Carboxy-terminal Degradation of Peptides using Perfluoroacyl Anhydrides : A C-Terminal Sequencing Method

Eur. J. Biochem. 228, 362-372 (1995) 0 FEBS 1995

Carboxy-terminal degradation of peptides using perfluoroacyl anhydrides A C-terminal sequencing method

Keiji TAKAMOT01,2, Masaharu KAMO', Kazuhiko KUBOTA', Kazuo SATAKE' and Akira TSUGTTA'

* Department of Pharmacology, Science University of Tokyo, Japan

(Received 11 October 1994) - EJB 94 1545/3

Research Institute for Biosciences, Science University of Tokyo, Japan

An accurate carboxy-terminal sequencing method has long been sought to complement the Edman degradation procedure for amino-terminal amino acid sequence analysis. The method presented here is a unique and simple method to partly fulfill the needs. Exposure of a polypeptide to perfluoroacyl anhydride vapor at -20°C for 0.5- 1 h causes sequential chemical degradation of the molecule from the C-terminus. Fast-atom-bombardment mass spectrometric analysis of the resultant mixture of C-terminally truncated molecules permits the determination of the C-terminal sequence by simple calculation of the mass differences in molecular ions. Experiments suggested that this C-terminal degradation proceeds by active inter- mediates such as oxazolone at the C-terminal carboxyl residues.

Keywords. Carboxy-terminal sequencing ; perfluoroacyl anhydride vapor ; fast atom bombardment MS ; chemically truncated fragments.

The amino acid sequence of a protein or peptide of interest is usually one of the first pieces of information required in today's molecular biology or biotechnology strategies, be it for gene cloning or synthesis of immunoreactive peptides. To date, amino-terminal sequencing using the Edman degradation procedure (Edman and Begg, 1967) has almost exclusively provided such data. Methodologies for sequencing proteins and peptides from their carboxy termini have remained relatively primitive, requiring much protein in return for little sequence information. Carboxypeptidase digestion is still the most widely used method despite its intrinsic limitations of substrate specific- ity and endoprotease contamination. Several stepwise chemical degradation methods have been developed (Stark, 1968 ; Yamashita, 1971 ; Bailey et al., 1993) including automatic equip- ment (Bailey et al., 1994; Boyd et al., 1994).

We reported that peptides subjected to the vapor of either 90 % aqueous pentafluoropropionic acid or heptafluorobutyric acid at 90 "C for several hours appeared to have amino acid residues successively cleaved from their C-termini (Tsugita et al., 1992a). By fast-atom-bombardment (FAB) or electrospray ionization mass spectrometry, the C-terminally successively truncated molecular ions were clearly observed and the peptide C-terminal amino acid sequence could be deduced from their molecular mass differences. The predicted reaction mechanism was the formation of the oxazolone rings at the C-terminal amino acids followed by removal of the C-terminal amino acid residues. As well as the C-terminal degradation, two specific cleavages of an internal peptide bond were observed C-terminal to aspartic acid and N-terminal to serine.

In this paper we present another C-terminal degradation method for peptides using perfluoroacyl anhydride vapor instead of perfluoric acid. This method is superior to the perfluoric acid

Correspondence to A. Tsugita, Research Institute for Biosciences, Science University of Tokyo, Yamazaki, Noda Japan, 278

Abbreviations. FAB, fast atom bombardment; (C,F,CO),O. heptafluorobutyric anhydride ; (C2F,CO),0, pentafluoropropionic anhydride ; CF,CO,H, trifluoroacetic acid; (CF,CO),O, trifluoroacetic anhydride.

vapor method since more extensive C-terminal degradation was achieved and no internal peptide bond cleavages were observed. We have preliminarily reported that pentafluoropropionic anhydride vapor at -20°C caused sequential degradation of a peptide from its C-terminus, resulting in a series of truncated peptides each with different C-termini (Tsugita et al., 1992b). Further in- vestigation confirmed that the C-terminal degradation reaction was suitable for other peptides (Tsugita et al., 1992c, 1993). In this paper, we present a detailed description of the C-terminal degradation method, including necessary precautions to ensure reproducibility.

