Matrix-assisted laser desorption/ionization mass spectrometry peptide sequencing utilizing selective N-terminal bromoacetylation

Article abstract of DOI:10.1016/j.ab.2011.11.022

In tandem mass spectrometric peptide sequencing, simplifying the mass spectrum is often desirable. The b-series ions were distinguished from the y-series ions in the MALDI TOF-TOF spectra by incorporating a bromine-tag to the N-terminal amino group through rapid and selective acetylation using bromoacetic anhydride without blocking the lysine and tyrosine residues. The 51:49 ratio of Br-79 and Br-81 isotopes facilitated identification of ions carrying the tag. With the Br-tag in the b-series ions, N-terminal sequencing of tryptic peptides from hemoglobin as well as model peptides was straightforward. When the b-ions were low in intensity, ions without the Br-tag were identified as y-ions and used for sequencing.

Full text of DOI:10.1016/j.ab.2011.11.022

Analytical Biochemistry 423 (2012) 269–276
Contents lists available at SciVerse ScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
Matrix-assisted laser desorption/ionization mass spectrometry peptide
sequencing utilizing selective N-terminal bromoacetylation
⇑
Jinsu Song, Hie-Joon Kim
Department of Chemistry, Seoul National University, Seoul, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 August 2011
Received in revised form 21 October 2011
Accepted 21 November 2011
Available online 1 December 2011
In tandem mass spectrometric peptide sequencing, simplifying the mass spectrum is often desirable. The
b-series ions were distinguished from the y-series ions in the MALDI TOF-TOF spectra by incorporating a
bromine-tag to the N-terminal amino group through rapid and selective acetylation using bromoacetic
anhydride without blocking the lysine and tyrosine residues. The 51:49 ratio of Br-79 and Br-81 isotopes
facilitated identification of ions carrying the tag. With the Br-tag in the b-series ions, N-terminal
sequencing of tryptic peptides from hemoglobin as well as model peptides was straightforward. When
the b-ions were low in intensity, ions without the Br-tag were identified as y-ions and used for
sequencing.
Keywords:
Bromoacetylation
N-terminal modification
MALDI TOF/TOF
Ó 2011 Elsevier Inc. All rights reserved.
Bromine-isotope tag
In both matrix-assisted laser desorption/ionization (MALDI)1
[1,2], and electrospray ionization (ESI) [3] methods, tandem mass
spectrometry (MS/MS) is widely used for structural characterization
[4], amino acid sequencing in particular. In tandem MS, multiple ions
are produced leading to complex mass spectra. To simplify spectral
analysis, the N-terminal amino group can be chemically modified
either to enhance or to suppress the b- or y-ion series. Fragmenta-
tion by electron transfer dissociation (ETD) [5] or electron capture
dissociation (ECD) [6,7] leads to c- and z-series ions. These methods
are used in the ESI mode and are not applicable to MALDI MS.
Sequencing of peptides using w-, y-, and x-ions generated by
157 nm laser needs specialized instrumentation [8].
In chemically assisted fragmentation, the N-terminus is often
sulfated to carry a negative charge so that the b-series ions are sup-
pressed [9–12]. In this case, since tryptic peptides with C-terminal
lysine or arginine are often used to take advantage of the strong
y-ion peaks, guanidination of the C-terminal lysine is necessary
to avoid sulfation of the C-terminus. Moreover, sulfation of the
peptide decreases the intensity of the parent ion to be analyzed
by tandem MS.
developing a chemical modification method for introducing a bro-
mine-tag to the N-terminal amino group even at the expense of
suppressed b-ions. The unique bromine isotopic ratio (50.69%
Br-79, 49.31% Br-81) was utilized to identify cysteine, [21] or to
monitor the reaction pathway of Br-containing compounds such
as bromobenzene with proteins [22,23]. Bromine-isotope tag
was also used in capillary electrophoresis detection by introduc-
ing positively charged compound to the N-terminal amino acid
of a peptide [24]. The bromine-tag had been used for various
applications [25–28]. We intended to selectively modify the
N-terminus using bromoacetic anhydride. Bromoacetylation with
bromoacetic anhydride is pH dependent and thus can be carried
out without protecting the lysine residue at pH around 6 [29].
This approach had been used in the past to introduce a bromo-
acetyl moiety to peptides and to use it as a linker to DNA or poly-
mers [29,30]. The unique isotopic abundance of bromine was
expected to produce a unique isotopic pattern and facilitate selec-
tion of the b-ion peaks in the presence of strong y-ion peaks in
both MS and MS/MS spectra. In particular, in peptides of 1000–
2000 Da molecular weight most useful in peptide analyses, the
first and the third isotopic peaks would stand out making selec-
tion of the tagged ions straightforward. In this paper, we show
that the bromoacetyl group can be selectively incorporated to
the N-terminal amino acid in the presence of lysine residues
and facilitate peptide sequencing using Br-tagged b-ions in the
MS/MS analysis of model peptides and a peptide mixture gener-
ated by tryptic hydrolysis of a protein.
There are several N-terminal modification methods that do not
involve suppression of b-ions [13–20]. We were interested in
⇑
Corresponding author. Address: San 56-1, Seoul National University, Daehak-
dong, Kwanak-gu, Seoul, Republic of Korea.
E-mail address: hjkim1@snu.ac.kr (H.-J. Kim).
Abbreviations used: ECD, electron capture dissociation; ETD, electron transfer
1
dissociation; MALDI, matrix-assisted laser desorption/ionization; ESI, electrospray
ionization; CHCA, a-cyano-4-hydroxycinnamic acid; TFA, trifluoroacetic acid.
0003-2697/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2011.11.022

