The document describes a novel method for C-terminal sequencing of proteins using partial acid hydrolysis and mass spectrometry analysis. Peptides or proteins are hydrolyzed with vapors of strong organic acids like trifluoroacetic acid or hepta-fluorobutyric acid at high temperatures. This results in successive degradation of the C-terminal residues as seen by mass spectrometry. The degradation is believed to occur via formation of an oxazolone ring at the C-terminal amino acid, followed by removal of the C-terminal residue. Specific cleavages also occur at the peptide bonds preceding aspartic acid and serine residues. This method allows efficient C-terminal sequencing of proteins in small quantities directly from mass

1. Eur. J. Biochem. 206,691 -696 (1992)
(c; FEBS 1992
C-terminal sequencing of protein
A novel partial acid hydrolysis and analysis by mass spectrometry
Akira TSUGITA, Keiji TAKAMOTO, Masaharu KAMO and Hiromoto IWADATE
Research Institute for Biosciences, Science University of Tokyo, Yamazaki, Japan
(Received January 23, 1992) - EJB 92 0088
Peptides or proteins were hydrolyzed by vapors of 90% pentafluoropropionic acid or hepta-fluorobutyric
acid at 90°C for various time periods. The hydrolyzate mixtures analyzed by both fast-atom-
bombardment and electrospray ionization mass spectrometry showed a series of C-terminal
successive degradation molecular ions. The degradation reaction may be due to the selective formation
of an oxazolone ring at the C-terminal amino acid, followed by hydrolytic removal of the C-terminal
amino acid. The major side reactions were cleavages of the peptide bonds at the C side of the internal
aspartic acid residue and the N side of serine residue.
Efficient and automatic methods of sequencing micro
quantities of proteins are the most demanded techniques in
biotechnology. Automated Edman degradation is a widely
accepted N-terminal sequencing method. However, chemical
C-terminal sequencing (Inglis et al., 1991) has not yet been
well established nor widely accepted.
Previously, we reported that the addition of trifluoroacetic
acid to hydrochloric acid, both in liquid (Tsugita and Scheffer,
1982) and vapour phases (Tsugita et al., 1987), increased the
efliciency of hydrolysis of peptides, suggesting a type of cleav-age
other than the conventional hydrochloric acid hydrolysis
of peptide bonds. Our work on partial acid hydrolysis using
high concentrations of strong organic acids have indicated the
cleavage specificity of peptide bonds to be different from those
of either Sanger's high-concentration-acid method (Sanger
and Thompson, 1953), or the conventional dilute-acid hy-drolysis
(Inglis, 1983). The present report summarizes the
results of a novel high-concentration organic acid hydrolysis
of peptides which essentially allows C-terminal protein
sequencing and C-terminal sequencing from specific cleavage
points (i.e. at the C side of aspartic acid and/or the N side of
serine). Analysis of these data was directly carried out by mass
spectroscopy (MS) of the hydrolyzate. Possible mechanisms
of the C-terminal successive degradation and the specific
cleavages are preliminarily discussed.
MATERIALS AND METHODS
Trifluoroacetic acid and F,C,COOH were the products
of Sigma Chemical CO. (USA); F7C3COOH and dithio-theritol
were obtained from Wako Pure Chemical Industry
(Osaka, Japan). 4-Vinylpyridine was obtained from Tokyo
Kasei Kogyo Co. (Tokyo, Japan). A peptide, LWMRFA, and
human des-Aspl-angiotensin I, RVYIHPFHL, were pur-chased
from Serva Feinbiochem (Heidelberg, FRG). Human
parathyroid hormone, fragment 69 - 84, EADKADVNVLT-Correspondence
to A. Tsugita, Research Institute for Bioscicnces,
Sciencc University of Tokyo, Yamazaki, Noda 278 Japan
ionization; m/z, molecular ion; MS, mass spectrometry.
