QualitativeAmino Acid Analysis of Small Peptides
Gary A. Mabbott' Colby College, Watewille, ME 04901
The combination of gas chromatography and mass spectrometry (GCiMS) is one of the most powerful analytical tools in modem instrumental lahoratories. The lower instrument costs that have accomoanied advances in comouter and spectrometer technology in the last few years have made GCIMS analvsis available to a wide varietv of chemical laboratories. ~ g e r e f o r e ,it is appropriate t&t undergraduate chemistry students gain experience operating instruments and interpreting G C M S data. The following experiments could he used either in an instrumental analysis course, a biochemistry course, a physical chemistry course, or an integrated lab. An interesting bioanalytical problem to which GCIMS can he applied is the characterization of peptides. There are many small peptides that play important biochemical roles. They commonly function as antibiotics, chemical messeneersl - . hormones. h e a w metal chelatine aeents..and detoxifving agents. Once a new active peptide is isolated, researchers are often faced with the task of determinine the structure of the substance. Finding its amino acid c&position is an toeether the puzzle. There are manv essential step . in oiecine . papers in the literaturethat describebfficient approaches t; this son of problem usinr G C / M S (1-8). Besides being appealing to students, the exercise described here gives them experience in derivatization methods that are &en necessary in order to make nonvolatile samples amenable to GC separations. There are also several asoects of GC/MS analvrtjs that can he demonstrated here that are not common tb conventional GC determinations. For example, both selected ion chromatograms and difference spectra of severely overlapping peaks can play an imoortant role in identifvine the comDonents in a mixture. Most of the work describe3 here canbe performed on standard capillam columns if these types of data manipulation ~~
~~~~~
~
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--
---
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The experiment starts with the hydrolysis of a peptide. ~ h i s o ~ e r & i odoes n not take much ofthe students'labtime, but it must bestartedat least 12 h inadvanceofthederivatization. Alternatively, if a student is given a simple amino acid mixture a t the beginning of the afternoon, the entire exoeriment can be oerformed in a single 4-h lab neriod. The . derivatization procedure described here is an adaptation of work bv Darbre and Islam ( 4 ) .The carhoxvlic acid nroup is first kethylated and then.free amino groups (as-weli as hydroxyl and thiol groups) are converted to amides (or esters) of trifluoroacetate.
R-CH-
I
NHz
P
-OH
CH?OH.HCI
R-CH-
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F
-O--CH3
NH2
Presented In part at the American Chemical Society meeting in New Orleans. August. 1987. ' Present address: College of St. Thomas. 2115 Summit Avenue, St. Paul. MN 55105.
There are other methods that are popular for the formation of longer chain esters or perfluorinaied amides (5-8). However, this particular scheme was chosen since the reactions given heri are fast and easy to carry out. They are also the same as the first steps in peptide sequencing strategies develooed hv Biemann's erouo . .I I.). Familiaritv with these reactions hasbeen an advantage for some &dents who have chosen to sequence a small peptide as a special project. ~
