Characterization of Nα-Fmoc-protected dipeptide isomers by electrospray ionization tandem mass spectrometry (ESI-MSn): Effect of protecting group on fragmentation of dipeptides

Article abstract of DOI:10.1002/rcm.5076

A series of positional isomeric pairs of Fmoc-protected dipeptides, Fmoc-Gly-Xxx-OY/Fmoc-Xxx-Gly-OY (Xxx = Ala, Val, Leu, Phe) and Fmoc-Ala-Xxx-OY/Fmoc-Xxx-Ala-OY (Xxx = Leu, Phe) (Fmoc = [(9-fluorenylmethyl) oxy]carbonyl) and Y = CH₃/H), have been characterized and differentiated by both positive and negative ion electrospray ionization ion-trap tandem mass spectrometry (ESI-IT-MSⁿ). In contrast to the behavior of reported unprotected dipeptide isomers which mainly produce y ₁⁺ and/or a₁⁺ ions, the protonated Fmoc-Xxx-Gly-OY, Fmoc-Ala-Xxx-OY and Fmoc-Xxx-Ala-OY yield significant b ₁⁺ ions. These ions are formed, presumably with stable protonated aziridinone structures. However, the peptides with Gly- at the N-terminus do not form b₁⁺ ions. The [M + H]⁺ ions of all the peptides undergo a McLafferty-type rearrangement followed by loss of CO₂ to form [M + H-Fmoc + H]⁺. The MS³ collision-induced dissociation (CID) of these ions helps distinguish the pairs of isomeric dipeptides studied in this work. Further, negative ion MS ³ CID has also been found to be useful for differentiating these isomeric peptide acids. The MS³ of [M-H-Fmoc + H]^- of isomeric peptide acids produce c₁^-, z₁ ^- and y₁^- ions. Thus the present study of Fmoc-protected peptides provides additional information on mass spectral characterization of the dipeptides and distinguishes the positional isomers. Copyright

Full text of DOI:10.1002/rcm.5076

Research Article
Received: 7 March 2011
Revised: 22 April 2011
Accepted: 2 May 2011
Published online in Wiley Online Library: 0
Rapid Commun. Mass Spectrom. 2011, 25, 1949–1958
(wileyonlinelibrary.com) DOI: 10.1002/rcm.5076
Characterization of Nα‐Fmoc‐protected dipeptide isomers by
electrospray ionization tandem mass spectrometry (ESI‐MSn):
effect of protecting group on fragmentation of dipeptides
1
1
1
2
*
2**
M. Ramesh , B. Raju , R. Srinivas , V. V. Sureshbabu , T. M. Vishwanatha and
H. P. Hemantha2
1National Centre for Mass Spectrometry, Indian Institute of Chemical Technology, Hyderabad 500 607, India
2Peptide Research Laboratory, Department of Studies in Chemistry, Central College Campus, Bangalore University, Dr. B. R.
Ambedkar Veedhi, Bangalore 560 001, India
A series of positional isomeric pairs of Fmoc‐protected dipeptides, Fmoc‐Gly‐Xxx‐OY/Fmoc‐Xxx‐Gly‐OY (Xxx = Ala,
Val, Leu, Phe) and Fmoc‐Ala‐Xxx‐OY/Fmoc‐Xxx‐Ala‐OY (Xxx = Leu, Phe) (Fmoc = [(9‐fluorenylmethyl)oxy]carbonyl)
and Y = CH3/H), have been characterized and differentiated by both positive and negative ion electrospray ionization
ion‐trap tandem mass spectrometry (ESI‐IT‐MSn). In contrast to the behavior of reported unprotected dipeptide
+
+
isomers which mainly produce y1 and/or a1 ions, the protonated Fmoc‐Xxx‐Gly‐OY, Fmoc‐Ala‐Xxx‐OY and
Fmoc‐Xxx‐Ala‐OY yield significant b1+ ions. These ions are formed, presumably with stable protonated aziridinone
structures. However, the peptides with Gly‐ at the N‐terminus do not form b1 ions. The [M + H]+ ions of all the
+
peptides undergo a McLafferty‐type rearrangement followed by loss of CO2 to form [M + H–Fmoc + H]+. The MS3
collision‐induced dissociation (CID) of these ions helps distinguish the pairs of isomeric dipeptides studied in this
work. Further, negative ion MS3 CID has also been found to be useful for differentiating these isomeric peptide
–
–
–
acids. The MS3 of [M–H–Fmoc + H]− of isomeric peptide acids produce c1 , z1 and y1 ions. Thus the present study
of Fmoc‐protected peptides provides additional information on mass spectral characterization of the dipeptides
and distinguishes the positional isomers. Copyright © 2011 John Wiley & Sons, Ltd.