270
MALDI MS peptide sequencing / J. Song, H.-J. Kim / Anal. Biochem. 423 (2012) 269–276
Fig.1. MALDI mass spectra showing pH dependence of bromoacetylation of DAEFRHDSGYQVHHQK in distilled water (a), 50 mM Hepes buffer, pH 7.0 (b), and 75 mM Tris
buffer, pH 8.8 (d). Panel c shows the MS/MS spectrum from the 2073.637 peak in b.
dissolved in 1 ml of dichloromethane in an Eppendorf tube and
157 l of diisopropylcarbodiimide (1 mmol) was added. After mix-
ing for 30 min using a vortex mixer, the mixture was filtered using
a 0.45 m filter (Millipore). When the solvent was removed using a
Materials and methods
l
Materials
l
Speed-Vac for 30 min, a light yellowish oily liquid of bromoacetic
Bromoacetic acid, diisopropylcarbodiimide, model peptides and
anhydride was obtained.
proteins,
a-cyano-4-hydroxycinnamic acid (CHCA), and trifluoro-
acetic acid (TFA) were from Sigma-Aldrich (St. Louis, USA). Dichlo-
romethane and acetonitrile were from Fischer (Pittsburgh, USA).
Distilled water was prepared using the Milli-Q system from
Millipore (Billerica, USA).
Bromoacetylation of model peptides
Model peptides were dissolved in 50 mM, pH 7.0, Hepes buffer
to give 5
dissolved in acetonitrile was kept cold in ice water at 0 °C, and
l of the model peptide solution was mixed with 10 l of the
cold bromoacetic anhydride solution and left cold for 10 min. Then
l water was added to terminate the reaction. After 5 min in ice
lM concentration. A 0.1% (v/v) bromoacetic anhydride
Preparation of bromoacetic anhydride
2
l
l
Bromoacetic anhydride was made by the published method
[30]. The amount of 0.278 g of bromoacetic acid (2 mmol) was
8
l

