Anal. Chem. 1990, 62, 2056-2058
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TECHNICAL NOTES Modification of Lysine Residues in Proteins To Improve Their Recovery When Using Derivatizing Reagents M a r y D. Oates a n d J a m e s W. Jorgenson*
Chemistry Department, University of North Carolina, Chapel Hill, North Carolina 27599-3290
INTRODUCTION Peptide and protein analyses require the ability to quantitatively determine the amino acids present in the molecule of interest. This is usually accomplished by using liquid chromatography with either a pre- or postcolumn derivatization of the amino acids (1-4). Derivatization is necessary so that all of the amino acids can be sensitively detected with a single detection method. Recently, o-phthalaldehyde (OPA), which reacts with primary amines in the presence of a thiol to form fluorescent products, has come into wide use for the analysis of amino acids (5-10). An analogue of OPA, naphthalene-2,3-dicarboxaldehyde(NDA), which improves upon several aspects of OPA (NDA derivatives are more stable and have higher quantum efficiencies of fluorescence than OPA derivatives) is beginning to find use for the same application (11-14). NDA has been used in this laboratory for the quantitative analysis of the amino acid content of subnanogram amounts of protein (15). The total sample volume present in these analyses is approximately 25 nL. A microinjection system developed in this laboratory is used to apply this volume directly onto the analytical column. A borosilicate glass micropipet containing the sample is inserted into the inlet end of the column and pressure is applied to force the sample onto the column. A difficulty is encountered with this injection method for compounds, such as lysine, which react twice with the derivatizing reagent. I t was found that doubly tagged lysine could not be recovered from the micropipet, perhaps due to adsorption to the glass. Lysine tagged only once with NDA does not exhibit this behavior, however. Modification of the protein prior t o hydrolysis to block the t-amine group of all the lysine residues would result in a lysine product on hydrolysis that could react only a t the a-amino site. This report describes the use of reductive alkylation to convert lysine residues in proteins to their t-dimethyl derivatives. While this procedure is known to protein chemists, it has not previously been used to modify lysine residues to prevent double tagging by derivatizing reagents, thus improving recovery of the lysine. A second problem with lysine that has been tagged twice with either OPA or NDA is a substantially reduced quantum efficiency of fluorescence when compared to singly tagged species (12, 13, 16-18). For example, the quantum efficiency of lysine tagged twice with NDA has been reported to be 0.02, while it was 0.8 for glycine and 0.75 for alanine under the same experimental conditions (13). Lysine doubly tagged with OPA was shown to fluoresce with one-tenth the intensity of other OPA-labeled amino acids (16). I t is thought that an efficient intramolecular relaxation process, resulting from hydrophobic interaction of the two aromatic rings, causes quenching of the fluorescence. The inclusion of surfactants in the derivatization solution has been found to partially reduce the quenching problem for OPA-lysine (16-18), although similar attempts for NDA-lysine have proven unsuccessful (12). Monosubstituted lysine, however, exhibits a fluorescence response equivalent to that obtained from other amino acids
for both OPA and NDA derivatives (13, 16). The protein modification procedure described here can also be used to prevent this problem from occurring.
EXPERIMENTAL SECTION Apparatus. The chromatographic system used in this work has been described previously (14, 23). A Waters 600E multisolvent delivery system was used to provide mobile phase to the column at a flow rate of 1 mL/min. A splitting system was used to divert the majority of the mobile phase to waste. A fused silica capillary column, 25 cm long and with a 42 pm inner diameter, packed with 5-pm porous spheres (C-8Spherisorb,Phase-Sep Co.), served as the analytical column (24). The electrochemical detector used has also been described previously (23, 25). A carbon fiber, 0.7 mm long and 9 pm in diameter, was inserted into the outlet end of the capillary column and served as the working electrode. All chromatograms were obtained in the amperometric mode with a working potential of 0.9 V vs a AgjAgC1 reference electrode. Data were acquired through the use of a microcomputer. A Model 427 current amplifier (Keithiey Instrument, Inc., Cleveland, OH) with a 300-ms rise time and a model 3341 low pass filter (Krohn-Hite Corp., Avon, MA), set at 10 Hz, were also used. The capillary electrophoresis system used in the fluorescence experiments has also been described elsewhere (26). The fused silica capillary had an inner diameter of 25 pm and was 55 cm in total length. The distance from injection to detection was 34 cm. The injections were made a t -5 kV for 5 s at the grounded end of the capillary. The voltage applied during runs was -15 kV. The running buffer was 25 mM sodium phosphate monobasic, pH 7.0. Detection was accomplished on-capillary with a heliumcadmium laser with an excitation wavelength of 442 nm. An emission filter with a cut-on wavelength of 500 nm was also used. This was not optimized for use with NDA derivatives. Reagents. HPLC grade acetonitrile (Fisher ScientificCo., Fair Lawn, NJ) was used as received. Amino acids and chymotrypsinogen were obtained from Sigma (St. Louis, MO), while reagent grade sodium cyanide was purchased from Aldrich (Milwaukee, WI). NDA was obtained from Molecular Probes (Eugene, OR) and used as received. The dimethyllysine standard was purchased from Chemical Dynamics Corp. (South Plainfield, NJ). All water was purified by a Barnstead water purification system prior to use. 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. Stock Solutions. Amino acid stock solutions were dissolved in water and stored in the refrigerator for up to 1 week. The protein stock solution was made daily by dissolving 5 mg of chymotrypsinogen in 1 mL of the pH 7.0 phosphate buffer. NDA solutions, made in acetonitrile, were prepared fresh daily, while the cyanide and borate buffers were used for up to 1 month. Protein Modification and Hydrolysis. Bovine chymotrypsinogen was modified prior to acid hydrolysis by using the method described by Jentoft and Dearborn (22). To 10 pL of a stock solution of 2 x M protein was added 1 pL of 0.3 M sodium cyanoborohydride. After mixing, 1 pL of 0.3 M formaldehyde was added and this was allowed to react at room temperature for 2 h.
