Analytical Biochemistry 428 (2012) 167172

Contents lists available at SciVerse ScienceDirect

Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio

Mass spectrometry-based sequencing of protein C-terminal peptide using

a-carboxyl group-specic derivatization and COOH capturing
Chihiro Nakajima, Hiroki Kuyama , Koichi Tanaka
Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Corporation, Kyoto 604-8511, Japan

a r t i c l e

i n f o

Article history:
Received 3 April 2012
Received in revised form 14 June 2012
Accepted 19 June 2012
Available online 27 June 2012
Keywords:
C-terminal sequencing
a-COOH-specic derivatization
COOH capture

a b s t r a c t
An approach to mass spectrometry (MS)-based sequence analysis of selectively enriched C-terminal
peptide from protein is described. This approach employs a combination of the specic derivatization
of a-carboxyl group (a-COOH), enzymatic proteolysis using endoproteinase GluC, and enrichment of
C-terminal peptide through the use of COOH-capturing material. Highly selective derivatization of aCOOH was achieved by a combination of specic activation of a-COOH through oxazolone chemistry
and amidation using 3-aminopropyltris-(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-propylamine). This amine component was used to simplify fragmentation in tandem mass spectrometry (MS/
MS) measurement, which facilitated manual sequence interpretation. The peptides produced after GluC
digestion were then treated with a COOH scavenger to enrich the C-terminal peptide that is only devoid
of COOH groups, and the obtained C-terminal peptide was readily sequenced by matrix-assisted laser
desorption/ionization (MALDI)MS/MS due to the TMPP mass tag.
2012 Elsevier Inc. All rights reserved.

The C terminus of protein has been reported to be of great

importance in determining various cellular functions [15]; hence,
its exact characterization provides keys to understanding diverse
events in cells. Mass spectrometry (MS)1 has been the method of
choice for analyzing the protein C terminus. However, even with
state-of-the-art technology, it is still difcult to reveal the C terminus of a protein of interest. In particular, conventional methods such
as peptide mass ngerprinting and peptide fragment ngerprinting
are incapable of clarifying the C-terminal sequence if the protein is
C-terminally processed. Therefore, an efcient and practical technique for characterizing protein C-terminal variation has been
sought [6].
A couple of years ago, we reported a protein C-terminal
sequencing method by MS [7]. Although this method is quite feasible, there remains the limitation that protein terminated with
lysine residue at the C terminus cannot be analyzed. We have since
Corresponding author. Fax: +81 0 75 823 2900.
E-mail address: kuyama@shimadzu.co.jp (H. Kuyama).
Abbreviations used: MS, mass spectrometry; MS/MS, tandem mass spectrometry; TMPP, tris(2,4,6-trimethoxyphenyl)phosphonium; Ac2O, acetic anhydride;
TCEP, tris(2-carboxyethyl)phosphine hydrochloride; MeCN, acetonitrile; HCOOH,
formic acid; PfpOH, pentauorophenol; TFA, triuoroacetic acid; MeOH, methanol;
Et3N, triethylamine; CHCA, a-cyano-4-hydroxycinnamic acid; MDPNA, methanediphosphonic acid; TMPP-propylamine, 3-aminopropyl tris(2,4,6-trimethoxyphenyl)phosphonium bromide; MALDI, matrix-assisted laser desorption/
ionization; TOF, time-of-ight; PSD, Post-source decay; CALM, calmodulin; SODM,
superoxide dismutase; CYC-c, cytochrome c; TPIS, triosephosphate isomerase.
1