Peptides are ubiquitous in living organisms and play various
essential roles such as protein folding, cell adhesion, cell
differentiation, and tumor metastasis.[1] Peptides are also
involved in important physiological and biochemical func-
tions such as neuro transmission, neuro modulation, and act
as hormones in receptor mediated signal transduction.[2]
Despite their suboptimal pharmacokinetic properties, pep-
tides demonstrate unparalleled potency and specificity
against a wide array of biological targets.[3] A major portion
of currently marketed drugs bear peptides in their core
structure. The increasing interest about the manifold actions
of the bioactive peptides has made their structural studies an
important aspect of research in pharmacology and medical
sciences.[4] During the drug development, identification of
an ’active’ part of a large peptide is important for further
improving its pharmacology. This involves synthesis of a
small segment, chemical modification and its biological
and structural evaluation. Mass spectral analysis, together
with other analytical tools, would aid the understanding
of complete structural aspects. In peptide synthesis, the
9‐fluorenylmethyloxycarbonyl (Fmoc) group is routinely
employed as amine protector due to the various synthetic
advantages associated with it.[5] Its stability over a range
of reaction environments, racemization tolerance and mild
deprotection conditions have made it a protecting group
of choice.
Tandem mass spectrometry (MS/MS) of protonated and
deprotonated organic and biological analytes including pep-
tides[6–11] in electrospray ionization (ESI) and matrix‐assisted
laser desorption/ionization (MALDI)[12–14] is now a well‐
established technique for structural elucidation and differentia-
tion of isomers.[15–25] Heerma and co‐workers[26–28] have
reported the high‐energy collision‐induced dissociation
(CID) of a variety of protonated dipeptides and tripeptides
and shown that fragmentation of the protonated species
leads to formation of the y1+ and/or a1+ ions. Isa et al.[29] have
carried out high‐energy CID studies of a series of protonated
dipeptides such as H‐Xxx‐Gly‐OH, H‐Gly‐Xxx‐OH, H‐Xxx‐
Leu‐OH and H‐Leu‐Xxx‐OH. They have shown that for the
* Correspondence to: R. Srinivas, National Centre for Mass
Spectrometry, Indian Institute of Chemical Technology,
Hyderabad 500 607, India.
E‐mail: srini@iict.res.in; sragampeta@yahoo.co.in
** Correspondence to: V. V. Sureshbabu, Peptide Research
Laboratory, Department of Studies in Chemistry, Central
College Campus, Bangalore University, Dr. B. R. Ambedkar
Veedhi, Bangalore 560 001, India.
+
observation of y1 ion, the proton affinity of the C‐terminal
amino acid should be greater than that of the N‐terminal
amino acid. Harrison and co‐workers[7,23] reported high‐
energy CID studies of series of protonated dipeptides.
E‐mail: hariccb@gmail.com
Rapid Commun. Mass Spectrom. 2011, 25, 1949–1958
Copyright © 2011 John Wiley & Sons, Ltd.

M. Ramesh et al.
They have shown that fragmentation of protonated species
control of Xcalibur software (Thermo Finnigan). The typical
source conditions were: spray voltage, 5 KV; capillary
voltage, 15–20 V; heated capillary temperature, 200°C; tube
lens offset voltage, 20 V; sheath gas (N2) pressure, 20 psi; and
helium was used as damping gas. For the ion‐trap analyzer,
the automatic gain control (AGC) setting were 2 × 107 counts
for a full‐scan mass spectrum and 2 × 107 counts for a full
product ion mass spectrum with a maximum ion injection
time of 200 ms. In the full‐scan MS2 mode, the precursor ion
of interest was first isolated by applying an appropriate wave
form across the end‐cap electrodes of the ion trap to
resonantly eject all trapped ions, except those ions of m/z
ratio of interest. The isolated ions were then subjected to a
supplementary alternating current (ac) signal to resonantly
excite them and so cause collision‐induced dissociation
(CID). The collision energies used were 15–38 eV. The
excitation time used was 30 ms. All the spectra were recorded
under identical experimental conditions for isomers, and
average of 20–25 scans. The methanolic solutions of samples
were infused into the ESI source at a flow rate of 5 μL/min by
using an instrument’s syringe pump.