Abbreviations. FAB, fast-atom-bombardment ; ESI, electrospray
KAKSE, and Xenopus luevis magainin 1, GIGKFLHSAGK-FGKAFVGEIMKS,
were the synthesized products of the
Peptide Institute Inc. (Minoh, Japan). Human glucagon was
obtained from Sigma Chemical Co. (USA) and hen egg white
lysozyme was from Seikagaku Kogyo Co. (Tokyo, Japan).
Acid degradation of peptide or protein
A small glass tube (6 mm x 40 mm), containing dried
peptide or protein, was placed in a large glass tube
(13 mm x 100 mm), which contained 100 pI aqueous organic
acid of a given concentration, and flame sealed under vacuum.
The tube was placed in an oil bath at 90°C for various time
periods. After the reaction, the tube was opened and the small
tube was placed in a vacuum desiccator to remove traces
of acid. Dithiotheritol (1 - 5%, mass/vol.) was added to the
hydrolysis acid in the large tube to prevent oxidation or oxi-dative
modification. Alternatively, the small sample tube was
placed in a middle-sized glass tube (10 mm x 30 mm) contain-ing
50 mg solid dithiotheritol, and the tubes were placed in
the large tube containing the acid and sealed under vacuum.
Analysis by MS
The dried hydrolyzate was disolved in 5% acetic acid and
mixed with glycerol and thioglycerol(1: 1, by vol.) as matrix
and put on the target. Fast-atom-bombardment mass spec-troscopy
(FAB-MS) was carried out with a HX-110 mass
spectrometer (JEOL, Japan), equipped with a DA 5000 data
system, employing an accelerating voltage of 10 kV and xenon
as an ionizing gas.
Electrospray ionization (ES1)-MS was carried out with a
SX-102 mass spectrometer with an electrospray ion source
(JEOL, Prototype, USA) at a flow rate of 0.5 pl/min, em-ploying
an accelerating voltage of 10 kV and nitrogen as a
drying gas. The sample was dissolved in 2% acetic acid/50%0
aqueous methanol.
Pyridylethylation
The protein in a small tube (6 mm x 40 mm) was placed
in a large tube (13 mm x 100 mm) which contained a mixture

2. 692
BE-
68-
48-
20-
1
a> , . 40-
m
a,
1-6 .- c .
- . 1-5
cc 20:
1
1-5
40 3
26-
J 881
-
1-6
1-3 1-1
1
1-6
I
"1
I
1-2 1-3 14
1 1 1-5
>
I 1-6
m/z(MH+)
Fig. 1. FAB-MS of the hydrolyzates of a hexapeptide, LWMRFA. The peptide (200 pmol) was hydrolyzed with 90% (by vol.) of various organic
acids containing 5% dithiotheritol in the vapor phase at 90°C. (a) Before hydrolysis; (b) trifluoroacetic acid for 4 h; (c) F5CzCOOH for 4 h;
(d) F7C3COOH for 4 h; (e) F5C2COOH for 24 h; (0 F7C3COOH for 24 h.
of 100 pl pyridine, 20 p1 4-vinylpyridine, 20 p1 tributyl-phosphine
and 100 pl water. The large tube was flame sealed
under vacuum and incubated at 60°C for 2 h. After reaction,
a small tube was taken out and dried in a vacuum desiccator
for 1 h. 5 pl water was added to a small tube and dried in the
vacuum desiccator for 1 h to further remove trace of reagents
(Yamada et al., 1991).
RESULTS AND DISCUSSION
A novel organic acid successive degradation
Trifluoroacetic acid, F5C2COOH and F7C3COOH were
chosen as test organic acids because of their volatility and low
pK,. To avoid contamination from these acids, the vapor
phase of the acids were used for the hydrolysis reaction as
described in Materials and Methods.
We have noticed different modes of acid hydrolysis with
high and low concentrations of organic acids in FAB-MS
molecular ions. Low acid concentrations resulted in non-specific
cleavages, but high acid concentrations gave C-ter-minal
successive degradation including specific peptide-bond
cleavages.
