Recent studies4"6 have demonstrated the feasibility of analysing 3-methyl-2-thiohydantoin (MTH) and 3-phenyl-2-thiohydantoin (PTH) derivatives by gas-liquid chromatography (GLC). We have now developed an additive Edman degradation procedure for small peptides in which GLC of MTHs is used to identify the terminal amino-acid released at each step of the degradation. After one cycle of Edman degradation, an aliquot of the reaction mixture is removed and the MTH derived from the N-terminal amino-acid is identified by GLC. At each successive step of the degradation the MTHs produced in the prior steps appear on the chromatogram together with the newly released derivative. Peptides of known sequence were obtained from Schwarz-Mann Laboratories, Orangeburg, New York. Three per cent OV-17 on 'Gas-Chrom Q' (100-120 mesh) column packing was obtained from Applied Science Laboratories, State College, Pennsylvania. All other reagents were redistilled before use as described previously4. The reactions were performed in 10x100 mm screw-capped tubes (teflon-lined caps) which had been treated with dimethyldichlorosilane to minimize adsorption of peptides and thiohydantoins to the glass.The peptide to be sequenced (0.05-0.5 jamol) is dissolved in 200 ul. 50% aqueous pyridine and 1-2 ul. of triethylamine is added to bring the pH to 9.5-10.0 (determined with /?H 9.0-13.0 indicator paper; Macherey, Nagel and Co., West Germany). To the peptide solution 5 ul. of methylisothio-cyanate in 50 ul. of pyridine is added; an identical addition is made after 15 min. The mixture is flushed with nitrogen and held at 50 C for 30 min; after 0.5 h the solvents are evaporated under a stream of dry nitrogen at 50. The residue is suspended in 0.2 ml. of ethyl acetate, which is then evaporated as above to ensure complete removal of the coupling solvents. Anhydrous trifluoroacetic acid (200 ul.) is added to the methylthiocarbamyl peptide in the tube and the N-terminal amino-acid is split off and simultaneously cyclized to the thiazolinone by heating at 50 C for 10 min. After evaporation of the trifluoroacetic acid at 25 C under nitrogen, ethyl acetate is added to the tube and a portion of the solution is removed for conversion of the thiazolinone to the MTH derivative. This is accomplished by evaporation of the ethyl acetate at 25 C under nitrogen, addition of 200 ul. of 1 M HC1 to the residue, and heating at 80 C for 10 min7. The peptide (minus one amino-acid) and most of the thiazolinone from the first cycle remain in the tube. This peptide is then subjected to successive cycles of Edman degradation as described above until the C-terminal acid is reached. Each cycle requires 50-60 min to complete.The MTHs (except the histidine and lysine derivatives) are identified by GLC of their trimethylsilyl (TMSi) derivatives as previously reported4. The TMSi derivatives of the MTHs of histidine and lysine are unstable and give smaller peaks than anticipated when determined by GLC4. We have recently found that the acetyl derivatives of these two MTHs are stable at 25 C for at least 2 days and upon GLC analysis give 3-4 times larger peaks than the corresponding TMSi derivatives. To form the acetyl derivatives the MTHs are dissolved in 100 |il. pyridine-acetic anhydride (1:1) and maintained at 25 C for 18 h; after dilution with 1 ml. of toluene the solution is evaporated to dryness at 80 C under nitrogen. The acetyl derivatives are dissolved in ethyl acetate and analysed on an OV-17 column. The oven temperature is programmed from 200 C to 260 C at 5/min and then held at 260 C for 10 min. The lysyl derivative eluted 14 min and the histidyl derivative 15 min after injection on the GLC column. Fig. 1 Gas chromatogram of TMSi, MTH derivatives after one cycle of additive degradation of a pentapeptide. The amino-acid composition of the peptide is (Ala, Asp, Phe, Ser, Val). 2.5 nmol of internal standard (IS=TMSi, MTH-Leu) was injected at a sensitivity of 3 x 10~10 A for full scale deflexion. Three minutes after injection of the sample, the column oven was programmed at 5/min.The sequence analysis by additive Edman degradation of the pentapeptide H2N-Phe-Asp-Ala-Ser-Val-OH is described in the following section. Initially, an amino-acid analysis of the peptide was performed by two different procedures. After acid hydrolysis, the amino-acids were analysed by the procedure of Roach and Gherke8 and as their MTH derivatives as previously reported4. These analyses confirmed that the peptide contained equimolar quantities of Ala, Asp, Phe, Ser and Val. 0.3 mg of peptide was sequenced by the additive procedure. After the first cycle of Edman degradation the mixture in the reaction vessel was dissolved in 0.5 ml. of ethyl acetate and 0.1 ml. of the solution was removed for conversion of the thiazolinone of the N-terminal amino-acid to its MTH derivative. Before conversion of the MTH to its TMSi derivative 25 nmol of internal standard (MTH-leucine) was added, and the TMSi derivatives were analysed on an OV-17 column4. The chromatogram obtained (Fig. 1) shows that Phe is the N-terminal amino-acid of the peptide. From the relative detector response of equimolar amounts of TMSi, MTH-Phe and TMSi, MTH-Leu, the yield of TMSi, MTH-Phe as calculated from the chromatogram was 48 nmol.Fig. 2 Gas chromatogram of TMSi, MTH derivatives after two cycles of additive degradation of a