MALDI MS peptide sequencing / J. Song, H.-J. Kim / Anal. Biochem. 423 (2012) 269–276
271
Fig.2. Bromoacetylation of various peptides at pH 7.0. (a) DRVYIFPH, (b) ASHLGLAR, (c) WHWLQLKPGQPMY, (d) FQpSEEQQQTEDELQDK.
water, solvents were removed using SpeedVac and the remaining
dried sample was dissolved in 1 l aliquot of 0.1% TFA solution.
MADLI-TOF/TOF MS
l
The amount of 700
ll acetonitrile containing 0.1% TFA was
mixed with 300 ll distilled water also containing 0.1% TFA and
Bromoacetylation of tryptic peptides
10 mg CHCA was dissolved in the mixture. Then the solution was
saturated with nitrilotriacetic acid and centrifuged. One microliter
Several milligrams of bovine hemoglobin or bovine serum
albumin was dissolved in 1 ml 50 mM ammonium bicarbonate
buffer, heat denatured, and digested with 7–10% (w/w) trypsin
for 6 h. Tryptic peptides produced were dissolved in 50 mM
of the matrix solution was mixed with 1 ll of the sample solution,
loaded on the sample plate, and dried. MALDI TOF/TOF MS was car-
ried out using Autoflex (Bruker Daltonics, Bremen, Germany). Mass
spectra were obtained in the positive reflector mode. Peptide stan-
dard from Bruker was used for mass calibration. MS/MS spectra
were obtained by averaging 1000 shots.
ammonium bicarbonate to 50
lM concentration and subjected
to bromoacetylation as above for model peptides.

272
MALDI MS peptide sequencing / J. Song, H.-J. Kim / Anal. Biochem. 423 (2012) 269–276
Fig.2 (continued)
Under optimal conditions, bromoacetylation by bromoacetic
Results and discussion
anhydride was immediate at room temperature and thus control
of lysine and tyrosine bromoacetylation was difficult. After
5–10 min at 0 °C, singly bromoacetylated peptide was obtained
as a major reaction product at optimal pH.
Fig. 1 shows the pH dependence of bromoacetylation, carried
out at 0 °C for 10 min, of a model peptide containing lysine and
tyrosine (DAEFRHDSGYQVHHQK). Bromoacetylation was incom-
Bromoacetylation
Acetylation is known to take place at lysine and tyrosine resi-
dues as well as at the N-terminus in a pH-dependent manner
[29]. The reaction is slow at low pH and thus site specific, whereas
it takes place at all sites at high pH. Therefore, it is important to
find reaction conditions for selective bromoacetylation at the
N-terminus.
plete when 2
mixed with 10
l
l
l of the peptide solution in deionized water was
l of bromoacetic anhydride solution in acetonitrile.
The bromoacetylation peak at 2073.122 with a characteristic bro-
mine isotopic pattern was the strongest; however, the control pep-
tide peak at 1953.251 without the bromine pattern was also strong
(Fig. 1(a)). The pH of the reaction mixture was below 5 due to
hydrolysis of bromoacetic anhydride to bromoacetic acid. When
Our preliminary test showed that bromoacetylation yield is low
when water is present in excess over acetonitrile. It is probably
because hydrolysis of bromoacetic anhydride is faster than bromo-
acetylation. Therefore, the reaction was carried out in excess
acetonitrile.