+
+
leads to formation of the y1 and/or a1 ions. Fonseca
et al.[20] also reported the formation of the y1 and/or
+
+
a1 ions in ESI‐MS/MS studies of dipeptide isomers.
Hiserodt et al.[24] demonstrated that dipeptides containing
an N‐terminal basic amino acid undergo a rearrangement
involving a cyclic intermediate to form the corresponding
protonated molecule of the respective basic amino acid.
They also reported that the [b1 + H2O]+ ion is formed from
+
A1A2 dipeptides whereas A2A1 dipeptides formed y1 ions.
Goel and Kenny[22] also reported the mechanism for the
formation of the b1‐1 and corresponding a1‐1 ions in the
product ion mass spectra of N‐para‐ferrocenylbenzoyl
dipeptide esters. A literature survey revealed that there
are very few reports on mass spectral studies of Fmoc
protected peptides/amino acids.[30–37] To examine the effect
of the Fmoc‐ group on the fragmentation of the dipeptides,
we have studied a series of Fmoc‐protected dipeptide
positional isomers using both positive and negative ion
ESI‐MS/MS.
EXPERIMENTAL
Materials
Mass spectrometry
Solvents used in the present study were purchased from
Merck (Mumbai, India), and used without further purifica-
tion. Stock (1 mM) solutions of peptides were diluted with
high‐performance liquid chromatography (HPLC)‐grade
methanol to achieve a final concentration of 10 μM each.
ESI mass spectra of peptides 1–24 (Scheme 1) were recorded
using a liquid chromatography quadrupole (LCQ) ion trap
mass spectrometer (LCQ Advantage Max, Thermo Finnigan,
San Jose, CA, USA). The data acquisition was under the
O
O
R1
H
N
O
N
OY
H
R2
O
R1
1. H
R2
Y
CH3
H
R1
13. H
R2
Y
CH3
CH2C6H5
CH2C6H5
H
CH3
2. H
CH3
14. H
H
3. CH3
H
CH3
H
15. CH2C6H5
16. CH2C6H5
17. CH3
CH3
H
4. CH3
H
H
5. H
CH(CH3)2
CH3
H
CH2CH(CH3)2
CH2CH(CH3)2
CH3
CH3
H
6. H
CH(CH3)2
18. CH3
7. CH(CH3)2
8. CH(CH3)2
9. H
H
CH3
H
19. CH2CH(CH3)2
20. CH2CH(CH3)2
21. CH3
CH3
H
CH3
H
CH2CH(CH3)2
CH3
H
CH2C6H5
CH2C6H5
CH3
CH3
H
10. H
CH2CH(CH3)2
22. CH3
11. CH2CH(CH3)2
12. CH2CH(CH3)2
H
H
CH3
H
23. CH2C6H5
24. CH2C6H5
CH3
H
CH3
Scheme 1. Structure of the studied Nα‐Fmoc protected peptides (1–24).
wileyonlinelibrary.com/journal/rcm
Copyright © 2011 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2011, 25, 1949–1958

Study of Nα‐Fmoc‐protected dipeptide isomers by ESI‐MSn
Synthesis of the peptides
RESULTS AND DISCUSSION
The synthesis and spectroscopic characterization of the com-
pounds studied in this work have been reported previously.[38]
A series of positional isomeric Fmoc‐protected (Fmoc = [(9‐
fluorenylmethyl)oxy]carbonyl) dipeptides studied in this
work are shown in Scheme 1. The positive ion ESI mass
spectra of all these peptides (1–24) show abundant [M + Na]+,
[2M + Na]+, and [M + H]+ ions, and low abundance [M + H–
Fme]+ (Fme = 9‐methylene‐9H‐fluorene), [M + H–Fmoc+ H]+,
and m/z 179 (9H‐fluoren‐9‐yl)‐methyl cation (C14H11+)) ions.
The formation of the [M+ H–Fmoc+ H]+ ions can be rationa-
lized in terms of a McLafferty‐type rearrangement involving a
γ‐hydrogen migration from the fluorenyl moiety to the
carbonyl oxygen in the FmocN moiety followed by the loss
of 9‐methylene‐9H‐fluorene (Fme, 178 Da) and subsequent
loss of CO2[31]from [M+ H]+ (Scheme 2). The formation of
[M + H–Fmoc + H]+ ions can also be explained by a stepwise
mechanism involving the loss of CO2 from [M + H–Fme]+
(Scheme 2), as evidenced by the MS3 spectra. Fragmentation
of these peptides can be explained by using the nomenclature
of Roepstorff, Fohlman, and Biemann.[42–44] To study the
mass spectrometric behavior of these Fmoc‐protected dipep-
tides and to investigate the possibility of distinguishing these
positional isomers, we have examined the MS/MS CID
spectra of the protonated positional isomers (1–24).