0003-2700/90/0362-2056$02.50/00 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62, NO. 18, SEPTEMBER 15, 1990
I N
G
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I
0.3nA
dimethyl
\,
Lys S
I
dim-Lys
I -
A U U
R
b
,/" 4 52
I LYs
TIME [min]
Flgure 1. Chromatogram of the NDA-tagged hydrolysis products of bovine chymotrypsinogen. The peaks are labeled according to their one-letter abbreviations.
After the modification reaction was completed, 5 pL of the modified protein solution was added to 10 p L of 6 N constant boiling HCl (Pierce, Rockford, IL) containing 1% phenol and 2 x lo-' M norleucine. The phenol improves the yield of tyrosine (27) while norleucine was added as an internal standard. The hydrolysis tube was evacuated for exactly 1 min and then placed in a 115 "C oil bath for 24 h. The hydrolysis mixture became a deep red color several hours after heating began. This occurred only if the modification reagents and phenol were both present and did not affect the amino acid analysis. The HCl was evaporated after hydrolysis by use of heat (80-90 "C) and a stream of nitrogen. The residue was dissolved in 10 p L of water. Derivatization. Standards and protein hydrolysates were derivatized with NDA by first adding 0.6 p L of 0.1 M cyanide to 1 p L of the amino acid mixture. Then 2.8 pL of 0.1 M borate containing 10% acetonitrile was added, followed by 0.6 p L of 0.1 M NDA. The reaction was allowed to proceed for 5 min at room temperature. The mixture was then diluted 1:l with the initial mobile phase. Sample injections were performed by use of a pneumatic micropipet (28). The pipet was made of borosilicate glass. After the tip was filled with approximately 20 nL of sample, the pipet was inserted into the inlet end of the column until a tight seal was obtained between the column and pipet. Several nanoliters of sample were then forced onto the column. Injections were accomplished with the aid of a Wolfe Selectra I1 stereomicroscope, a Brinkman micromanipulator, and an Oriel micropositioner.
RESULTS AND DISCUSSION Reductive alkylation has been used by protein chemists since the late 1960s to convert amino acid groups in proteins to their alkylamine derivatives (19). The reaction is often performed using formaldehyde as the alkylating reagent and sodium cyanoborohydride as the reducing agent (20,21). This results in the formation of a dimethyl product, as shown in eq 1 (22). The reaction is both quantitative and very specific; only the terminal amine group and lysine residues are modified (20, 2!+31).
GHCHO + 3Prot-NHz + 2NaCNBH3 3Prot-N(CH&
-
+ 2HCN + 2NaH,B03
(1)
Figure 1 demonstrates the use of this modification procedure. This is a chromatogram resulting from the microinjection of the NDA-tagged hydrolysis products of bovine chymotrypsinogen, which contains 14 lysine residues. Mobile phase A was 85% pH 7 phosphate buffer/l5% tetrahydrofuran, while mobile phase B was 65% acetonitrile/20% methanol/l5% 0.01 M phosphate buffer. The gradient was from 95 A/5 B to 67 A133 B linearly in 30 min to 50 A150
4
8 TIME[min]
12
16
Flgure 2. (a) Injection of NDA-labeled tdimethyllysine, norleucine, and lysine from a solution volume of 1 mL. (b) Microinjection of a few nanoiiters of the same solution as in part a.