MATERIALS AND METHODS

Reagents. His-Pro-Phe-His-Leu-Leu-Val-Tyr was purchased from Sigma Chem. Co. and Met-Arg-Phe-Ala was from Serva Feinbiochemie. The other peptides were purchased from the Peptide Institute Inc. (Minoh, Japan). Trifluoroacetic anhydride [(CF,CO),O], pentafluoropropionic anhydride [(C,F,CO),O], heptafluorobutyric anhydride [(C,F,CO),O] and acetonitrile were obtained from Nacalai Tesque (Kyoto, Japan).

Perfluoroacyl anhydride degradation. Most operations were done in a glove box (1100X600X810mm) that was continuously flushed with dry nitrogen gas. The sample peptide (2-20 pg) was dried in a small sample tube (6x40 mm) in a vacuum desiccator and then transferred to the reaction vessel (19X100mm, Pierce). (C,F,CO),O (10% or 30%) or (C,F,CO),O (15%) in acetonitrile was used for the reagent solution. These reagents were obtained in 300-pl ampoules from Nacalai Tesque (Kyoto). The reagent solution (100-500 pl) was added to the reaction vessel but outside of the sample tube(s), whilst dry nitrogen gas was continuously flushed into the reaction vessel which was also cooled with liquid nitrogen. Care must be taken to maintain dry conditions when cooling the reagent as moisture in the air easily condenses and hydrolyses the acid anhydride. At liquid-nitrogen temperature, the reaction vessel was evacuated (1 Pa) and sealed. The reaction vessel was

Takamoto et al. (Eur: J. Biochem. 228) 363

"1 I

Fig. 1. FAB mass spectra of C-terminal degradation products of a dodecapeptide, Ala-Arg-Gly-Ile-Lys-Gly-Ile-Arg-Gly-Phe-Ser-Gly by the effect of acetonitrile or water. The reactions on the peptide (10 pg) were performed at -20°C for 1 h with the vapor of (A) 30% (C,F,CO),O acetonitrile solution (300 pl); (B) only (C,F,CO),O (90 pl); (C) (C,F,CO),O (90 pl) and acetonitrile (210 pl) in separate tubes; (D) 300 pl (C2F,C0),O/water mixture (10: 1, molar ratio) without acetonitrile; (E) the same mixture with acetonitrile (100 PI).

364 Takamoto et al. ( E m J. Biochem. 228)

transferred to the reaction bath (Histo-bath, Neslab Instrument Inc., Newlngton, USA) set at -20°C when the reaction proceeded for various reaction times. After the reaction, the reaction vessel was again transferred to liquid nitrogen to stop the reaction. The sample tube was removed from the reaction vessel, and then dried under vacuum.

Water vapor treatment. The water vapor treatment was performed after the degradation reaction. The sample after degradation was dried under vacuum and exposed to the vapor of 10% aqueous pyridine at 100°C for 30 min. The treated product was dried again to remove traces of the reagent and analysed by mass spectrometry.

Esterification of the degraded peptides with propanol vapor. The reacted peptide with the vapor of perfluoroacyl anhydride in acetonitrile solution was dried under vacuum and immediately exposed to the vapor of propanol at 60°C for 30 min. The product was dissolved in 2 p1 67% acetic acid; 1 p1 of this solution was mixed with matrix and subjected to mass spectrometry.

FAB mass spectrometry. The sample was dried under high vacuum and then dissolved in 2 p1 dimethylformamide or 67% (by vol.) acetic acid; 1 pl of the sample was mixed with the same volume of the matrix mixture and subjected to FAB mass spectrometry. Spectra were obtained with a JEOL HX-110 mass spectrometer (Tokyo) equipped with a DA5000 data system. An accelerating voltage of 10 kV was employed and xenon was used as the ionizing gas. The matrix was a mixture of glycerol/ thioglycerollm-nitrobenzyl alcohol (1 : 1 :1, by vol.) for both positive and negative ionization for the standard conditions, un- less specified. These matrix reagents were obtained from Wako Pure Chemicals.