When high concentrations of organic acids were used,
oxidation and oxidative modifications of side chains in the
peptide were observed. Addition of a reducing reagent,
dithiotheritol, was found effective to avoid the oxidative reac-tions
of methionine, tryptophan and tyrosine residues.
Fig. 1 shows a comparison of the molecular ions produced
by trifluoroacetic acid, FJ2COOH and F7C3COOH hy-drolysis
of a hexapeptide LWMRFA in FAB-MS. In Fig. lb,
trifluoroacetic acid at 90°C for 4 h shows several modified
molecular ions, indicated by higher-mass molecular ions than
that of the original peptide residues 1-6, (m/z of 823). The

3. 693
I 00
90
80
70
a, 60 2
2
m
-50 50
.a>-, 40
m
a,
-c
oc
30
20
I0
0
I (1-22) +4
300 350 400 450 500 550 600 650 700 750 800 850 900
m/Z =(M+nH?/n
-. _. 1 5 10 15 ZU 23
Gly-Ile-Gly-Lys-Phe-Leu-His-Ser-Ala-Gly-Ly~-Phe-Gly-Lys-Ala-Phe-Val-Gly-Qlu-Ile-Met-LyE-Ser
I
(1-7)+ 2 I
I
+2 I
I
t I (8-18)
t i (1-23)*y
I I (1-22)+3
I I (1-21)+3
I I (1-20)+3
+3 I I (1-19)+3
I I (1-18) +3
I I (1-17)+ 3
I I (1-16) +3
I I (1-15)+3
I I (8-23) +3
t I (8- 32'2)
t I (8-21) +3
I i (1-23)-*
+4 I I (1-22)+ 4
Fig. 2. EN-MS of the hydrolysate of a peptide, GIGKFLHSAGKFGKAFVGEIMKS. The peptide (125 pmol) was hydrolyzed with 90%
F5C2COOH in the vapor phase in the presence of solid dithiotheritol(50 mg) for 2 h. The dried hydrolyzate was disolved in 25 pl2% acetic
acid in 50% aqueous methanol solution.
successive C-terminal degradation ions were observed as 1 -
5 (m/z of 752), 1-4 (m/z of 605), 1-3 (m/z of 449) from
the original peptide in addition to the modified molecules.
Nonspecific cleavages were also observed, such as residues
4-6,3-5 or 3-6.
On the contrary, both FSC2COOH (Fig. lc) and
F&COOH (Fig. Id) hydrolysis for 4 h at 90°C showed more
clear-cut C-terminal degradation molecular ions. Fig. 1 e and
f show the successive C-terminal degradation molecular ions
for both F,C,COOH and F7C3COOH after 24 h hydrolysis,
respectively. No preference was shown between F5C2COOH
and F7C3COOH at 4 h hydrolysis, but F5C2COOH gave more
clear C-terminal degradation ions than F,C,COOH for the
longer 24 h reaction time, leading to the use of F5CzCOOH for
the following experiments. Of the various acid concentrations
tested (50 - 98%) for several peptides, around 90% were

4. 694
100
80
60
40
20
I Y Y -
z
1-9
1-9
40-
1-8
m/z (MH+)
Fig. 3. FAB-MS of the hydrolyzates of a nonapeptide, RVYIHPFHL. The peptide (ZOO pmol) was hydrolyzed with 90% F7C3COOH containing
5% dithiotheritol in the vapor phase at 90°C. (a) Before hydrolysis; (b) F7C3COOH for 4 h; (c) F7C3COOH for 24 h. The conditions for MS
are same as in Fig. 1.
found out to be optimal for this specific C-terminal degradati-on.