MALDI MS peptide sequencing / J. Song, H.-J. Kim / Anal. Biochem. 423 (2012) 269–276
273
Fig.3. MALDI mass spectra from bovine hemoglobin tryptic peptides before (a) and after (b) bromoacetylation showing 120 Da mass shift.
Table 1
Hemoglobin tryptic peptides carrying bromoacetylation tag. Most lysine-containing peptides are one bromoacetylated.
Start
–
End
Observed
Sequence
1 Br
2Br
MS/MS
76b
8b
–
–
–
–
–
–
–
–
–
–
–
–
–
–
81b
16b
40a
740.3807
950.4689
HLDDLK
AAVTAFWGK
MFLSFPTTK
LRVDPVNFK
LHVDPENFK
VDEVGGEALGR
VVAGVANALAHR
LLVVYPWTQR
VKVDEVGGEALGR
EFTPVLQADFQK
VGGHAAEYGAEALER
TYFPHFDLSHGSAQVK
FFESFGDLSTADAVMNNPK
AVEHLDDLPGALSELSDLHAHK
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
32a
91a
95b
19b
132b
30b
17b
120b
17a
41a
40b
69a
1071.5255
1087.5941
1098.4916
1101.6016
1177.6766
1274.7255
1328.7214
1422.7166
1529.7492
1833.9187
2089.9834
2367.2246
99a
103b
29b
143b
39b
29b
131b
31a
56a
58b
90a
o
o
o
o
o
o
o
o
o
the peptide dissolved in 50 mM Hepes, pH 7.0, buffer was used, the
bromoacetylation peak was the strongest and the doubly bromo-
acetylated peptide ion showed a small peak at 2193.555, while
the control peptide peak almost disappeared (Fig. 1(b)). MS/MS
analysis of the mono-bromoacetylated peptide ion at 2073.637
showed b-series ions consistent with tagging at the N-terminus
only (Fig. 1(c)), indicating that bromoacetylation at the N-terminus
was strongly favored. At pH 8.8 in 75 mM Tris buffer, doubly and
triply bromoacetylated peptides were dominant, suggesting that
both lysine and tyrosine residues as well as the N-terminal amino
group were tagged. Both doubly and triply tagged peptides were
observed when the reaction was carried out at room temperature
for 5 min at pH 7. These results show that selective bromoacetyla-
tion at the N-terminus can be achieved by pH and temperature
control.
produced mono-bromoacetylation product in a peptide with one
tyrosine (DRVYIFPH, Fig. 2(a)), in a peptide with one lysine in the
middle and a C-terminal tyrosine (WHWLQLKPGQPMY, Fig. 2(c)),
and in a phosphopeptide with a C-terminal lysine (FQpSEE-
QQQTEDELQDK, Fig. 2(d)). They all showed a mono-bromoacetyla-
tion peak as the major product. Also several peptides with different
N-terminal amino acids were synthesized and tested for the influ-
ence of the N-terminal amino acids on tagging efficiency. Some
variation was observed; however, single N-terminal bromoactyla-
tion was predominant as verified by MS/MS analyses (supplemen-
tary material).
Mono-bromoacetylation was also demonstrated using a mix-
ture of tryptic peptides. If the N-terminal amino groups of all
peptides are preferentially tagged before lysine residues, a mass
shift of 119.92 Da should be observed in all tryptic peptides.
Fig. 3 shows mass spectra from control (Fig. 3(a)) and bromoacet-
ylated (Fig. 3(b)) bovine hemoglobin tryptic peptides. Most hemo-
globin tryptic peptides have C-terminal lysine. Yet practically all
To establish that the selected reaction conditions general apply,
bromoacetylation of 10 additional peptides was tested. Fig. 2
shows that bromoacetylation for 10 min at 0 °C and pH 7.0

274
MALDI MS peptide sequencing / J. Song, H.-J. Kim / Anal. Biochem. 423 (2012) 269–276
Fig.4. MALDI mass spectra from WHWLQLKPGQPMY before (a) and after (b) bromoacetylation. Br-tag was observed only in the b-series ions.
Fig.5. MS/MS spectrum from one of hemoglobin tryptic peptides.
tryptic peptides were singly tagged except the chain A 17-31 pep-
tide which showed double tagging as well. Fig. 3(b) shows little
unmodified peptides without the Br-tag, except a small A 17-31
peptide peak. The result indicates that bromoacetylation is almost
complete regardless of the N-terminal amino acid. The N-terminal
amino acids of hemoglobin tryptic peptides that left no observable
unmodified peptide signal include histidine, alanine, methionine,
leucine, valine, glutamic acid, threonine, and phenylalanine as