Sequence analysis from mass spectra. In mass spectra the abscissa axis represents the mass number ([M+H]+ or [M-HI-) and the ordinal axis is the relative abundance of the molecular ion. The principal degraded fragment ions of a peptide are de- noted by the residue numbers and acyl groups. For example, 1 - l2facyl (Fig. 2C) indicates the ion of acyl-Ala-Arg-Gly-Ile- Lys-Gly-Ile-Arg-Gly-Phe-Ser-Gly. Numbers in parentheses indicate mass (in Da). The sequence of a peptide is deduced from the mass differences between consecutive peaks of the principal degraded ions. The principal degraded ions of mass m are accompanied by ions of mass m-18 and m-46 shown by -H,O and *, respectively. The ions of mass m-I were also observed but not marked on the spectrum to avoid complication. The spectra were analyzed by an in-house sequencing program.

HPLC separation of C-terminal degradation fragments. Peptide fragments were separated by reverse-phase HPLC. The columns used are noted in the figure legends and the HPLC systems employed were either the model 600E multisolvent de- livery system (Waters-Millipore, USA) or the SMART HPLC system with a pPeak monitor (Pharmacia, Biotech). The column temperature was maintained at 40°C. Fragments were eluted by a linear gradient of acetonitrile in trifluoroacetic acid (CF,CO,H) solution. The detailed conditions are described in the figure legends.

RESULTS

Reaction with perfluoroacyl anhydride vapor. In the previous experiments (Tsugita et al., 1992b,c and 1993), the degradation reaction was conducted only in the presence of acetonitrile. However, when the same degradation reaction was performed in the dry glove box, we observed that acetonitrile was not essential for the reaction. Comparison of FAB mass spectra of the reaction products of the dodecapeptide, Ala-Arg-Gly-Ile-Lys-

Gly-Ile-Arg-Gly-Phe-Ser-Gly, under the usual reagent conditions of a vapor from 30% (C,F,CO),O in acetonitrile solution (Fig. 1 A), (C,F,CO),O in the absence of acetonitrile (Fig. 1 B), and by incubation with vapors from separated (C,F,CO),O and acetonitrile (Fig. 1 C), clearly showed that acetonitrile was not necessary for the reaction to proceed. To clarify this seemingly contradictory data, we performed the degradation reaction on the same dodecapeptide using the vapor from 300 p1 (C,F,CO),O/ water (lO:l, molar ratio), in the absence of acetonitrile (Fig. 1 D) and in the presence of acetonitrile (100 pl) (Fig. 1 E). Without acetonitrile, the reaction did not proceed in the presence of water. However upon inclusion of acetonitrile, the reaction proceeded as normal. We think that acetonitrile must absorb the moisture/water in the air ensuring that the degradation reaction can take place.

Three kinds of perfluoroacyl anhydride vapors [(CF,CO),O, (C,F,CO),O and (C,F,CO),O] were tested on peptides for successive degradation. Vapors were generated from 100 p1 10% perfluoroacyl anhydride/acetonitrile solutions and the reactions were performed at -20°C for 1 h. Even under such mild conditions, both (C,F,CO),O and (C,F,CO),O successively degraded the peptide and all the degraded peptides were identified as their protonated molecular ions of acyl derivatives (data not shown). Treatment of the peptide with (CF,CO),O yielded degraded peptide peaks accompanied with other modified molecular ions such as the diacyl product. Treatment of the peptide with (C,F,CO),O gave a similar result to that obtained by reaction with (C,F,CO),O without the modifications. The differences of molecular ion masses of pentafluoropropionyl residues (146 Da) and trifluoroacetyl residues (96 Da) have similar masses to the phenylalanyl residue (147 Da) and prolyl residue (97 Da), respectively ; this can lead to misinterpretation of C-terminal sequences. The mass of the heptafluorobutyryl residue (196 Da) is dissimilar to any natural amino acid residue so we employed the vapor of (C,F,CO),O. Acylation by this anhydride may also be the most suitable among the three anhydrides tested for FAB mass spectrometry because it is the most hydrophobic residue (Naylor et al., 1986). In fact, it gave the cleanest backgrounds in mass spectra.