0003-2697/$ - see front matter 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ab.2012.06.016

been intensively seeking a general method of protein C-terminal

analysis by MS.
We recently demonstrated that a protein C-terminal sequence
analysis can be performed by specic derivatization of a-COOH
and proteolytic digestion [8]. Virtually any type of protease can
be used for this technique, and the C-terminal sequence of a
protein incorporating lysine at its C terminus can be analyzed. In
addition, this technique has no enrichment or recovery steps for
C-terminal peptide, which eliminates time-consuming operations.
The C-terminal peptide peak is discerned by its strong intensity
(sometimes the strongest) in the mass spectrum. If the target peak
is weak and buried among other nontarget peaks, it can be discriminated by tandem mass spectrometry (MS/MS) analysis owing to
the characteristic fragment peak of the tris(2,4,6-trimethoxyphenyl)phosphonium (TMPP) mass tag [9], which is attached only
to the target C-terminal peptide. For protein digestion, practically
any type of protease can be used in the technique, which is an
advantage over other reported approaches. However, in cases
where the target C-terminal peptide is hardly discerned even by
MS/MS analysis, an orthogonal means such as isolation or
enrichment of the target peptide is required. Therefore, we studied
a C-terminal analysis method incorporating a-COOH-selective
derivatization and C-terminal peptide enrichment.
Here we report an approach to C-terminal peptide enrichment
from protein and its MS-based sequence analysis using a combination of selective attachment of a TMPP group [10,11] to the C

168

MS sequencing of protein C-terminal peptide / C. Nakajima et al. / Anal. Biochem. 428 (2012) 167172

terminus [9] and enrichment of the C-terminal peptide using a

COOH scavenger after GluC digestion.
Materials and methods
Calmodulin (bovine), superoxide dismutase (Escherichia coli),
cytochrome c (pigeon), triosephosphate isomerase (rabbit), endoproteinase GluC, endoproteinase AspN, acetic anhydride (Ac2O),
and iodoacetamide were purchased from Sigma (St. Louis, MO,
USA). Galanin (rat) was obtained from Peptide Institute (Osaka, Japan). Tosylhydrazide glass (Si-Tosyl Hydrazine, loading 0.8 mmol/
g, particle size 4063 lm) was purchased from SiliCycle (Quebec
City, Quebec, Canada). Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was obtained from Fluka Chemie (Buchs, Switzerland).
ZipTip C18 was obtained from Millipore (Bedford, MA, USA). Acetonitrile (MeCN), formic acid (HCOOH), pentauorophenol (PfpOH),
toluene, triuoroacetic acid (TFA), methanol (MeOH), and triethylamine (Et3N) were obtained from Wako Pure Chemical Industries
(Osaka, Japan). a-Cyano-4-hydroxycinnamic acid (CHCA, high-purity MS grade) was purchased from Shimadzu (Tokyo, Japan). Methanediphosphonic acid (MDPNA) [12] was obtained from Tokyo
Chemical Industry (Tokyo, Japan). 3-Aminopropyl tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-propylamine) was
prepared according to the reported protocol [8].
Derivatization of C-terminal a-carboxyl group with
TMPP-propylamine and subsequent GluC digestion
Disulde bonds in protein were reduced and alkylated by the
successive treatment with TCEP and iodoacetamide according to
the standard protocol at a scale of 500 pmol. After precipitating
with acetone or desalting with ZipTip C18, the S-carbamidomethylated protein pellet or solution was dried using a vacuum centrifuge. The resulting material was dissolved in a mixture of
HCOOHAc2OPfpOH (12 ll, 1:1:1, in volume) and incubated at
60 C for 20 min, followed by evaporation using a vacuum concentrator. This step was repeated twice. To further conrm the activation, the dried material was then mixed with 14 ll of a 5:2 mixture
of toluene and PfpOH and was evaporated to dryness. Then 4 ll of
aqueous TMPP-propylamine solution (1 lmol/ll) and 4 ll of Et3N
MeOH (1:1, in volume) were added to the activated protein, and
the resulting mixture was sonicated for 5 min and left standing
at 60 C for 60 min. The solution was evaporated using a vacuum
centrifuge, and the residual material was dissolved with H2O
(10 ll), followed by acetone precipitation (40 ll at 0 C). The collected precipitation, after spinning down to the tube bottom, was
washed twice with 20 ll of acetone at 0 C and was dried in vacuo.
The dried protein pellet was dissolved in 3 ll of phosphate buffer
(50 mM, pH 7.9) containing urea (8 M), and then the solution
was diluted with 4 ll of the buffer. After that, 1 ll of aqueous solution of protease (100 ng/ll) was added to the solution, which was
then incubated for 4 to 12 h at 37 C. If needed, Asp-N solution was
added for a second digestion (1 ll, 100 ng/ll).
Enrichment of C-terminal peptide using COOH capture material
Tosylhydrazide glass (10 mg) was prewashed twice with 100 ll
of 1% aqueous sodium dodecyl sulfate (SDS) solution1 mM HCl
(1:1). Then 10 ll of 1 mM HCl, 5 ll of N-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (EDC) aqueous solution
(30 mg/ml), and 1 ll of digest solution (corresponding to 100 pmol
of starting protein) were added to the glass beads, and the mixture
was held at 50 C for 60 min. The C-terminal peptide was often observed not in the supernatant but rather in the formic acid extract
(twice with 20 ll).