General procedure for the preparation of Nα‐Fmoc
dipeptide methyl esters
To a stirred suspension of an amino acid methyl ester
hydrochloride salt (1.0 mmol) and activated Zn dust (1.0
mmol) in tetrahydrofuran (THF) (3.0 mL) was added a
solution of Fmoc amino acid chloride (1 mmol) followed by
activated Zn dust (1.0 equiv) in THF and the mixture was
stirred for 10–15 min at room temperature. After completion
of the reaction (analyzed by thin‐layer chromatography
(TLC) analysis), the solution was filtered and the filtrate was
evaporated. The residue was diluted with CH2Cl2 (15 mL),
washed successively with 5% HCl (10 mL × 2), 5% aq.
NaHCO3 (10 mL × 2), water (10 mL × 2) and dried over
anhydrous Na2SO4. After evaporation of the solvent, the
residue was purified by column chromatography (EtOAc/
hexane, 1:3) to obtain the dipeptide methyl ester as a white
solid (see Supporting Information).[39]
Preparation of Nα‐Fmoc amino acid chlorides
The ESI‐MS/MS spectra of [M + H]+ ions (m/z 383) of
positional isomeric peptides Fmoc‐Gly‐Ala‐OCH3 (1) and
Fmoc‐Ala‐Gly‐OCH3 (3)[34] are distinctly different from
each other (Fig. 1). Both the isomers show product ions at
m/z 351 ([M + H–CH3OH]+), 205 ([M + H–Fme]+), 187 (loss
of (9H‐fluoren‐9‐yl)methanol), 179 (9H‐fluoren‐9‐yl)‐methyl
cation (C14H11+)), 173 ([M + H–Fme–CH3OH]+), 161 ([M + H–
Fmoc+ H]+), and 129 ([M + H–Fmoc+ H‐CH3OH]+). Isomer 1
shows additional peaks at m/z 323 ([M + H–CH3OH–CO]+)
and 145 ([M + H–Fme–CH3OH–CO]+) which are absent for 3,
whereas isomer 3 shows product ions at m/z 339 ([M + H‐CO2]+),
294 (b1+) (Scheme 3), 133 ([M + H–Fmoc + H–CO]+), and 116
(b1+–Fme). These fragmentation pathways have been con-
firmed by MSn experiments. Loss of CH3OH from [M + H]+
Under an argon atmosphere, Fmoc amino acid (1.0 mmol)
was suspended in CH2Cl2 (5 mL), SOCl2 (1 mL) was added
and the mixture was sonicated at room temperature for
20–30 min. Solvent and excess of SOCl2 were removed
in vacuo and the residue was dissolved in CH2Cl2 (1.0 mL).
Addition of hexane (10 mL) precipitated pure acid chlorides.
The resulting crystals were filtered and dried (see Supporting
information).[40]
General procedure for hydrolysis of methyl esters
A solution of Nα‐Fmoc peptide ester (1.0 mmol) in iPrOH/
H2O (7:3, 10.6 mL) was treated with NaOH (1.2 mmol)
and 0.8 M CaCl2. The reaction mixture was stirred for 3–4
h (TLC analysis). When the reaction was complete, the
hydrosylate was neutralized with 1 N HCl (10 mL) and
the solid residue was dissolved in MeOH (15.0 mL).
Addition of H2O (30.0 mL) resulted in a precipitate which
was filtered and extensively washed with H2O. The crude
product was purified on silica gel (eluant: CH2Cl2/
MeOH/AcOH, 1–4% MeOH, 1% AcOH) gave the pure
peptide acid as a white crystalline solid (see Support-
ing Information).[41]
and [M + H–Fmoc + H]+ ions can be explained by
a
plausible mechanism involving an intramolecular nucleo-
philic attack by the protonated amide carbonyl on the
carbonyl carbon of the C‐terminal ester group, forming a
five‐membered cyclic ring with the extrusion of
+
CH3OH.[6,45] The formation of the b1 ion in 3 containing
alanine at the N‐terminus is in contrast to the fragmentation
behavior of the protonated unprotected dipeptides, H‐Ala‐
+
+
Gly‐OH, which mainly produce y1 and/ or a1 ions.[7,29] It
+
can be noted that the absence of the b1 ion for isomer 1,
H
O
R
H
R
H
H
O
C
H
R
McLafferty type
rearrangement
HC
O
H
N
- CO2
H
C
H2N
- Fme
H
O
N
H
[M+H-Fmoc+H]+
[M+H-Fme]+
[M+H]+
Scheme 2. Proposed McLafferty‐type rearrangement.