To simplify the identification of the degraded molecular ions in the mass spectrum, the reaction mixtures resulting from the perfluoroacyl anhydride degradation were treated with the water vapor from 10% aqueous pyridine. This caused the oxazolone peaks (shown as -18 Da peaks) to shift to the corresponding degraded principal molecular ions. Also pad of the acylated peaks (probably 0-acyl) disappeared. It can be seen that, after the water vapor treatment, there is a reduction in the -18-Da mass peak (compare Fig. 2B, before the treatment, with Fig. 2C, after the treatment).

The effect of temperature on the successive C-terminal degradation of the dodecapeptide, Ala-Arg-Gly-Ile-Lys-Gly-Ile- Arg-Gly-Phe-Ser-Gly, with (C,F,CO),O for the I-h reaction is shown at both 0°C and -20°C (Fig. 2, A and B). The principal successive degradation molecular ions were each accompanied by the corresponding -1-Da, -18-Da and -46-Da molecular ions. Differences in the amounts of molecular ions at different temperatures were mainly due to the different extents of acylation. At -2O"C, di-acyl peptides were partially observed (Fig. 2B) while, at O"C, the acylation reaction became stronger and the serine residue was occasionally dehydrated to dehydroalanine. Although the internal seryl peptide bond was observed to be stable at -2O"C, as shown by the 1-11 molecular ion (see Fig. 2B), partial cleavage at the amino side of this peptide bond was observed at 0°C as indicated by the decrease in the 1 - 11 ion peak (Fig. 2A). The successive degradation rate was enhanced at the higher temperature as demonstrated by the

Takamoto et al. (EUK J. Biochem. 228) 365

r f

P . . , . 1 *

1 - 2 *w

1 :+ I II li I -

sM*

---AcyI-AR 1 G I- I

I

, I - 4 + 4

1 - 1 \ 1 - S*Acyl

4 i O O

l . l P * A c y l I

t 1 e00 a I0 -SAG’ [M+H]’

Fig. 2. FAB mass spectra of C-terminal degradation fragments of Ala-Arg-Gly-Ile-Lys-Gly-Ile-Arg-Gly-Phe-Ser-Gly at various temperatures. The degradations were performed on a dodecapeptide (2 pg) with vapor of 10% (C,F,CO),O acetonitrile solution (100 p1) for 1 h at (A) at 0°C and (B) -20°C. (C) The sample in B was further treated with a vapor of 10% aqueous pyridine (300 pl) for 30 min at 100°C.

366 Takamoto et al. (EUK J. Biochem. 228)

0L-7 r- i-

1- (I

I

P 5 y / e0

0 = i I

rn

1 .

1- I *rn 1

I I

Q 604 I I l l I

. . . . . 500. 1000 1500 . . 2000 250a . . . . 3000

[M+H]'

Fig.3. FAB mass spectra in time course of C-terminal degradation. C-terminal degradations were performed with vapor of 10% (C,F,CO),O acetonitrile solution (1 00 pl) at -20°C for various time periods on a synthetic peptide Gly-Ile-Gly-Lys-Phe-Lys-His-Ser-Ala-Gly-Lys-Phe-Gly-Lys- Ala-Phe-Val-Gly-Glu-Ile-Met-Lys-Ser (2.5 pg). The degradation products were immediately treated with vapor of 10% aqueous pyridine. FAB mass spectra were obtained from the peptide treated for (A) 10 min, (B) 30 min, (C) 1 h and (D) 5 h.

Takamoto et al. (Em J. Biochem. 228)

407

i (*) 35

[M+H]'

i- lO+Acyl 1-1 J+ACyl I

1

367

30

Fig. 4. Positive and negative ionizations in FAB mass spectra of the C-terminally degraded products of the peptide Tyr-Gly-Gly-Phe-Leu- Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln . The peptide (5 pg) was exposed to a vapor of 10% (C,F,CO),O acetonitrile solution at -20°C for 1 h. The degraded product was treated with pyridine/water vapor. The product was dissolved in 3 p1 67% acetic acid and 2 p1 solution was mixed with same volume of matrix composed of glycerol, thioglycerol and m-nitrobenzyl alcohol. Then 1 p1 of the solution was applied to the FAB mass spectrometer in both positive (A) and negative (B) mode using the same product solution and the same matrix.

reduction of the original peptide ion peaks. Based on these observations, we now use a reaction temperature of -20°C.

