QuantitativeDetermination of the Amino Acid composition of a Protein Using Gas Chromatography-Mass Spectrometry Christian S. Hamann. David P. Myers. . Karla J. Wile, Edward F. Wirth, and Owen A. Moe, Jr. Lebanon Valley College. Annville, PA 17003 Open tubular column gas chromatography-mass spectrometrv (GC-MS) is one of the most powerful analytical ~. tools available for the chemical analysis of complexmixtures. The use of a mass s~ectrometeras a detector in GCMS significantly enhances'the capabilities of gas chromatogranhv in that: (a) the mass spectrometer is auniversal detector; (b) the specific inform&on provided in the mass spectrum also makes the mass spectrometer a highly selective detector that can be used forhualitative analysis and structural determination; (c) the mass spectrometer is among the most sensitive chromatographic detectors, having a detection limit on the order of picograms; and (d) the mass spectrometer provides the canacitv to effect spectral resolution of chrom&ugraphirally inresilved components through the use of selected ion monitoring (SIM) (1.2). G C M S , an essential tool i n research in organic chemistry, in environmental trace analysis, and in dmgtesting, is growing in importance in pharmaceutical and biomedical analysis (3).The recent development of reliable, affordable, easily maintained instrumentation has made GC-MS accessible to undergraduate education. Of the 224 public and private nondoctoral chemistry departments surveyed in the directory Research in Chemistry a t Undergraduate Institutions, 97 (43%) ~ - -, had acauired GC-MS instrumentation bv the end of 1988 (4). However, few published experiments employing GC-MS in the undereraduate laboratorv curriculum cur." rently exist ( 5 , 6 ) . The determination of the amino acid composition of proteins has been greatly facilitated by modern chromatogriphic instrumentation. Methods have been developed for the rapid and accurate determination of amino acids-using highperformance liquid chromatography (HPLC) (7,8),GC (9), and GC-MS (10-12). The GC-based methods require derivatization of the amino acids to produce volatile adducts, while the HPLC methods often use derivatization to enhance the sensitivity of detection. For the GC-MS method, individual internal standards are used for each amino acid since the amino acids differ in the efficiencies of derivatization reactions. in the on-column stabilities of their volatile derivatives, i d in mass spectral detector responses (10). The abilitv of MS to discriminate on the basis of mass allows deuterated forms of the amino acids to be used as internal standards (10). The exp&nent presented here uses GC-MS analysis of volatile N-trifluoroacetvl-n-hutylester (TAB) derivatives of amino acids (Fig. I J to-determine rhe amino acid composition of cytocbromec from bovine heart, a protein containing ~~~
438
O
H
II I
H
I
O
II
CF3-C-N-C-C-O-(CH2)3-CH3 I
Figure 1. General chemical structure of the Ntrifiuwoacefyi-c-butyl ester derivative of an amino acid.
~
A oresentation deaiina with this work was aiven in Seotember. 1989 Owen A Moe.. .Ir..-christian S. Hamann..~ a n d~-~ David P. ~ v. e r .s : ~ . "Amino Acid Analysis by Gas Chromatography-Mass Spectrometry," Symposium on NSF-Catalyzed Innovations n the Undergraduate Laboratory. American Chemical Soclefy luational Meet ng, Miami Beach. FL. September 12. 1989. ~~~
TAB Derivative
.