Fig.2 shows an ESI-MS of the 90% F5CzCOOH hy-drolyzate
of a peptide, magainin 1, consisting of 23 amino
acids. The multi-charged degradation molecular ions of both
the intact peptide and a fragment specifically cleaved at the
eighth serine residue, (8 - 23) were observed. The + 5 molec-ular
ion was the maximal chargeable ion corresponding to the
N-terminal amino group and four internal lysine residues in
the peptide. The absence of further degraded ions in the series
of f 4 and + 5 molecular ions was possibly due to the charge
at Lys22. The longest successive C-terminal degradation ions,
of eight residues, were detected in the + 3 molecular-ion series.
It should be noted that the amino acid analysis of the
hydrolyzate mixtures detected the amino acids which corre-spond
to the degradation shown in the MS data and further
more, the data were able to distinguish the amino acids of
identical masses such as isoleucine and leucine and lysine and
glutamine (data not shown).
Susceptible and resistant peptide bonds
The peptide bonds at both sides of aspartic acid have been
known to be labile in dilute acid (Inglis, 1983). The bond at
the N side of serine was found to be labile for a concentrated
hydrochloric acid hydrolysis (Sanger and Thompson, 1953).

5. 69 5
16-29
600 800 1000 1200 1400 1600 1E a0
1 5 10 15 20 25 29 m/z
H S Q Q T P T S D Y S K Y L D S R R A Q D F V Q W L U I T
1-9 (MHt979) I I 16-29 (Mw1753)
1-8 (Mw865) I I 16-28 (MH+1652)
1-7 (Mp778) 1 I 16-27 (MP 1538) - 1-6 (Mp677) 16-21 (Mp733) - 1-5 (Mv530) 16-20 (MP677)
H 16-18 (Mw530) - 16-19 (MH'618)
120-126 1-14 1-15
d
@a0l ' ' ' l 0 b 0 ' ' ' 1200 ' ' ' 14'0 0 ' ' . 16'0 0 l a 0 0 2000 2 a0
mlz
1 5 10 15 20 25
K V P G R C E L A A A M K R H G L D N Y R G ~ S L ~G . . . .
I I 1-18 (MH'2109)
I I 1-17 (MH+1993)
1 I 1-16 (MH+188O)
I i 1-15 (MH:1823)
I I 1-14 (MH 1686)
.... 110 la o 129 I G U N A W V A W R I R C K G T D V Q A W I R G C R L
I I- 1120-129 (MW1308)
I 120-128 (Mp1194) 120-127 (M'l1038)
120-126 (MP 830)
Fig. 4. FAB-MS of the hydrolyzates of proteins. (a) Human glucagon (100 pmol) was hydrolyzed with 90% F,C,COOH containing 5%
dithiotheritol in the vapor phase at 90°C for 4 h. The cleavage and molecular ions are schematically illustrated. The conditions of MS were
same as in Fig. 1. (b) Pyridylethylated hen egg white lysozyme (100 pmol) was hydrolyzed with 90% FsCzCOOH in the vapor phase and with
solid dithiotheritol at 90°C for 4 h. The conditions of MS were same as in Fig. I.

6. 696
Under the present conditions, the peptide bond only at the C
side of the internal aspartic acid and the peptide bond at the
N side of serine were selectively cleaved. In the experiments
performed on various test peptides, the N side of threonine
and both sides of glycine were observed to be occasionally
cleaved (data not shown).
The peptide bond of Pro-Xaa is partially resistant to the
present successive degradation. A nonapeptide, RVYIHP-FHL,
was hydrolyzed for 4 h with 90% F7C3COOH at 90°C.
Analysis of the hydrolyzate indicated resistance to hydrolysis
at the peptide bond between residues 6 and 7. Prolonged
hydrolysis showed further successive degradation ac-companying
a small extent of nonspecific cleavage (Fig. 3 b
and c).
It should be noted that under the present conditions little
cleavage of the amide groups of acidic amino acids, except for
the C-terminal a-amide bond, was observed.