MALDITOF MS
Matrix-assisted laser desorption/ionization (MALDI) mass spectra were acquired on an AXIMA Performance reectron timeof-ight (TOF) mass spectrometer (Shimadzu/Kratos, Manchester,
UK) equipped with a nitrogen laser (337-nm wavelength). All of
the measurements were performed in the positive ion reectron
mode. Post-source decay (PSD) mode was used for the MS/MS
experiments.
The matrix used in this experiment was CHCA, which was dissolved to saturation in 50% aqueous MeCN containing 0.05% TFA.
We used MDPNA [12], which has been proven to be useful for
MALDI analysis of salt-containing samples, as a matrix additive.
MDPNA was used as a 1% aqueous solution. An aliquot (0.5 ll) of
sample solution was mixed with an equivalent volume of matrix
and matrixadditive solutions on the MALDI target plate and analyzed after drying.
The scale of the m/z value in the spectra was externally calibrated with the monoisotopic peaks of human angiotensin II (human) and human ACTH fragment (1839).

Results and discussion

As mentioned in the introductory paragraphs, we recently submitted a method for C-terminal sequence analysis of protein using
selective amidation at a-COOH of protein and enzymatic digestion
[8]. This method can be applied to the analysis of a protein having
lysine residue at the C terminus, which cannot be analyzed by a
method using Lys-C digestion [7]. In addition, the use of protease
is not conned to one type; any proteolytic enzyme can be used.
The peak intensity by MS and TMPP mass tag peak determined
by MS/MS serves as the indicator of the target C-terminal peptide
peak. Therefore, the target peak can be distinguished without
C-terminal peptide enrichment, and the amino acid sequence can
be claried by MS. This is a time-saving and fairly versatile technique, although the C-terminal peptide must be isolated or enriched when the target peak cannot be readily pinned down,
albeit with the loss of the simplicity of the protocol. This prompted
us to investigate incorporating an enrichment step in the above
method. A chemical means of xing target (positive selection) or
nontarget molecules (negative selection) onto a solid support
was envisaged for enrichment.
In this study, the COOH group was employed as the xation
functional group. A protein of interest was rst derivatized at the
a-COOH and then digested using GluC, which cleaves the carboxyl
termini of Glu and Asp. Each peptide resulting from the digestion
contains two COOH groups, with the exception of the C-terminal
peptide that is devoid of COOH groups. Tosylhydrazide glass beads
successfully trapped COOH groups in the peptides, whereas the Cterminal peptide did not bind to the beads and remained in the
solution. The overall scheme is presented in Fig. 1 (route a). In this
study, four proteins (calmodulin [CALM], superoxide dismutase
[SODM], cytochrome c [CYC-c], and triosephosphate isomerase
[TPIS]) and one peptide hormone (galanin) were used for the C-terminal analysis. The results of enriching the C-terminal peptide for
CALM, SODM, and galanin are presented in Fig. 2A, C, and E, and
their PSD spectra are depicted in Fig. 2B, D, and F. In the case of
SODM, a C-terminal peptide peak (AAARFAAKK-NHC3H6-TMPP,
m/z 1561, where an asterisk indicates formylation) was detected
but very weakly, whereas a peak of m/z 1672 was the strongest
in intensity. Its identity was determined by the PSD spectrum as
Pyr-AAARFAAKK-NHC3H6-TMPP (where Pyr indicates pyroglutamate residue; Fig. 2D). A comparison of the mass spectrum of the
GluC digest with that of the enrichment indicates that newly
formed Glu at the N terminus of an atypical cleavage product