Experlrnental A kev t o successful derivatization is keeoine the reaeents. . sample, and glassware dry. For best res;ltsUdry the clean elassware in an oven for 1h a t 140 "C and cool ih a desiccator prior to use. Since the reagents are reactive and uolatile, students are directed to work in a hood and to wear latex gloues. (Note that both acetyl chloride and trifluoroac~tic anhydride ore extremely destructive to tissue. Extensiue inhalation can be fatal. Mahvlene chloride is an irritant and suspected carcinogen. Exposure can cause nausea, dizziness and headaches. The gloues are protection against spills but should be washed immediately if they contact the reagents to auoidpossible penetration.) Peptlde h!vdrolysis (Optlonal) A small amount of peptide (S10mg) is dissolved in 1-2 mL of 6 M HCI and transferred to a orescared am~ule . with a Pasteur .oioet. The bottom of theampule iscooled briefly in a beaker of rce, and the open end is attsched to an aspirator (via a trap, and sealed with a propane torch. Iris then placed inan oven at 11O0Cfur at leapt 12 h. Before derivatization the solution is transferred to a 25-mL roundbottom flask and evaporated to dryness using a rotary evaporator. Traces of water are removed by adding about 1mL of methylene chloride (HPLCmade) to the flask and evaporatinato wain - dryness using the rotary evaporator. ~~~~
~
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Derivatizatlon Procedure A fresh 3%methanolic HCI solution is prepared by mixing methsno1 and acetyl chloride in a 20:l ratio immediatelybefore use (9).To do this, 10 mL of methanol (HPLC grade) in a graduated cylinder are chilled in a beaker of ice. (Warning: adding the aeetyl chloride too rapidly to the methanol cnn cause splattering.) A Pasteur pipet is used to add 0.5 mL acetyl chloride a few drops at a time to the methanol. The reagent is mixed by stirring. About 1mL of methanolie HC1is usedto dissolve the sample. (For individual amino acids Z5 mg of each is plenty.) The sample solution is sealed in a vial with a Volume 67 Number 5 May 1990
441
conical bottom and a Teflon-linedscrew cap and mixed gently. The reaction is allowed to proceed in an oven at 70 'C for 30 min. After cooling, the solution is transferred to a clean, dry 25-mL roundbottom flask. The excess reagent is removed by evaporating to d m e s s at 40 'Con a rotary evaporator. In order to remove traces of water, about 1 mL of methylene chloride is swirled gently in the flask and evaporated to dryness again with the rotary evaporator. The residue is taken up in 0.2-0.5 mL of trifluoroacetie anhydride and transferred to a clean, dry ampule using a Pasteu~pipet. The bottom of the ampule is cooled briefly in a beaker of ice, and the open end is attached to an aspirator (via a trap) and sealed with a propanetorch. Theampuleisplaced intoa beaker and set in an oven at 140 OC for 10min. The ampule is allowed to cool toroom temperature before opening. (If the instrumental work is to be carried out on another dm. the samnle will keeo well in the sealed amode.) The sealiahmkk;l and thhexcess reaeent hv* directin; " a stream -~~~~ ~~-~~~~ ~ ~ removed . . of dry Nlthrough a Pasteur pipet onto the liquid aurface. Note that the final product is usually an oil. About 100 rcL of methylene chloride ia used todissolv~the product and transfer it toavial witha screw cap. ~
~~
~~~~
~
GC/MS Analysis Derivatized samples (0.5-1 fiL) can be injected into the GC/MS with a split ratio of 70 (or greater). An injection port temperature of 220 OC is recommended. The column temperature is programmed from 30 to 1W O C at a rate of 15 deglmin and then 100 to 220 'Cat 6 deelmin. Seoarationsin this lab have been oerformedoneither a 15m phenyl cyanopropyl capillary column (DB-2?5.J & W Scientific) or on a 25-mchiral capillary column (Chiral Val-Ill. Alltech Associates).The instrument used here was a Hewlett Packard 589UA GC with a 5970B mass selective detector.