Reaction mechanism
The successive degradation is due to the formation of an
oxazolone five-membered ring at the C-terminal amino acid
(Matsuo et al., 1965, Inglis et al., 1991), followed by hydrolysis
of the C-terminal amino acid in the oxazolone. This mecha-nism
was indicated by the observations of oxazolone molec-ular
ions (- 18 from the respective peptide) in the MS, as
shown in Fig. 4. The two internal cleavage reactions are at the
C side of aspartic acid and the N side of serine. The internal
aspartic acid forms another type five-membered ring, followed
by hydrolysis of the succinylimine group (Inglis, 1983). Serine
undergoes an N+O peptidyl shift for the cleavage of the
peptide bond (Sanger and Thompson, 1953). The slow down
of the prolyl peptide-bond cleavage may be explained by a
steric hindrance for the formation of oxazolone ring. The
establishment of the above reaction mechanism needs further
experiments.
Application to protein sequencing
Application of this method to proteins is still preliminary.
Glucagon, a peptide consisting of 29 amino acids (200 pmol),
was hydrolyzed with 90% F,C&OOH at 90°C for 1 h. The
result gave three series of C-terminal successive degradation
molecular ions; one ion series is from the C-terminal peptide
fragments, 16-29, 16-28 and 16-27, where the cleaved
Deatide bond between residues 15 and 16 is Asa-Ser and the
other is that of the N-terminal peptide fragments of residues,
1 - 9, 1 - 8, 1 - 7, 1 - 6 and 1 - 5, where the cleaved peptide
bond between residues 9 and 10 is Asp-Tyr. Further hydrolysis
gave an additional ion series of peptides 16 - 21,16 - 20,16 -
19 and 16- 18 (Fig. 4a), where the cleaved peptide bond be-tween
residues 21 and 22 is Asp-Phe.
The FAB-MS molecular ions of the hydrolyzates of
lysozyme are shown in Fig.4b. The protein was pyridyl-ethylated
as described in Materials and Methods in a vapor
phase (Yamada et al., 1991) to protect the cysteine residues.
Two series of molecular ions were observed, the C-terminal
sequence of residues, 120- 129, 120- 128, 120- 127 and
120- 126, and the C-terminal degradation of an N-terminal
fragment composed of residues, 1 - 18, 1 - 17, 1 - 16, 1 - 15
and 1 - 14. The direct amino acid analysis of the hydrolyzate
mixtures of both proteins were carried out and the data con-firmed
the above MS observations.
Analysis by FAB-MS has limitations, such as molecular
size or selectivity of ionization of peptides. The present specific
peptide cleavage reactions for aspartic acid and serine may
favor this type of FAB-MS. ESI-MS is also useful to analyze
the present hydrolyzate. Programs to calculate the successive
degradation molecular ions have been developed and will
become available for handling the complexed multi-charged
molecular ions for ESI-MS. In conclusion, the present method
of partial hydrolysis, including C-terminal successive degra-dation,
may permit useful C-terminal analysis of medium-sized
peptides.
REFERENCES
1. Inglis, A. S. (1983) in Methods Eiriymol. 91,324-332.
2. Inglis, A. S., Moritz, R. L., Begg, G. S., Reid, G. E., Simpson, R.
J., Graffunder, H., Matschull, L. & Wittmann-Liebold (1991) in
Methods in protein sequence analysis (Joernvall, H., Hoeoeg, J.-
0. & Gustavsson, A.-M., eds) pp. 23 - 34, Birkhaeuser Verlag,
Basel.
3. Matsuo, H., Fujimoto, Y. & Tatsuno T. (1965) Tetrahedron Lett.
39,3465 - 3461.
4. Sanger, F. & Thompson, E. 0. P. (1953) Biochem. J. 53,353-366.
5. Tsugita, A. & Schemer, J. J. (1982) Eur. 1. Biochem. 124, 585-
588.
6. Tsugita, A., Uchida, T., Mewes, H. W. & Ataka, T. (1987) J.
Biochem. 102,243 - 246.
I . Yamada, H., Moriya, H. & Tsugita, A. (1991) Anal. Biochem. 198,