Table I. Comparison of a Large Injection and Microinjection of e-Dimethyllysine and Lysine, Both Tagged with NDA" ratio to norleucine dimethyllysine lysine
large injection microinjection
0.957 0.974
0.832 0.166
"The data were obtained from Figure 2. B linearly in 50 min. The separation was optimized for lysine, resulting in the overlap of valine with methionine and isoleucine with phenylalanine. Further changes in the gradient used could have resolved all of the amino acids. The purpose here was to emphasize the quantitative recovery of lysine. The modification procedure is apparently quantitative. The numbers of lysine residues found in three separate hydrolyses were 15.0, 15.2, and 12.5. The average for these three experiments is 14.2, which matches the correct value of 14. Quantitation was accomplished using a calibration curve with norleucine as an internal standard by a method described previously (14). Many samples, particularly of biological origin, provide very small analysis volumes, requiring a microinjection method (32, 33). The technique developed in this laboratory for microinjection uses a borosilicate glass micropipet to directly apply a few nanoliters of sample to the column (28). While analyzing small volumes of protein hydrolysates, we found that lysine, after being tagged twice with NDA, could not be quantitatively recovered from the micropipet. This is demonstrated in Figure 2. Both chromatograms show the separation of NDA-tagged t-dimethyllysine, norleucine, and lysine, in that elution order. The first two are tagged mce with NDA, while lysine is labeled twice. Mobile phase A in this experiment was 0.05 M phosphate buffer, pH 7.0, while mobile phase B was acetonitrile. The gradient was a linear increase from 65 AI35 B to 50 AI50 B in 10 min. The upper trace is the result of a large injection; i.e. 1 mL of solution was used to fill the tee in which the column was placed. A splitting system then diverts all but a few nanoliters of the sample to waste (14,23). The sample is introduced into the tee by using a 1-mL glass syringe. The lower chromatogram was obtained by microinjecting a portion of the same solution as in Figure 2a using the glass micropipet.
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groups. An even more useful modification procedure would block only the t-amine group of free lysine residues, leaving all other free amine groups unaltered.
dimethyl-Lys
ACKNOWLEDGMENT We thank Dr. R. G. Hiskey of the University of North Carolina in Chapel Hill for suggesting the alkylation procedure for the modification of lysine residues in proteins. We also thank Waters Associates (Milford, MA) for the gift of the W E multisolvent delivery system. Registry No. L-LYS,56-87-1; NDA, 7149-49-7; NDA-Lys, 128388-13-6; dimethyl-Lys, 2259-86-1; NDA-dimethyl-Lys, 128388-12-5;chymotrypsinogen, 9035-75-0.
LITERATURE CITED Spackman, D. H.; Stein, W. H.; Moore, S . Anal. Chem. 1958, 30,
I
3
1190-1206. TIME ( m i d
Figure 3. Capillary zone electrophoresis with laser-induced fluorescence detection of loa M dimethyllysine, labeled once with NDA, and M lysine, tagged twice with NDA.
Table I compares the ratios of the area of each lysine peak to the area of the norleucine peak in that run. The ratio for dimethyllysine actually increased slightly in the microinjection, while the ratio for doubly tagged lysine fell by a factor of 5 in going from the large injection to the microinjection. Lysine labeled twice with NDA must in some way be adsorbing to the borosilicate glass of the micropipet. The amount of doubly labeled lysine recovered is not constant from injection to injection, ranging from none to about 50% of the amount of lysine present. This makes quantitation of unmodified lysine in protein hydrolysates impossible with the ultramicroscale techniques developed in this laboratory. However, if the protein modification described here is performed prior to hydrolysis, quantitation of the dimethyl derivative is easily accomplished, as demonstrated in Figure 1. To confirm the usefulness of this modification procedure for the prevention of fluorescence quenching, a mixture of lo4 M lysine tagged once with NDA and lod M lysine tagged twice with NDA was injected onto a capillary electrophoresis system equipped with laser-induced fluorescence detection. Figure 3 shows this electropherogram. The peak area for the singly tagged lysine was 1024, while the peak area for the doubly tagged lysine was 211. Peak area is reported in arbitrary units. Therefore, for an eqimolar solution of the two species, the dimethyllysine would give a fluorescence response approximately 50 times larger than that for lysine which is tagged twice with NDA. The modification procedure described here would result in lysine residues following protein hydrolysis which could be labeled only once with any amine derivatizing reagent, resulting in increased fluorescence over the doubly labeled derivative. The method described in this report has several important limitations. One is that the a-amine group of the terminal amino acid of the protein is modified along with the lysine residues, so that the terminal amino acid is lost to the analysis. The greatest limitation is that the modification can only be applied to lysine residues in proteins (not free lysines), because the reaction will cause the methylation of all free amine
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RECEIVED for review January 11,1990. Accepted May 31,1990. Support for this work was provided by the National Institutes of Health under Grant Number GM39515.