Time course experiments were performed on a large peptide having 23 amino acid residue (Gly-Ile-Gly-Lys-Phe-Leu-His- Ser-Ala-Gly-Lys-Phe-Gly-Lys-Ala-Phe-Val-Gly-Glu-Ile-Met- Lys-Ser) at -20°C with (C,F,CO),O vapor; the reaction products were treated with water vapor. After 10 min, most of degraded ions could be observed ; peptide acylation had occurred only to a small extent (Fig. 3A) but increased slowly with reaction time. The extent of degradation at 30 min (Fig. 3B) was almost the same as that at 1 h (Fig. 3C) and only progressed slowly after the 5-h reaction time (Fig. 3D). Thus we routinely use 30 min or 1 h. We avoid the short time reaction, e.g. 10 min, because of complexity due to partial acylation.

Ion mode of FAB mass spectrometry. In addition to the reaction itself, attention to the ion mode used in FAB mass spectrometry is important to maximize the amount of sequence information obtained in the analysis. We degraded the peptide, Tyr-

Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-T~-Asp- Asn-Gln, followed by the water vapor treatment and mass spectrometry of the degraded peptide in both positive and negative mode. Positive mode analysis, as shown in Fig. 4A, suggested that degradation was only achieved until the peptide 1-6, while the negative mode analysis demonstrated that the reaction had proceeded further to the peptide 1-4 (Fig. 4B). This difference was caused by the loss of the positive charges in the degraded peptides including acylation. Many degradations appear to stop in the positive mode earlier than really achieved. Therefore mass spectrometry in both positive and negative modes is needed to ensure all the sequence information has been retrieved, although positive mode analysis is more sensitive than negative mode, in general.

Evidence for chemical degradation. Evidence that the reaction described is actually a C-terminal chemical degradation was supplied by separation of the degraded peptides by HPLC. A tetrapeptide Met-Arg-Phe-Ala was degraded by (C,F,CO),O

368 Takamoto et al. (Eul: J. Biochern. 228)

0.05 -

0.04 -

Lo v

2 - 0.03.

3

O . O 2 1

I 1 I I I 2 5 30 3 5 4 0 4 5 50 (min)

Fig. 5. FAB mass spectrum and HPLC profile of the C-terminally degraded dodecapeptide. A dodecapeptide, Ala-Arg-Gly-Ile-Lys-Gly-Ile- Arg-Gly-Phe-Ser-Gly (20 pg), was degraded with the vapor of 30% (C,F,CO),O acetonitrile solution at -20°C for 1 h followed by water vapor treatment. After the reaction, an aliquot (1120) of the product was analysed by FAB mass spectrometry. The rest of the product was separated by reverse-phase HPLC. (A) FAB mass spectrum of the degraded product mixture; (B) elution profile of the product by reverse-phase HPLC, under the following conditions: system, 600E (Waters-Millipore, USA); column, TSK gel (4.6X250 mm Tosoh, Japan); flow rate, 0.8 mumin; CF,CO,H and 0.1 % CF,CO,H in 80% aqueous acetonitrile; gradient system, a linear 0-48% acetonitrile gradient for 60 min. The chromatographic peaks with numbers were analysed by FAB mass spectrometry. The results are summarized in Table 1 . The most of the peaks without numbers are amino acid acyl derivatives from the truncated C-termini.

vapor and treated with aqueous pyridine vapor. The amino acid composition and FAB mass spectra of five acylated peptides showed the sequences of the peptides to be 1-4 (16%), 1-3 (50%) and 1-2 (10%) where percentages in parentheses indicate the yields of the acylated peptides from the compositions. Two acyl peptides for 1 -4 and 1 - 3 with identical compositions were observed, indicating optical isomers at their C-terminal amino acids due to formation of oxazolone.

Another peptide, Ala- Arg-Gly-Ile-Lys-Gly-Ile- Arg-Gly-Phe- Ser-Gly (20 pg), was reacted with (C,F,CO),O vapor and an aliquot was analysed by FAB mass spectrometry to check that

the peptide was degraded (Fig. 5A). The remainder of the degraded peptide was fractionated by HPLC (Fig. 5B). The iso- lated fractions were subjected to FAB mass spectrometry and identified as the acylated products corresponding to the respective sequential degradation products. In addition to the acylated sequences, -1-Da peaks were also observed (Table 1). These peaks are due to the cleavages at the amino groups resulting in the acid amides (Fig. 8A), not to cleavages at the peptide bonds, which stops further successive degradation.