MS sequencing of protein C-terminal peptide / C. Nakajima et al. / Anal. Biochem. 428 (2012) 167172

COOH
D

TMPP amidation

CONHC3H6-TMPP
D

(a) GluC

(b) GluC-AspN

digestion

E,

E,
D
CONHC3H6-TMPP

CONHC3H6-TMPP

COOH scavenging

CONHC3H6-TMPP
or

CONHC3H6-TMPP

Sequence analysis by MS
Fig.1. Normally, the protein of interest is analyzed through route (a). However,
when Asp residue remains in the C-terminal peptide due to missed cleavage, a
second digestion using AspN is carried out to yield a C-terminal peptide in which
Asp residue is located at the N terminus. The C-terminal peptide terminated with
Asp is not bound to COOH-capturing material (route b).

(EAAARFAAKK-NHC3H6-TMPP) readily cyclizes to form pyroglutamate residue in the enrichment step (data not shown). The
a-COOH group of galanin is amidated; hence, the TMPP amidation
did not occur. After enrichment, the a-NH2 group was derivatized
using TMPP-Ac-OSu [10,11] for manual sequencing. The PSD spectrum was measured with the N-terminally TMPP-derivatized peptide (Fig. 2F). The difference in the TMPP derivatization site reects
that of the fragmentation pattern [9,10].
The ratio of HCOOH and Ac2O in the reagent for the selective
derivatization of a-COOH was studied previously [13]. Under the
ratio used (HCOOH/Ac2O = 1:1), Ac2O can be completely converted
into acetic formic anhydride, which can react with COOH in peptide to form the mixed anhydride with HCOOH. Then the nucleophile (TMPP-propylamine) preferentially attacks the less
hindered carbonyl group derived from HCOOH, which allows the
COOH in side chain to remain unchanged [13,14].
The selective reactivity of TMPP-propylamine toward a-COOH
has been tested in this work using four proteins whose C-terminal
amino acid variety is Lys and Gln, and in the previous studies the
reactivity was veried with ve proteins terminated with Lys,
Gln, Glu, Arg, or His [8] and four peptides incorporating Tyr, Gln,
Glu, or Har (homoarginine) at the C terminus [9]. The study of
TMPP amidation of peptides [9] demonstrated that functional
groups (including COOH) on the side chains were not involved in
the amidation reaction except for the e-amino group of lysine residue. The results of TMPP amidation obtained so far are summarized in Table 1.