~
~
Journal of Chemical Education
104 amino acid residues of known sequence and composition (13). The TAB derivatization method was selected because established literature methods were available (10, 12). becaude the derivatization process is straightforward, and hecause the TAB derivatives are included in the h'ational Bureau of Standards Mass Spectral Library (Revision E) used for computer searching. This paper provides tested methods for protein hydrolysis, for TAB derivative formation, and for separation and auantitation of amino acid derivatives. The experiment presented here asks students ro carry out the separation, identification, and quantitation of 12 amino acids resulting from the hydrolysis of a protein. Close structural similarities exist among the analytes, making component seoaration and analvsis a ditl'icult task. This exoerim i n t is hesignednot mereiy to&ow students to u s e t h e ' ~ C MS system, but to place the students in a situation where they must fully utilize the capabilities of the instrumentation in order to achieve success. This amino acid analysis experiment is suitable for use in upper level instrumental analysis or biochemistry laboratory courses. ~~~
~
~
Materials A mixture of deuterated algal amino acids (an algal deuterated amino acid mixture is obtained by purifying and hydrolyzing protein from aleae e r o m in deuterated media) was obtained from MSD Isotopes, a'bivkon of Merck Frosst ~ & d a Inc. (Montreal). An enact analysis of the composition of the algal mixture is supplied by Merck Frosat Canada, Inc. upon request. An amino acid calibration standard mixture (2.5 pmollmL) and a 1.0 mL vacuum hydrolysis tube was purchased from Pierce (Rockford,IL).Cytochrome c from bovine heart (Type V-A) and butylated hydroxytoluene (BHT) were obtained from Sigma (St. Louis, MO). Anhydrous dicbloromethane, anhydrous 1-hutanol, acetyl chloride, trifluoroacetic anhydride, and 1.0 mL Wheaton V-vials with Teflon-faced ruhber screw cap liners were purchased from Aldrich (Milwaukee,WI). Protein HyUrolysk The rime requirements for this step are 1-2 h in the laboratory and an unatlended reaction runningov~rnight.A7-12-mg sampleof cyrochrume c is dissolved in 300 g1. of 6 11 HCI, and the resulting
qualitative analysis of amino acids, the reader is referred to a recent article hy Mahhott in this Journal (6). For quantitative analysis, however, a small number of scans per peak means that the true peak maximum is often missed by a spectral scan and that the peaks are non-Gaussian.Integrated areas of such peaks have considerable error, making the SCAN mode unsuitable for quantitation. For quantitative measurements, the mass spectrometer is operated in the SIM (selected ion monitoring) mode in which only certain ion fragments are monitored within any given time window. For example, the TAB derivatives of both ALA and GLY (amino acid three-letter codes are given in Tahle 1)elute in the time range of 12.0 min to 14.5 min. Within this time range, SIM analysis monitors one ion fragment from protonated ALA (140 m u ) , one ion fragment from deuterated ALA (144 amu), one ion fragment from protonated GLY (126 amu), and one ion fragment from deuterated GLY (128 amu). Limiting the number of ion fragments greatly increases the scan rate. In the case of ALA and GLY, only four ion fragments are monitored and the scan rate is 10 scansls. Thus, a peak which is 2-4s wideis comprised of 20-40 spectralscans and has a Gaussian peak shape that can be integrated accurately. The retention times of the amino acid derivatives determined using the SCAN mode are used to develop a SIM data acquisition program. Tahle 1 summarizes a SIM program that we developed for the separation of amino acid TAB derivatives on a 25-m HP-I column. The SIM program includes the time windows used, the amino acid derivatives that elute within each window. the ion fmemenrs monitored within each window, and the massspectral scan rate within enrh window. The protonnted and deurprated iun frapmenu selected fur monitoring in the SIM mode were has& on previous work by 1.eimer er al. 1121. Quantitation is accomplished by creating and integrating single ion rhn,matugrama for each of the ion fragments listed in l'ahle I. Single-ion chromatagrams are constructed through postchromatographic plotting of the SIM data using data station software. For example, in the ease of ALA, two single ion chromatograms are created and integrated: one for the TAB derivative of protonated ALA in which only the signal from the 140 amu ion fragment is plotted versus time in the time range 12.0-14.5 min: and one for the deuterated internal standard in which only the signal from the 144 amu ion fragmentis plotted in the same time window. The use of the integrated areas in determining amino acid composition is explained in the Results section.