Evidence that the direct degradation products are oxazolones (or mixed anhydrides) was provided by converting them to the

Takamoto et al. (EUK J. Biochem. 228) 369

& ---

80-

a, u 2 60-

Table 1. FAB mass spectrometric identification of the HPLC-separated fragments of C-terminally degraded products. The truncated degradation products were separated by reverse-phase HPLC (Fig. 5 B). Fractions numbered in the HPLC profile were identified by FAB mass spectrometry. Degraded products were acylated except 1-12 (peak 7); sequences with an asterisk are the -1-Da products.

Peak Observed mass Sequence (calculated mass)

Da (Da) 1 391.3 1-2 * ( 391.1) 2 449.2 1-3 ( 499.2) 3 392.2 1-2 ( 392.1) 4 1220.3 1-10 (1220.6) 5 746.3 1-6 * ( 746.4) 6 689.3 1-5 * ( 689.4) 7 747.3, 1218.5 1-6 ( 747.4), 1-12" (1218.7) 8 690.4, 747.3 1-4 ( 690.4), 1-6 ( 747.4) 9 1072.1 1-9* (1072.6)

10 561.2, 746.3, 1015.5 1-4* ( 561.2), 1-6" ( 746.4), 1-8* (1015.5)

11 1016.4, 1073.5 1-8 (1016.5), 1-9 (1073.6) 12 1016.4, 1073.3 1-8 (1016.5), 1-9 (1073.6) 13 1017.5, 1074.5 1-8 (1016.5), 1-9 (1073.6) 14 562.5, 859.5 1-4 ( 562.2), 1-7* ( 859.4) 15 562.2 1-4 ( 562.2) 16 562.1 1-4 ( 562.2) 17 860.5, 1364.5 1-7 ( 859.4), 1-12 (1364.7) 18 860.5, 1308.0 1-7 ( 859.4), 1-11 (1307.7) 19 1219.8 1-10* (1220.6) 20 1219.7 1-10* (1220.6)

Not acylated.

corresponding propyl esters. The degraded octapeptide His-Pro- Phe-His-Leu-Leu-Val-Tyr was esterified by propanol vapor and the products analysed by FAB mass spectrometry (Fig. 6). The principal degraded products were shown to be acylated inter- mediate compounds such as oxazolones or mixed anhydrides which are easily converted to their propyl esters.

The -46-Da peak was thought to be due to decarboxylation of the C-termini of the degradation products. This was tested by

degrading the tetrapeptide Met-Arg-Phe-Ala under the standard conditions and fractionating the degraded products by HPLC. Fig. 7A shows the chromatographic profile at 280 nm. It is known that the A,,, of a phenyl group attached to an aryl group is 280 nm, whilst that of a phenyl group alone is 254 nm with a low absorption coefficient. Therefore the major peak in the chromatogram at 280 nm corresponds to the degraded peptide 1-3, acyl-Met-Arg-NH-CH=CH-C,H,, (Fig. 8 C). FAB mass spectrometry of this peak showed a protonated molecular ion peak of 553 Da (Fig. 7B) which corresponded to the calculated mass of the degraded compound (552.9 Da). Taken together, this experiment confirmed the nature of the -46-Da mass peak.

DISCUSSION

Reproducibility of the present C-terminal degradation is achieved when the following precautions are taken. (a) The moisture of the air is easily absorbed by the reagent at -20°C or -196"C, and the reagent anhydride instantly hydrolysed to the corresponding acid; so the entire experiment must be performed under dry nitrogen gas in a glove box. (b) The reaction proceeds at -20°C with the vaporized reagent under re- duced pressure in a relatively short period so the reaction vessel containing the reagent must be kept at liquid nitrogen temperature before and after the reaction. (c) The perfluoroacyl anhydride reagents are extremely volatile and evaporate when the reaction vessel is evacuated. So the anhydride must be diluted with acetonitrile and cooled with liquid nitrogen. So far, acetonitrile is the only solvent we have found to be inactive towards the perfluoroacyl anhydrides. The advantage of using acetonitrile solution is that it does tolerate up to 10% (molar ratio) water/(C,F,CO),O in the reaction mixture without interfering with the reaction, although acetonitrile is not essential for the reaction.