169

Protease GluC cleaves only at the glutamyl bond in ammonium

acetate (pH 4.0) or bicarbonate (pH 7.8), whereas it cleaves at both
the glutamyl and aspartic bonds in phosphate (pH 7.8). However, it
is suggested that this protease highly prefers the glutamyl bond as
the cleavage site [15]; hence, the aspartic bond is often not cleaved
even with phosphate buffer.
In this study, CYC-c did not produce its expected C-terminal
peptide (LIAYLKQATAK-NHC3H6-TMPP) when cleaved with
GluC; rather, it produced a miss-cleaved peptide (RADLIAYLK
QATAK-NHC3H6-TMPP). The C-terminal peptide produced in this
way reacted with the COOH-capturing glass beads and then was
eliminated from the solution due to the remaining aspartic acid
residue in the sequence. To address this issue, we tried endoproteinase AspN digestion, by which two residues (RA) in the C-terminal peptide were cleaved off to produce an Asp-terminated
sequence, DLIAYLKQATAK-NHC3H6-TMPP. This idea came from
the C-terminal enrichment of SODM (Fig. 2C), when an atypical
C-terminal peptide having Glu residue at the N terminus was successfully recovered in the enrichment step (Fig. 2D). The MS/MS
analysis revealed that the Glu residue at the N terminus was
transformed to pyroglutamate in the enrichment step and that
this is why it survived during the scavenging step. The results
from CYC-c demonstrated the successful enrichment of the C-terminal peptide incorporating Asp residue at the N terminus (DLIAYLKQATAK-NHC3H6-TMPP; Fig. 3A), and the sequence was
claried by MS/MS analysis (Fig. 3B). In the mass spectrum after
the enrichment (Fig. 3A), a peak of m/z 1944 is discernible but
very weakly so. This is the dehydration (18) peak of the C-terminal peptide, DLIAYLKQATAK-NHC3H6-TMPP, and MS/MS analysis
claried that the dehydration occurred at the N-terminal Asp
residue (data not shown). In this case, it is very likely that the
dehydration is derived from cyclization; therefore, this indicates
that the cyclization had occurred on the N-terminal Asp residue,
but it was a very limited amount and most of the Asp residue
remained intact. The reason for the reluctancy to cyclization is
that the highly strained four-membered (b-lactam) ring formation
is not favorable.
The COOH in a peptide should be protonated for the facile
reaction of COOH and tosylhydrazide glass. Hence, the pH of
the capture reaction is set to 3. The reason why the peptide having Asp at its N terminus was not captured with the solid-phase
scavenger may be the highly acidic nature of the COOH group on
the N-terminal Asp. The elevated acidity might be ascribed to the
fact that COOH and protonated a-NH2 on N-terminal Asp are in
close proximity, leading to a form of tight ion pair (Fig. 4). Thus,
the COOH is kept deprotonated in the reaction condition, working
against binding to the scavenger. This hypothesis is supported by
the results of scavenging experiments using a three-peptide mixture (MKRPPGFSPFR [peptide 1], RPKPQQFFGLM-NH2 [peptide 2],
and DVGIKLSGAQYQQHGRAL-NH2 [peptide 3]) and a protein (TPIS
from rabbit). In the three-component mixture, only peptide 1 was
trapped onto the tosylhydrazide glass beads; peptide 3, which
incorporates Asp residue at its N terminus, escaped the binding
to the beads (data not shown). In the case of TPIS, GluC digestion
produced FVDIINAKQ-NHC3H6-TMPP as the C-terminal peptide.
The carboxyl side of the Asp residue located at the seventh position from the C terminus was not cleaved, and thus a C-terminal
peptide incorporating Asp residue at the third position from the N
terminus was obtained, which was similar to the result of GluC
digestion of CYC-c. Therefore, a second digestion was performed
using AspN to produce DIINAKQ-NHC3H6-TMPP. The resulting
target peptide was successfully enriched (Fig. 3C), followed by a
sequence analysis by MS/MS (Fig. 3D). Given these results, the
combined use of GluC and AspN is generally practical (Fig. 1,
route b) when the cleavage at the aspartic bond using GluC is less
than expected.

