phe, 63-91-2;Leu, 61-90-5;NDA, 7149-49-7;cyanide, 57-12-5.
LITERATURE CITED Cohen, S. A.; Bidlingmeyer, B. A.; Tarvin, T. L. Nature 1986, 320, 769-770. Hill, D. W.; Waiters, F. H.; Wilson, T. D.; Stuart, J. D. Anal. Chem. 1979, 57, 1338-1341. Lindroth, P.; Mopper, K. Anal. Chem. 1979, 57, 1667-1674. bjong, c.; Hughes, G. J.; van Weringen, E.; Wilson, K. J. J. ChromatOgr. 1982, 247,345-359. Rubenstein, M.; Chen-Kiang, S.;Stein, S.; Undenfriend, S. Anal. Blochem. 1979, 95,117-121. Koop, P. R.; Morgan, E. T.; Tarr, G. E.; Coon, M. J. J. Biol. Chem. 1982, 257,8472-8480. Roth, M. Anal. Chem. 1971, 43,880-882. Joseph, M. H.; Davies, P. J. J. Chromafogr. 1983, 277, 125-136. Allison, L. A.; Mayer, G. S.;Shoup, R. E. Anal. Chem. 1984, 56, 1089-1096. de Montigny, F.; Stobaugh. J. F.; Givens, R. S.;Carlson, R. G.; Srlnivasachar, K.; Sternson, L. A.; Higuchi, T. Anal. Chem. 1987, 59, 1096-1101. Oates, M. D.; Jorgenson, J. W. Anal. Chem. 1989, 67, 432-435. Kennedy, R. T.; Jorgenson, J. W. Anal. Chem. 1988, 60, 1521-1524. Kennedy, R. T.; St. Claire, R. L.; White, J. G.; Jorgenson, J. W. Mlkrochim. Acta 1986, 7987(II),37-45. Flurer, C.; Borra, C.; Beale, S.;Novotny, M. Anal. Chem. 1988, 60, 1826- 1829. Hskh, Y.yBeale, S.C.; Wiesler, D.; Novotny, M. J. Microcolumn S e p . 1989. 7 . 96-100. Knecht, L. A.; Guthrie, E. J.; Jorgenson, J. W.Anal. Chem. 1984, 56, 479-482. St. Claire. R. L. Ph.D. Thesis, University of North Carolina at Chapel Hill, 1986. White, J. G.; St. Claire, R. L.: Jorgenson, J. W. Anal. Chem. 1988, 56, 293-298. Dupont, D.; Keim, P.; Chui, A,; Bozzini, M.; Wilson, K. J. Appl. Bkasysf. User Bull. 1988, 2. Stobaugh, J. F. Personal communication, Universlty of Kansas, 1988. Pelletier. S.W.; Chokshi, H. P.; Desai, H. K. J. Nat. prod. 1986. 49, 892-900. Lehninger, A. Principles of Biochemistry: Worth: New York, 1982; p 127. Oates, M. D.; Jorgenson, J. W. Unpublished results.
ACKNOWLEDGMENT
RECEIVED for review December 27,1989. Accepted April 30,
We thank Waters Associates (Milford, MA) for the gift of the 600E Multisolvent Delivery system.
1990. Support for this work was provided by the National Institute of Health under Grant No. GM39515.
Axial-Beam On-Column Absorption Detection for Open Tubular Capillary Liquid Chromatography Xiaobing Xi and Edward S. Yeung* Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011 A novel approach has been developed for absorptlon detection In open tubular caplllary liquld chromatography. The axial coupling of source Hght wfth the caplllary columns and the use of optical waveguide capillary columns made It posslble to utilize the full length of the sample bands Inside the capillary columns as the path length for absorbance measurement. Compared with the cross-beam arrangement, the axlal-beam on-colwnn absorption detectlon provides an Increase of up to 1000 tlmes In path length and In associated improvements on the concentration limits of detectlon. This allows lnvestlgation of substantially lower sample concentrations and quantlties than those conventionally feasible for absorption detectlon In open tubular capillary liquid chromatography.
INTRODUCTION Recently, the subject of open tubular capillary liquid
chromatography (OTCLC) has received considerable interest (1-3). Using “separation impedance”, a criterion introduced by Bristow and Knox (41,to assess the theoretical performance of conventional and capillary columns in liquid chromatography, Knox (5) predicted that OTCLC could have the smallest theoretical plate height and the best performance. It is generally accepted that OTCLC with inner diameters of less than 10 pm and employing detectors with low nanoliter volumes and high sensitivities will produce high separation efficiencies in a reasonable time with superior limits of detection (6-8). The advantages of OTCLC with respect to separation speed and efficiency can be exploited only when the external contribution to peak broadening is minimized (6,9). Since the inner diameters of OTCLC columns are normally 10 Fm or less, the injection and detection volumes have to be substantially reduced compared with conventional liquid chromatography to maintain the column efficiency. As far as the
0003-2700/90/0362-1580$02.50/00 1990 American Chemical Society