MALDI MS peptide sequencing / J. Song, H.-J. Kim / Anal. Biochem. 423 (2012) 269–276
275
Fig.6. MS/MS spectra from FQpSEEQQQTEDELQQK before (a) and after (b) bromoacetylation showing y-ions only.
summarized in Table 1. Most of the peptides were singly tagged,
while 3 of the 14 peptides were doubly tagged.
It must be pointed out that sensitivity of detection is compro-
mised by bromoacetylation of the basic N-terminal amine group.
Fig. 5 shows an example of partial sequencing using the b-ions
and full sequencing using both b- and y-ions. When one of the
tryptic peptides from bovine hemoglobin was subjected to MS/
MS analysis, both b- and y-ions were observed in about equal
abundance. Interestingly, the y-ions were not necessarily stronger.
Anyhow, the Br-tag was particularly useful when identification of
the peaks was difficult due to the low signal intensity. MS/MS data
for other peptides are provided in the supplementary material.
These results show that the b-ions obtained after bromoacetylation
can be used for de novo sequencing of peptides.
When tryptic peptides equivalent to 400 fmol hemoglobin in 1 ll
were loaded on the plate, the strongest peak with the Br-tag was
observed with S/N of about 70, which was enough to perform
MS/MS experiment. This corresponded to about 3-fold reduction
in signal intensity upon bromoacetylation. We believe that the
advantages of the Br-tag could offset the reduced signal in many
circumstances, particularly if more powerful mass spectrometers
are used.
MS/MS analysis without b-series ions
MS/MS analysis of b-series ions with Br-tag
In an extreme case, the b-ions could be completely suppressed
by the y-ions and not observed at all. In that case, most of the
observed peaks will be from y-ions and carry no Br-signature.
Fig. 6 shows mass spectra from a beta-casein phosphopeptide
(FQpSEEQQQTEDELQDK). The negatively charged phosphate group
is near the N-terminus. Moreover, there are many acidic amino acids
in the peptide. Therefore, the b-ion peaks with negatively charged
groups are expected to be low in intensity in the positive ion mode
in the presence of y-ions with positively charged, basic C-terminal
residues. In fact, all the fragment ions observed before and after
bromoacetylation were identical except the mass-shifted y16
mother ion. Interpretation of the common ions as y-ions enabled
sequencing of nine consecutive residues in the phosphopeptide.
Since the Br-tag is introduced to the N-terminus, the Br signa-
ture is expected in the b-series ions. Fig. 4 shows the MS/MS
spectra from a model peptide (WHWLQLKPGQPMY) before and
after bromoacetylation. Distinction between b- and y-series is
not straightforward without the Br-tag. In de novo sequencing,
the b- and y-ion pair is searched based on the fact that their sum
should match the mother ion mass. In that case, which is b-ion
and which is y-ion is not straightforward. The assignment is possi-
ble upon analysis of the entire spectrum.
With Br-tag, assignment of the b- and y-series ions is straight-
forward. The peaks in Fig. 4(b) carrying the Br-tag could be easily
assigned as b2, b3, b4, b5, b6, b7, b8, b9, and b10 ions. Moreover,
peaks without the Br-tag could be assigned as y-ions, which were
also observed without bromoacetylation (Fig. 4(a)).
Conclusion
Admittedly, the Br-tag at the N-terminus could lead to a-ions as
well as b-ions complicating the analysis. Another concern about
using the Br-tag is that some of the b-ions may not be detected
from tryptic peptides, where the y-ions are stronger due to the
lysine and arginine residues at the C-terminus.
The N-terminal amino group of peptides can be selectively
bromoacetylated by a simple reaction with bromoacetic anhydride
for 10 min at 0 °C and pH 7.0. MS/MS analysis of bromoacetylated
peptide shows Br-tag in the b-ions, but not in y-ions. This distinction

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MALDI MS peptide sequencing / J. Song, H.-J. Kim / Anal. Biochem. 423 (2012) 269–276
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ab.2011.11.022.
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