170

MS sequencing of protein C-terminal peptide / C. Nakajima et al. / Anal. Biochem. 428 (2012) 167172
X100

1554.9

100
95
90

100

FVQMMTAK*-NH-C3H6-TMPP

95
90

85
85

y2

80
80
75
75
70

70

65

65

60

60

55

55

50

y7

50

45

45

40

40

35

35

30

30

25

25

20

20

15

15

10

tag

y1

y5

y4

y3

y6

w6

10

0
1000

1100

1200

1300

1400

1500

1600

1700

1800

1900

2000

2100

2200

2300

2400

2500

2600

2700

2800

2900

3000

550

600

650

700

750

800

850

900

950

1000

1050

m/z

1150

1200

1250

1300

1350

1400

1450

1500

1550

X50

1672.0

100

1100
m/z

95
90

100
95

Pyr-AAARFAAK*K*-NH-C3H6-TMPP

90

85
85

80
80

75
75

70

70

65

65

60

60

55

55

50

50

45

y6

45

40

40

35

35

30

y4
tag

30

1561.1

25
20

20

15

15

10

10

y7

y8

y3

y1

25

y5

y2

0
1000

1100

1200

1300

1400

1500

1600

1700

1800

1900

2000

2100

2200

2300

2400

2500

2600

2700

2800

2900

600

3000

700

800

900

1000

1100

1154.3

100

1200

1300

1400

1500

1600

1700

m/z

m/z

95
90

X5

100
95
90

85
85

TMPP-Ac-K*HGLT-NH2

80
80

75
75

70

a5+2

70

65

65

60

60

55

55

50

50

45

45

40

40

35

35

30

30

25

25

20

20

15

15

10

10

1000

1100

1200

1300

1400

1500

1600

1700

1800

1900

2000

2100

2200

2300

2400

2500

2600

2700

2800

2900

3000

tag

a2

a1

a3
550

600

650

700

750

800

850

900

a4
950

1000

1050

1100

1150

m/z

m/z

Fig.2. Enrichment and PSD analysis of calmodulin (A, B), superoxide dismutase (C, D), and galanin (E, F). An asterisk () indicates formylation, and Pyr indicates
pyroglutamate residue.

Table 1
TMPP propylamidation of a-carboxyl group.

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

Sample

Proteolytic enzyme

TMPP-propylamidated peptide

Ref.

Calmodulin (CALM_BOVIN)
Cytochrome c (CYC_COLLI)
Cytochrome c (CYC_COLLI)
Cytochrome c (CYC_COLLI)
Superoxide dismutase (SODM_ECOLI)
Superoxide dismutase (SODM_ECOLI)
Triosephosphate isomerase (TPIS_RABIT)
Triosephosphate isomerase (TPIS_RABIT)
Glyceraldehyde-3-phosphate dehydrogenase (G3P_RABIT)
Hemoglobin subunit alpha (HBA_BOVIN)
Hemoglobin subunit beta (HBB_BOVIN)
Ac-SQNY
Ac-SRPLSDQE
Pyr-PLPDCcamCcamRQ
Ac-ADQLTEEQIAEFJ

GluC
AspN
Trypsin
GluC
Trypsin
GluC
AspN
GluC
AspN
AspN
AspN

FVQMMTAK-NHC3H6-TMPP
DLIAYLKQATAK-NHC3H6-TMPP
ADLIAYLKQATAK-NHC3H6-TMPP
RADLIAYLKQATAK-NHC3H6-TMPP
FAAKK-NHC3H6-TMPP
Pyr-AAARFAAKK-NHC3H6-TMPP
DIINAKQ-NHC3H6-TMPP
FVDIINAKQ-NHC3H6-TMPP
DLMVHMASKE-NHC3H6-TMPP
DKFLANVSTVLTSKYR-NHC3H6-TMPP
DFQKVVAGVANALAHRYH-NHC3H6-TMPP
Ac-SQNY-NHC3H6-TMPP
Ac-SRPLSDQE-NHC3H6-TMPP
Pyr-PLPDCcamCcamRQ-NHC3H6-TMPP
Ac-ADQLTEEQIAEFJ-NHC3H6-TMPP

This
This
[8]
[8]
[8]
This
This
[8]
[8]
[8]
[8]
[9]
[9]
[9]
[9]

work
work and [8]

work
work

Note. This table summarizes the results obtained for TMPP propylamidation toward a-COOH of proteins and peptides. K, formylated lysine; Ccam, S-carboxamidomethylcysteine; Pyr, pyroglutamic acid; J, homoarginine.