-
Discussion Flame sealing the reaction mixture in a n ampule for the derivatization orocess is somewhat hazardous and complicates the procehure. A reasonable modification is t o run t h e trifluoroacetvlation steo at a lower temperature in a capped vial. ~ x ~ e r i e in k ethis laboratory shows that forminithe amides a t room temperature lowers the yields for some amino acids. (Derivatives of cysteine, hydroxyproline, leu-
TIME
IN
cine, phenylalanine, tyrosine, and valine were about half as abundant: those for arzinine and histidine were not detected when the samples were not heated.) Also, this modification of the procedure yielded a high chromatographic background after the column reached 165 O C (after the elution of hydroxyproline and before the emergence of methionine). However, this modified approach still permits students to identify most amino acids in simple mixtures. Very good separation, including resolution of D- and Lisomers, is obtainable with a 25-m chiral capillary column (see Fig. 1). However, a less expensive, standard capillary column with a polar stationary phase will separate most of the amino acid derivatives satisfactorilv. (See Fie. 2.) In the latter case some components do co-eluie. 1n cases where the overlao is not exact. the distinct soectra for two comoonents can b e observed i n t h e MS scans taken on the leading and trailinn- edees - of the chromatoeraohic oeak. Figure 3 shows spectra from different portions or t h e p e a k at-13.2 min for the standard column revealing methionine and glutamic acid. There may still be some mixing of the spectra using this strategy. I n those cases Ghosh and Anderreg have shown that taking the difference in two spectra within a chromatographic peak produces a plot of the fragmentation pattern for one substance in the neeative direction and the other in the positive direction making both possible to interpret (10). Fieure 4 shows the soectra for threonine and isoleucine rev k e d by taking the hifferencb in two spectra from the peak emereine a t 6.2 min from the standard column. Note that sincebo'th compounds produce some of the same fragment masses these ion peaks will be distorted in the difference spectrum. Students are given a table of important ion fragments for the amino acid derivatives. (The table below gives mainly ions that are a t least 5% as intense as the base peak for masses meater than 58.) With this table thev do not reallv need t o o b t a i n retentidn times for s t a n d a d materials order to analyze their mixtures (although i t is emphasized
MINUTES
Figure 1. TrifluoroaceUMldemethyl ester derivativesof standard amlno acas separated on a 25-m capillary chlral column
TlME
IN
MINUTES
Flgura 2. Trifluoroacetamidemethyl ester derlvatlvea of standard amino acids separated on a 1% capillmy wlumn with a bonded phenyl cyanopropyl statlonary phase.
442
Journal of Chemical Education
Figure 3. identlflcstlond sample wmponents by examining s p e m from the Outer edas of SBvereIy overlapping chromatographic peaks at 13.2 mi" in Figure 2. (ion intellsnies lessthan 2% were omined.)
that running standards under the same conditions provides an important corroboration that is part of any careful analysis). They must rely on the mass spectra inorder toverify the presence of a given amino acid. Althouch sometimes Door ieaction yields and/or dirty samples obscure impoitant chromatographic peaks, the peaks of interest stand out when the chromatogram is replotted for a single ion corresponding w a prominent fragment in the spectrum of the amino acid derivative in question. Comparing two such ion chromatograms can focus the search for a particular amino acid even more finely. This type of search procedure saves time that otherwise might be wasted in examining the full spectrum of each peak in a busy chromatogram. The spectra of the methyl ester derivatives have been discussed in the literature (11). Manv of the imoortant ions for each amino acid derivative c& be rationalized using basic framentation mechanisms that are covered in the lecture section of the instrumental analysis course. (Many of the principles from Chapters 2-4 from McLafferty's text (12) are discussed in class.) In their reports the students are required to discuss the mechanisms leading to a t least three major ions for each amino acid found in their samples. For example, consider the spectrum for the cysteine derivative. Althouah verv few of these derivatives show a sienificant molecuiar ioi abundance, most show a fragment [on a t 59 mass units less than the molecular ion. This mieht be rationalized as the loss of the carhonyl plus methoxfgroup initiated by induction a t the carbonyl.
Chemical supply houses, such as Sigma Chemical Co., offer several small peptides at costs that are reasonable for this experiment. We have used synthetic glycyl-leucyl-alanine, tyrosyl-glycine, glycyl-leucyl-tyrosine. and the commercial sugar substitute, Nutra Sweet (aspartame), which is a methyl ester of aspartyl-phenylalanine. Figure 5 shows a chromatogram for a Nutra Sweet product known as Equal obtained from the grocery. (The product contains dextrose and produces some other chromatographic peaks that may be the result of hydrolysis of the carbohydrate. For example, the Deak at 3.5 min amears to be 5-methvl-Z(3H)furanone.) . . . Fig;re 6 shows a c&bmatogram of the amino acids from Gramicidin S, a readily available antibiotic produced hy a bacterium. Gramicidin S is a cyclic decapeptide of (L-leucylL-ornithyl-L-valyl-L-propyl-D-phenylalanyl2 Comparing the retention time for the phenylalanine derivative found in the Gramicidin S with standards shows that it is definitely the more unusual D isomer. We have also used Gramicidin D, another natural product that actually contains four peptides
PHE
ASP
I
Another common inductive cleavage removes the trifluoroacetyl group. 2
4 ' 6
8
10"12
14°K
TIME IN MINUTES Figure 5. Chromatogram of derlvatlzd prducts horn the hydrolysate of the mificial sweetener. Equal (a Nutra-Sweet preparation). A DB.225 column was wed.