deuterated amino acid) is used to construct the single-ion chromatomam shown in Fieure 5(h) and the 240 amu fraement (proionated amino a i d ) is uskd to plot the single-& chromatogram shown in Figure 5(c). Corresponding single ion chromatograms using the 155 amu (deuterated) and 148 amu (protonated) ion fragments of P H E are shown in Figure 5(d) and Figure 5(e), respectively. Figure 5 demonstrates that it is possible t o combine the chromatographic and spectral capabilities of G G M S to completely resolve the TAB derivatives of the deuterated and protonated forms of closely eluting amino acids. T o quantitate the amino acids, single ion chromatograms such as those shown in Figure 5 are constructed for each
THR ALA CLY
SER VAL
PRO LYS TYR
~-
Results Analysis of Standard Amino Acid Mixture A total ion chromatomam of a derivatized calihration mixt&e of standard protonated amino acids and deuterated internal standards is shown in Finure 3. This chromatopam was carried out in the SIM mode using the acquisition parameters aiven in Tahle l. In nome canes excellent chromatographic r&olution of the protonated and deuterated forms of closely eluting amino acids is achieved. Resolution of the structurally similar pair LEU and ILE can be seen in Figure 4 in a n expanded-scale portion (18.0-19.0 min) of the total ion chromatogram from Figure 3. In other cases, such a s in the separation of ASP and PHE (25.9-26.5 min in Fig. 3) or in the separation of LYS, TYR, and GLU (28.5-28.9 min in Fig. 3), significant chromatographic overlap of protonated and deuterated components occurs. I n the latter cases, single-ion chromatograms are constructed and used to resolve spectrally the chromatographic mixtures. An example of the use of single-ion chromatograms to resolve the ASP, PHE group is shown in Figure 5. In this figure, the original chromatographic data collected in the SIM mode yields the total ion chromatogram (expanded scale) shown in Figure 5(a). Recall that this portion of the total ion chromatogram is produced by the measurement and summation of the sign& from ion fragments 240 m u , 243 amu, 148 amu, and 155 amu (see SIM program in Tahle 1). I t can he seen from Figure 5(a) that the protonated and deuterated forms of ASP and P H E are only partially resolved. I n the case of ASP, the 243 amu ion fragment (from the
440
Journal of Chemical Education
ia
38
Retention Time, min
Figure 3. Total ion chromatogram fw the separation of TAB derivativesof me protonated and deuterated amino acids in a standard calibration mixture. OC MS system is operated In the selected ion monitoring (SIM)mode, according to the parameters given In the text and in Table 1.
Retention Time. min Figure 4. Total Ion chromatogram (expandedtime scale)showing the separation of the protonated and deuterated formsof LEU and I1E.
protonated amino acids and deuterated internal standards which are then used to calculate nH, the number of moles of protonated amino acid in the protein: nH = [AREA (H)/ AREA (D)]/[Rlno]. A molecular weight for cytochrome c from hovine heart of 12,327 glmol (13) is used to calculate the moles of protein hydrolyzed.
amino acid and its corresponding deuterated internal standard. The single ion chromatograms that result are then integrated to yield areas of the deuterated and the protonated amino acid derivatives. Integrated single-ion chromatographic data is then used to calculate the response factor, R, for each amino acid:
In this equation the ratio of the integrated areas of the ~rotonatedand deuterated standards is divided by the ratio of the known numbers of moles of protonated and deuterated standards. Sample data for the determination of amino acid response factors are given in Table 2. We have found the determination of response factors to he highly reproducible: a study involving 16 replicate determinations of R for each amino acid in Table 2 eave an averaee relative standard deviation in the mean R d u e of f6%; Experimentally measured R values are needed to evaluate nH, the number of moles of each amino acid in the protein hydrolysate. Determination of the Compositionof Cytochrome C
Total ion chromatograms (not shown) for derivatized mixtures of hydrolyzed cytochrome c containing deuterated internal standards are identical in form to the chromatogram shown in Figure 3. Integration of single-ion chromatographic data from cytochrome c hydrolysate yields the areas of the
Table 2.
(4
240 amu: ASP (HI
-
Determlnatlonot Amlno Acld Resvonse Factors
Amino Acid
Area (H)'
ALA GLY THR SER VAL LEU I E PRO ASP PHE LYS GLU
3085449 1352915 2794041 2265192 2662149 1414226 1347495 5921434 1200063 2233602 2612078 1261454
148 amu: PHE (H)
Retention Time, min
Figure 5. Total ion chromatogram and single ion chromatograms (expanded time scale) lor Me separation ol the protonated and deuterated forms of ASP and PHE. Slngie Ian chromatagrams are for the amino acid form and ion hagment indicated.
injected into the ~olurnn
Table 3.