171

MS sequencing of protein C-terminal peptide / C. Nakajima et al. / Anal. Biochem. 428 (2012) 167172
X50

1962.1

100
95
90

X10

100
95
90

85
85

DLIAYLK*QATAK*-NH-C3H6-TMPP

80

y2

80

75
75

70

70

65

65

60

60

55

55

50

50

45

45

40

40

35

35

30

30

1944.0

25

25

20

20

15

15

10

10

y11
tag

y4

y1

y6

y5

y3

y9

y7

y10

y8

5
0

0
1000

1100

1200

1300

1400

1500

1600

1700

1800

1900

2000

2100

2200

2300

2400

2500

2600

2700

2800

2900

600

3000

700

800

900

1000

1100

1200

1400

1500

1600

1700

1800

1900

2000

X50

1400.4

100

1300
m/z

m/z

95
90

100
95
90

DIINAK*Q-NH-C3H6-TMPP

85
85
80
80
75
75
70

y6

70

65

65

60

60

55

55

50

50

45

45

40

40

35

35

30

30

25

25

20

20

15

15

10

10

z4
y3

tag

y5

y4

y2

y1

0
1200

1300

1400

1500

1600

1700

1800

1900

2000

2100

2200

2300

2400

2500

2600

2700

2800

2900

3000

550

600

650

700

750

m/z

800

850

900

950

1000

1050

1100

1150

1200

1250

1300

1350

1400

m/z

Fig.3. Enrichment and PSD analysis of cytochrome c (A, B) and triosephosphate isomerase (C, D). An asterisk () indicates formylation.

HOOC

H2N

CO

LIAYLK*QATAK*-NHC3H6-TMPP

pH~3

Acknowledgments

-O

tide having Asp residue at the N terminus. This type of peptide,

although it has COOH, did not bind to the solid-phase scavenger
and was successfully enriched; thus, this technique has become
practical.
In the current status, there is no general method for C-terminal
sequencing analysis of protein, due partly to its highly varied nature; hence, versatile techniques for such purposes have been
strongly sought. The method described here can be used complementarily with previously reported protocols for protein C-terminal analysis.

N
H3

CO

LIAYLK*QATAK*-NHC3H6-TMPP

Fig.4. Hypothetical arrangement of side chain COOH and a-NH2 on the Nterminally located Asp residue. These functional groups may form a tight ion pair.
An asterisk () indicates formylation.

For scavenging COOH-containing peptide, we tested a couple of

commercially available products, including aminoalkyl group or
hydrazide group, on solid-phase material. Of the materials tested,
only tosylhydrazide glass satisfactorily scavenged COOH-containing peptides. Thus, we used the glass beads throughout this work.

Conclusion
An approach to protein C-terminal analysis employing specic
derivatization of a-COOH and C-terminal peptide enrichment has
been described. C-terminal-specic derivatization was performed
through a-COOH-specic activation by oxazolone chemistry, and
the C-terminal peptide was enriched by the treatment using tosylhydrazide glass material after double digestion using GluC and
AspN. This double digestion sometimes produces a C-terminal pep-