L-ORN , , ;D
L-LEU
I
IL-PRO
One might account for the fragment ion at mlz 184by radical site initiation starting from the nitrogen as the point of ionization. 444
Journal of Chemlcal Education
TIME
IN MINUTES
Figure 6. Chromamgram of tha derlvatized components from the hydrolysate of QramlcldR Son a chiral column.
relative ion intensities are not going to match the tabled (and is sometimes referred to merely as gramicidins). The values. principal species represents 87% of the mixture and has a structure of HC0-(~)-Val-Gly-(~)Ala-(~)Leu-(~)AlaAcknowledgment (D)V~~-(L)V~~-(D)V~~-[(L)T~~-(D)L~U]~-(L)T~~-NHCH~-
CH20H.Despite comments in the literature about the loss of tryptophan during hydrolysis (Z),a strong signal was observed for each of the amino acids for this peptide. Finally, afew comments on the limitations of the methods described here are appropriate. The work in this lah has focused on qualitative analysis only. Other workers recommend the oreoaration of the correspondinz n-butvl esters for quant&t&e analysis (7). In f a k the signal intensity decreases with ace of the derivatives for several amino acids in our experience. Also, arginine and cystine only occasionally produce derivatives with recognizable spectra. MacKenzie out that moisture andlor contaminants are the most likely causes for poor yields (2). One should also keep in mind that asparagine and glutamine form the same derivatives as aspartic acid and glutamic acid, respectively, in any of the reactions that have been widelv used for GC analvsis. In interpreting the spectra, the relative ion intensities are often auite im~ortant.(See the tabled intensities for leucine and isileucine; for example.) However, since intensities vary with ionization source volta~eand pressures, the table should he used cautiously. Wehave noted daily variations of a few percent for our instrument in some cases. Also, students often do not recognize when an ion intensity has gone off scale. Whenever the instrument truncates the signal,
The GC/MS instrument used here was purchased with the assistance of a grant from the National Science Foundation College Science Instrumentation Program (CSI 8552183). Amino acid standards and the chiral GC column were purchased for work supported by a grant from the National Science Foundation Research at Undergraduate Institutions program (DMB 8410015). The author thanks Wayne Smith for his critical reading of the manuscript. Lnerature Clted 1 . Anderegg, R. J.: Biemenn. K.: Buchi, G.; Cuahman, M. J. Am. Chem. Sor. 1976.98, %.lnL"*"" """"
2. MacKenzie, S. L. "Amino Acids and Peptide" In Coa Chromfogrophy/Moas SpectlomPtryApplieatiom in Microbiology; Odham, G.; Lamn, L.; Mardh. P-A,; Ed% Plenum: New York,1964: Chapter 5. 3. Keltor, P.B.;Carr.J.D. J. Chsm.Edue. 1933,60,437-438. 4. Dsrbre,A.: Is1am.A. Biorham. J. 1368,106,923. 5. Gelpi,E.;Koenig, W.A.;Gibert, J.:Oro,J. J. Chromologr.Sri. 1969, 7 . M 1 3 . 6, Leimer,K. %Rice, R.H.: G8hrke.C. W. J. Chramorogr. 1971,141,121-144. 7. Kaiser.F.E.;Gehrke.C. W.:Zumwalt,R. W.;Kuo,K.C. J.Chromnfogr. 1974,94, I l b 133. 8. MacKeneie,S. L.:Hogge,L.R. J. Chromolagr. 1977,132,485-493. 9. Fie~er.L.A.:Fciser,M.Reaganlr/or OlgonicSynfhesis; Wi1ey:New York,1967:Val. 1.
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