Amino Acid
26.6
26.0
EXP 1
EXP
2
Representative Amlno Acld Composltlon Data for Cytochrome C
E ~ P
3
Moi AA oer Moi Cvtochrame C E ~ P E ~ P
4
5
EXP
6
Mean Value iSD
Known Value*
ALA GLY THR SER VAL LEU I E PRO ASP PHE LYS
au Ave. Enorb ~~
values taken horn cytochrorne o amino acid sequence given in ref 13. aThe average percent e m is calculated as desnibed in text. a
Volume 66
Number 5 May 1991
441
Representative data from six determinations of the composition of cytochrome c using the method in this paper is presented in Table 3. Each of the six experiments in Table 3 is hased on a single derivatization of a calibration mixture and a single derivatization of an unknown mixture from the cytochrome c hydrolysate. Four different cytochrome c hvdrolysates were usedamong the six experiments. Measurdd amino acid compositional values (mol AAImol protein) are listed in Tahle 3 for each of the six experiments. For each experiment in Tahle 3 anaverage error of analysis was determined bv averaeine - the individual errors for each of the amino acids analyzed. The individual errors are percent errors hased on the differences between the measured comDositional values and the known literature values (13). The average error of analysis for each experiment is included in Tahle 3. The mean compositional values for each amino acid are also included in Tahle 3.
-
Dlscusslon The method of rotei in deeradation emdoved in this experiment, acid-catalyzed hGdrolysis, is the-method most commonlv used in amino acid composition analvsis (14). There are certain limitations, howevdr, imposed h i acid catalysis: tryptophan (TRP) is completely destroyed, SER, THR, cysteine (CYS) and MET are partially degraded, and the amides, glutamine (GLN) and asparagine (ASN), are converted to their respective free acids, GLU and ASP (15). In our work, we did find complete loss of T R P and suhstantial -Inns of MET. We found. however. no sienificant loss in - - ~ either SER or THR. Additional limitations are imposed bv the use of the d e a l de"t&ed amino acid mixture.;rhe availability of the commercial algal deuterated standard mixture makes this experiment financially possible for undergraduate use. Using the algal mixture is 10-15-fold less expensive than preparing a mixture by purchasing individual deuterated amino acids. Unfortunately, however, the algal mixture contains no CYS and contains low amounts of three amino acids, TYR, histidine (HIS), and arginine (ARG). The absence of CYS in the deuterated mixture makes its determination impossible. A combination of a poor TAB derivatization yield for HIS and ARG and the low amounts of these two amino acids in the deuterated internal standard mixture makes an accurate determination of HIS and ARG difficult. In our studies. TYR gave high and widely fluctuating values (-7-30) for the resoonse factor. R (see Tahle 2 for R values for other amino acihs). These ~'val"es indicate that the TAB derivatives of nrotonated and deuterated TYR mav have differing stahilities during the chromatographic separation. In order to make this experiment consistent with the limitations in time end budget of an undergraduate laboratory, we omit determinations of the amino acids destroyed hg acid hydrolysis (TRP and MET), we do not attempttodetermine those amino acids (CYS, ARG, HIS, and TYR) whose quantitation is made impossible or difficult by their low levels in the deuterated algal mixture, and we determine total glutamate (GLU plus GLN) and total aspartate (ASP plus ASN). For the 12 amino acids that are determined in this experiment. the accuracv of analvsis is verv. .. eood: the ranee in the average error of analysis for the six individual experiments in Table 3 is 5.9mo LO 12.9%. These errors compare favoral~lv with average errors of 20.0%, 8.4%, and 8.2% reported f i r single analyses of superoxide dismutase, calcitonin, and human growth hormone (IGF-2) by HPLC methods (8). In ~
~~~
~
442
Journal of Chemical Education
addition to cytochrome c, we have also used the method described in this . DaDer . to successfullv carrv out determinations of the amino acid composition of the protein insulin. Thisexperiment todetermine theamino acid comoosition of a pro& can he used in several ways. The instrubtor can determine the retention times of the ~rotonatedand deuterated amino acids in advance, p1epare.a SIM program for the chromatographic system to he used, and allow students to carry out the quantitative aspects of the experiment including protein hydrolysis, derivatization, and GC-MS analysis in two laboratory periods. If the instructor also prepares the hydrolyzed protein in advance, a student can complete the experiment in a4-h laboratory period. Studentscan begiven this experiment as a small laboratory project in whch they are a-ked to identifv amino acid elution oeaks.. assien retention times, create a SIM program, and carry out the quantitative aspects of the ex~eriment.Such a ~ r o i e c would t require -3-4 laboratory pkriods. While the ex~erimentpresented here determines onlv 12 amino acids, the experimental protocol could well serve as the basis of a longer term independent study project in amino acid analysis. A more complete amino acid analysis could he achieved by spiking the algal deuterated mixture with individual deuterated samples of ARG, CYS, HIS, TRY, and MET. In cases where the determination of TRP is essential, a second method of hydrolysis (e.g., hase-catalyzed) could be used (14). T o determine CYS and MET, 1e.s.. conversion of these amino acids to acid-stahle adduct. - ~ ~ ~ oxidation by performic acid to form cysteic acid and methionine sulfone) can he carried out prior to acid hydrolysis (16). GLN and ASN are usually determined together with GLU and ASP as total glutamate and total aspartate. In cases where it is essential to determine GLN and ASN, enzymatic methods of protein hydrolysis can he used (In.