This research was supported by the Japan Society for the

Promotion of Science (JSPS) through its Funding Program for
World-Leading Innovative R&D on Science and Technology (FIRST
Program). The authors are grateful to Takashi Nakazawa of Nara
Womens University and Osamu Nishimura of Osaka University
for their valuable suggestions and constant encouragement.
References
[1] W.J. Henzel, T.M. Billeci, J.T. Stults, S.C. Wong, C. Gremley, C. Watanabe,
Identifying proteins from two-dimensional gels by molecular mass searching
of peptide fragments in protein sequence databases, Proc. Natl. Acad. Sci. USA
90 (1993) 50115015.
[2] J.J. Chung, S. Shikano, Y. Hanyu, M. Li, Functional diversity of protein C-termini:
more than zipcoding?, Trends Cell Biol 12 (2002) 146150.
[3] J.J. Chung, H. Yang, M. Li, Genome-wide analyses of carboxyl-terminal
sequences, Mol. Cell. Proteomics 2 (2003) 173181.
[4] F.X. Gomis-Ruth, Structure and mechanism of metallocarboxypeptidases, Crit.
Rev. Biochem. Mol. Biol. 43 (2008) 319345.
[5] J.H. Cox, R.A. Dean, C.R. Roberts, C.M. Overall, Matrix metalloproteinase
processing of CXCL11/I-TAC results in loss of chemoattractant activity and
altered glycosaminoglycan binding, J. Biol. Chem. 283 (2008) 1938919399.
[6] F. Impens, N. Colaert, K. Helsens, K. Plasman, P. Van Damme, J.
Vandekerckhove, K. Gevaert, MS-driven protease substrate degradomics,
Proteomics 10 (2010) 12841296.
[7] H. Kuyama, K. Shima, K. Sonomura, M. Yamaguchi, E. Ando, O. Nishimura, S.
Tsunasawa, A simple and successful C-terminal sequencing analysis of proteins
by mass spectrometry, Proteomics 8 (2008) 15391550.

172

MS sequencing of protein C-terminal peptide / C. Nakajima et al. / Anal. Biochem. 428 (2012) 167172

[8] C. Nakajima, H. Kuyama, T. Nakazawa, O. Nishimura, C-terminal sequencing of

protein by MALDI mass spectrometry through the specic derivatization of the acarboxyl group with 3-aminopropyltris(2,4,6-trimethoxyphenyl)phosphonium
bromide, Anal. Bioanal. Chem. 404 (2012) 125132.
[9] C. Nakajima, H. Kuyama, T. Nakazawa, O. Nishimura, S. Tsunasawa, A method
for N-terminal de novo sequencing of N-blocked proteins by mass
spectrometry, Analyst 136 (2011) 113119.
[10] Z.-H. Huang, J. Wu, K.D.W. Roth, Y. Yang, A picomole-scale method for charge
derivatization of peptides for sequence analysis by mass spectrometry, Anal.
Chem. 69 (1997) 137144.
[11] Z.-H. Huang, T. Shen, J. Wu, D.A. Gage, J.T. Watson, Protein sequencing by
matrix-assisted
laser
desorption
ionizationpostsource
decaymass
spectrometry analysis of the N-tris(2,4,6-trimethoxyphenyl)phosphineacetylated tryptic digests, Anal. Biochem. 268 (1999) 305317.

[12] H. Kuyama, K. Sonomura, O. Nishimura, Sensitive detection of

phosphopeptides by matrix-assisted laser desorption/ionization mass
spectrometry: use of alkylphosphonic acids as matrix additives, Rapid
Commun. Mass Spectrom. 22 (2008) 11091116.
[13] H. Kuyama, C. Nakajima, T. Nakazawa, O. Nishimura, S. Tsunasawa, A new
approach for detecting C-terminal amidation of proteins and peptides by mass
spectrometry in conjunction with chemical derivatization, Proteomics 9
(2009) 40634070.
[14] M. Yamaguchi, M. Oka, K. Nishida, M. Ishida, A. Hamazaki, H. Kuyama, E. Ando,
T.-A. Okamura, N. Ueyama, S. Norioka, O. Nishimura, S. Tsunasawa, T.
Nakazawa, Enhancement of MALDIMS spectra of C-terminal peptides by
the modication of proteins via an active ester generated in situ from an
oxazolone, Anal. Chem. 78 (2006) 78617869.
[15] B. Keil, Specicity of Proteolysis, Springer-Verlag, Berlin, 1992.