C-terminal degradation of peptides having an a-carboxyl amide group did not occur upon exposure to perfluoroacyl anhydride vapor under the present conditions (data not shown). The peptides employed in this study contain almost all of the common amino acid residues and their C-terminal degradation ex-

1 -7+Acyl+Prop

//

Fig. 6. FAB mass spectrum of the esterified product of the C-terminally degraded His-Pro-Phe-His-Leu-Leu-Val-Tyr with (C$,CO),O vapor. The peptide ( 5 pg) was exposed to the vapor of 15% (C,F,CO),O acetonitrile solution (300 pl) at -20°C for 30 min. The product was dried in vucuo and reacted with the vapor of n-propanol at 60°C for 15 min. The reaction product was dried in vucuo again and analysed by FAB mass spectrometry. In this figure, Prop indicates propyl ester.

370 Takamoto et al. (Eul: J. Biochern. 228)

0 .08

0.06

0.04 2

0 . 0 2

0.00

0.0 5 . 0 10.0 15.0 2 0 . 0 2s .o Time (min )

1-3+acyl-46 (553.0)

ZE0 300 400 500 600 700 800 900 1000 [M+H]+

Fig.7. The HPLC elution profile of C-terminally degraded Met-Arg-Phe-Ala at 280 nm and FAB mass spectrum of the fractionated major peak. C-terminal degradation of the peptide (20 pg) was performed with a vapor of 30% (C,F,CO),O acetonitrile solution (100 pl) for 1 h at -20°C. HPLC used the SMART HPLC system under the following conditions: column, pRPC C2/C18 PC 3.2/3 (2.1 m m X I O O m m , Pharmacia); flow rate, 0.2 mumin; solvents, 0.1% aqueous CF,CO,H and 0.1 % CF,CO,H acetonitrile solution. A linear gradient of 5-60% acetonitrile was made from 5 rnin to 17.5 min followed by an isocratic elution from 17.5 rnin to 20 min. The chromatogram was monitored at both 215 nm (data not shown) and 280 nrn (A). Peaks were fractionated and collected. The major fraction marked by * was analysed by FAB mass spectrometry (B).

hibited no problems. In an attempt to determine the relative ease of cleavage, we investigated degradation yields, roughly calculated from the peak height of mass spectra. Table 2 summarizes the results from more than 50 experiments. The values are not quantitative so they only indicate the relative ease of bond cleavages. Partial oxidation of peptides under the present reaction conditions was observed for methionine and tryptophan residues and unmodified cysteine residues. Cysteine residues may be pyridylethylated (Amons, 1987) before the reaction.

In FAB ionization, a, b and c series ions are observed, and x, y, and z series ions with the N-terminal fragments (Biemann, 1988). Fragment ions similar to the latter N-terminal fragment ions were not observed in the present experiments. However, the present C-terminally degraded molecular ions (-46-Da, - 18- Da and -1-Da ions) are the same as the FAB fragmented ions of a (-46 Da), b (- 18 Da) and c (-1 Da), respectively. BIE and B'IE linked scans (Gaskell et al., 1979; Boyd and Beynon 1977) allow detection of fragment ions and parent ions, respectively. Analysis of selected molecular ion peaks from the perfluoroacyl anhydride reaction mixture failed to show either the fragment or parent ions. This indicated that the original degradation spectrum was mainly caused by chemical reaction.

Some of the asparaginyl and glutaminyl residues were observed to be dehydrated and converted into their corresponding nitriles (Fig. 8B). Most of -18-Da peaks, such as C-terminal oxazolone derivatives, disappeared after water treatment but this

nitrile -18-Da mass reduction is not recoverable (Fig. 4A). The unrecoverable - 18-Da mass was also occasionally observed by the dehydration of serine residues to dehydroalanine and by formation of a five-membered ring and a six-membered ring from internal aspartic acid and glutamic acid residues, respectively (also see Fig. 8B). These -18-Da mass reductions may not be involved in the factors stopping the successive degradation reaction.