pentapeptide. Conditions for analysis were the same as in Fig. 1. After the second cycle of Edman degradation, 0.4 ml. of ethyl acetate was added to the reaction tube and 0.1 ml. of solution was removed for conversion of the thiazolinones to the MTH derivatives. Analysis of the TMSi, MTH derivatives by gas chromatography on an OV-17 column provided the chromatogram shown in Fig. 2, which demonstrates that aspartic acid is the amino-acid in the peptide adjacent to phenylalanine. The yield of TMSi2, MTH-Asp was 48 nmol. Because 2-methylamino-4-phenyl thiazolinone was still present in the reaction mixture in addition to the peptide, this thiazolinone obtained from Asp. Thus, the MTH derived from it appears as the TMSi, MTH derivative in the chromatogram (Fig. 2).Fig. 3 Gas chromatogram of TMSi, MTH derivatives after three cycles of additive degradation of a pentapeptide. Conditions for the analysis were the same as in Fig. 1. After another cycle of Edman degradation and conversion of 0.3 of the thiazolinones to the MTH derivatives, the chromatogram shown in Fig. 3 was obtained. The next amino-acid in the sequence is thus Ala. The yield of TMSi, MTH-Ala was 34 nmol, 71 % of the yield of TMSi, MTH-Phe obtained after the first step of Edman degradation. The area of the phenylalanine peak relative to that of the internal standard is 75 % less than the area of the phenylalanine peak in the first chromatogram. This decrease in peak size was observed with the other derivatives also and can be attributed to the instability of the thiazolinones in the reaction tube. After four cycles of Edman degradation, the chromatogram shown in Fig. 4 was obtained, indicating that Ser is the next amino-acid in the sequence. The low yield of MTH-Ser, 15 nmol, is characteristic of this derivative.With the sequence elucidated so far, and knowledge of the amino-acid composition of the peptide, one can predict that the C-terminal amino-acid is valine. This prediction was confirmed by a final cycle of Edman degradation (Fig. 5) in which 16 nmol of TMSi, MTH-Val was obtained. This represents 33 % of the yield of the TMSi, MTH-Phe obtained in the first reaction cycle. Only 6 h were required to perform all the operations described. To test the general applicability of the additive Edman degradation, several other peptides were sequenced (Table 1). During the sequencing of the peptide H2N-Trp-Met-Asp-Phe-CONH2 our previously described procedure had presented some difficulty4, presumably because of cyclization of aspartic acid2. This problem was eliminated with the procedure outlined above, as were difficulties associated with extensive cyclization of glutamine residues. Peptides which contain more than one residue of the same amino-acid (for example, the hexapeptide containing three glycine and two proline residues shown in Table 1) can be sequenced with the aid of quantitative GLC. After each of the first three cycles of Edman degradation of the octapeptide shown in the table, the thiazo-linones were completely extracted from the dried peptide with 0.2 ml. of ethyl acetate. The remaining pentapeptide was sequenced by the additive method.Fig. 4 Gas chromatogram of TMSi, MTH derivatives after four cycles of additive degradation of a pentapeptide. Conditions for the analysis were the same as in Fig. 1. Table 1 Peptides sequenced by the Additive Edman DegradationH2N- Leu- Trp- Met- OH H2N- Trp- Met- Asp- Phe- CONH2H2N- His- Ser- Gin- Gly- Thr- Phe2 OH H2N-Gly- Pro- Gly- Gly- Pro- Ala- OHH2N- Phe- Val- Gin- Trp- Leu- Met- Asn- Thr- OH______After each of the first three cycles of Edman degradation of the octapeptide, the thiazolinones were completely extracted from the dried peptide with 0.2 ml. of ethyl acetate. The remaining pentapeptide was sequenced by the additive method. Using the method described, about 25 nmol per amino-acid (or 0.15 umole of hexapeptide) is required to sequence a peptide with 5 or 6 amino-acids. For analysis by GLC only 1-2 nmol of derivative is needed, however. It is conceivable that the contaminating by-products seen as background in Fig. 5 could be reduced significantly by more careful purification of reagents and by use of an optimal quantity of methylisothio-cyanate for quantitative reaction with the peptide. (The present procedure uses a large excess of this reagent.) With these refinements less peptide would be needed for analysis.Fig. 5 Gas chromatogram of TMSi, MTH derivatives after five cycles of additive degradation of a pentapeptide. Conditions for the analysis were the same as in Fig. 1. The results presented in this paper demonstrate the usefulness of the additive Edman degradation for sequencing small peptides and the C-terminal region of longer peptides. The method is fast and sensitive, and takes advantage of the inherent accuracy and reliability of gas chromatographic analysis. The procedure which has been described may provide a more practical and financially feasible approach to the elucidation of peptide sequences than other techniques currently being tested such as mass spectrometry9, automated solid phase degradations10 and analysis of peptides by a sequencer11.This investigation was supported by grants from the National Institutes of Health. D. E. V. is a postdoctoral fellow of the National Institutes of Health; and D. S. F. is the recipient of a Public Health Service career development grant.