~~-
~~~~
- -
,-~-~.
Acknowledoment The authors gratefully acknowledge support from the National Science Foundation (Grant CSI-8750577 from the College Science Instrumentation Program). Literature Cned -~~~~ 1. Willsrd,H.H.;Menit,L.L., Jr.:Desn.J.A.;Seftle,F.A.,Jr.InstrumrntolMrfhodsa/ Analysis, 7th ed.; Wadswarth: Belmont. CA. 1988: pp 569571. 2. Lee. M. L.: Yanp, F. J.: Bartie. K. D. Open Tubular Column Gos Chromatography: Wiley: New York, 1984; pp 143-148. 3. Gloss Copiilory Chmmorogrophy in Clinirol Medicine nndPhormoeology: Jaeger. H.. Ed.:Dekker: New York, 1965. 4. Research in Undergraduate Inaiilulionr, 4th ad.; Andreen, 8.; Wubbls, G. G., Eds.: Council on Undergraduate Research: 1990. 5. Hil1.D. W.;McSharry,B.T.;T~zupek,L.S. J C h m ~ E d u c 1988,65,907-910. . 6. Mahboft. G. A. J.Chom.Educ. 1 9 9 0 . 6 7 . 4 4 1 4 6 7. Aitken, A ; Geisow. M. J.; Findlay, J. B. C.; Holmes. C.: Yanuaod, A. In Protein Sequencing A Pmclicol Approarh; Findlay. J. B. C.; Geisow, M. J., Ed..; IRL: Oxford. 1889; pp 4 3 4 1 . 8. Bergman, T.: Csrlquist, M.; Jornvall, H. In Aduanced Method8 in Protein Mieroszquence Analysis: Wiftman-Liohold, B.;Sslinkow, J.; Erdmsnn. V.A., Edr; Springer Berlin, 1986; pp 45-55. 9. Ksiser,F.E.;Gehrke.C. W.;Zumwslt,R.W.:Kuo.K.C. J. Chrom. 1974.94. 133. 10. Rafter. J. J.; Ingleman-Sandberg. M.; Gustafmon. J. A. Aclo Eiol. Med. Germ. 1979, 1R. ~J21-381 , ~
-
~
11. Epstein, P.; Kaine, C. RioChromotogrophy 1990.5, 97-LW. 12. Leimer, K. R. Rice, R.H.; Gehrke,C. W. J. Chrom. i977,141.121-144. 13. Yssunobu, K. T.: Nakashima, T.: Higa, H.; Mstsuhara, H.; Banson. A. Riochim. Biophys. Act0 1963.78.191-794. 14. Mshler, H. R.:Cordes. E. H. Biologirol Chemistry, 2nd ed.: Harper and Raw: New "-7.
To"..-
-" ,&.
15. Hunt, S . In Chamistry ond Biochemistry of the Amino Aeidr: Bsrrett, G.C., Ed.; Chapman and Hail: London, 1985; pp 376398. 16. Hirs, C. H. W. InMefhodsinEnrvmolo#y;Hirs.C. H. W.,Ed.: Academic: Now York, 1967: Val. XI, pp 5 M 2 . 17. Tower, D. B. In Methods olEnrymology; H i m C. H. W., Ed.; Academic: New York, 1967:Vol. XI, pp 7 6 9 3 .
~
-