The factors stopping the reaction are not fully understood. A candidate may be the heterogeneous vaporlsolid reaction which causes limited accessibility of the reagent vapor to the peptides. The formation of the acid amide at the C-terminus, as observed by a mass reduction of 1 Da (Fig. 8A and Table 1) is another stopping factor. The formation of the -46-Da molecular ion(s), due to decarboxylation of the respective C-terminal a-carboxyl group (Figs 7B and 8C), is another stopping possibility.

At - 20 "C the acylation is slower than the above degradation reaction (Fig. 3). This difference in the two reactions was clearly shown by ninhydrin analysis, where acylation was observed following the release of the ninhydrin-positive amino groups (data not shown). Possible acylation sites are the a-NH, group of the N-terminus, the E-NH, groups of lysine residues, the OH group of the C-terminal oxazolone (enol form) and the OH groups of internal serine residues. Among them, a-NH,-acylation is the most abundantly observed, while e-NH2-acylation is not common under the present conditions. Both oxazolone 0-

371 Takamoto et al. (Em J. Biochem. 228)

Table 2. Degradation of peptide bonds by perfluoroacyl anhydride vapor. The first column lists the amino side residues of peptide bonds; the values are the percentage degradation of bonds to the indicated carboxy side residues. Degradation was roughly calculated from the peak height in mass spectra.

N residue

Degradation of C-terminal bond to

D N T S E Q P G A V M I L Y F H K R W

%

D 99 99 9 96 94

T 22

E 68 76 94

N 55 95 98 99 80

S 95 42 93 87 91

Q 87 94 50 P 91 65 89 40 12 90 14 G 99 97 85 29 50 65 84 95 96

V 69 75 27 M 99 65

64 84 I 23 60 99 70 81 90 90 L

Y 94 21 96 75 75 F 98 89 94 93 96 22 85 60 78 83 H 87 56 98 K 91 97 41 75 95 88 63 93 71 66 73 77 R 14 86 58 82 42 50 23 42 42 20 W 98

A 33 89 68 96 47 80

91 38 90

(A) -1 mass ion peak Acid uiiiide -NH.AH-$-NH-CH-C-OH Ri 8 2 -NH-CH-$-NH2 71

0 4 8 0

(8) -18 mass ion peak Oxazolone

were only observed in reactions above 0°C or with extended reaction times (more than 5 h).

Successive C-terminal degradation can also be achieved by conventional carboxypeptidase digestion. However carboxy- peptidases have, like other enzymes, strict degradation specifi- cities. The present chemical degradation procedure thus far has no specific limitations, even prolyl peptide bonds being degraded.

In summary, we propose the following conditions for C-terminal successive degradation of peptides: (a) exposure of solid peptides to the vapor of 15% (by vol.) (C,F,CO),O in acetonitrile solution for 0.5-1 h (to complete acylation) at -20°C (to avoid side reactions); (b) following the reaction, exposure of product to 10% (by vol.) pyridine/water vapor at 100°C for 30 min (in order to simplify interpretation of the mass spectrum).

ASP, (GINo 5(6)-membered ring This work was supported by the Ministry of Education, Science and II Hz Culture, a Grant-in-Aid for Specially Promoted Research.

CH, fC\ OH 7 1 - H 2 O ,c. 0 - -NH-CH F' 9' \ N-CH-E-

0 REFERENCES C' 8

(C) -46 mass ion peak Decarboxylated-Phe

0 Phe

I

?1 II -NH-~WC-NH-CH-C-OH - -NH-CH-C-NH-CH

8 8 8 R i 7% -46

Fig. 8. Possible schemes in the reaction of perfluoroacyl anhydride vapor on peptides.

acylation and serine 0-acylation disappear with water vapor treatment.

The possible side reactions during the course of degradation are cleavages of the internal peptide bonds at the carboxyl side of aspartic acid and the amino side of serine. These cleavages

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Carboxy-terminal Degradation of Peptides using Perfluoroacyl Anhydrides : A C-Terminal Sequencing Method