Rapid Commun. Mass Spectrom. 2011, 25, 1949–1958 Copyright © 2011 John Wiley & Sons, Ltd.
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M. Ramesh et al.
161
129
100
80
60
40
20
0
100
80
60
40
20
0
(a)
(a)
351
205
+
383
y1
104
101
179
173
129
161
143
145
150
323
187
200
250
m/z
300
350
60
80
100
120
140
160
m/z
179
90
100
80
60
40
20
0
100
80
60
40
20
0
(b)
(b)
133
+
y1
161
205
129
133
129
383
351
350
+
b1
294
+
161
160
x1
116
187
116
339
173
143
140
101
100
150
200
250
m/z
300
60
80
120
m/z
Figure 1. ESI‐MS/MS spectra of [M + H]+ ions (m/z 383) of
(a) 1 and (b) 3 at 23 eV.
Figure 2. ESI‐MS3 spectra of [M + H–Fmoc + H]+ ions (m/z
161) of (a) 1 and (b) 3 at 27 eV.
containing glycine at the N‐terminus, may be attributed to
the unstable aziridinone structure (Scheme 3).[46,47]
significantly different from that of 3. Both the isomers
show product ions at m/z 143 ([M + H–Fmoc + H–H2O]+),
129 ([M + H–Fmoc + H–CH3OH]+) and 101([M + H–Fmoc +
H–CH3OH–CO]+). In addition, isomer 1 shows an ion at m/z
104 (y1+) (Scheme 4),[8] whereas isomer 3 shows product ions
To probe further the fragmentation of the dipeptides, we
have examined the MS3 CID of the common ions of m/z 161
([M + H–Fmoc + H]+) of 1 and 3, which were formed via
sequential losses of Fme and CO2 from the [M + H]+ precursor
ions. It can be seen from Fig. 2 that the MS3 spectrum of 1 is also
O
R1
R1
O
H
N
H
N
H3N
OY
Fmoc
N
OY
H2
R2
O
O
R2
proton transfer
O
H2
N
proton transfer
R1
O
H2
N
R1
H2N
OY
Fmoc
N
OY
OY
H
R2
O
O
R2
O
O
H2
N
H2
N
H
H
H
OY
N
Fmoc
R2
N
O
R2
O
HN
R1
R1
O
O
H2N
H
R1
OY
O
R2
H3N
Fmoc
OY
N
O
R2
+
+
R1 b1
y1
+
+
Scheme 3. Proposed mechanism for the formation of the b1
Scheme 4. Proposed mechanism for the formation of the y1
ion from [M + H–Fmoc + H]+ ions.
ion from [M + H]+ ions.
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Copyright © 2011 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2011, 25, 1949–1958

Study of Nα‐Fmoc‐protected dipeptide isomers by ESI‐MSn
(a1+),[23,33,45] a characteristic ion for the presence of valine at
at m/z 133 ([M + H–Fmoc + H–CO]+), 116 (x1+) and 90 (y1+). The
formation of the y1+ ion from [M + H–Fmoc + H]+ ion of 1 and 3
is in agreement with earlier reports of unprotected dipep-
tides.[7,29] Thus, the positional isomers 1 and 3 can be clearly
distinguished from each another by their characteristic
fragmentation in both MS2 and MS3 CID spectra.
+
the N‐terminus. The formation of the y1 ion for 5 and the
immonium ion (a1+) for 7 is in agreement with higher proton
affinity of valine compared to that of glycine.[7,29] Thus, the
MS3 spectra of 5 and 7 are also useful in distinguishing the
presence of glycine or valine at the N‐terminus.
Similarly, another two pairs of isomeric peptides, 9/11
(Fmoc‐Gly‐Leu‐OCH3/Fmoc‐Leu‐Gly‐OCH3) and 13/15
(Fmoc‐Gly‐Phe‐OCH3/Fmoc‐Phe‐Gly‐OCH3), can be clearly
distinguished from one another from their characteristic
fragmentation in both MS2 and MS3 CID spectra (Table 1
and also Supporting Information).
Similarly, another pair of positional isomers, Fmoc‐Gly‐
Val‐OCH3 (5) and Fmoc‐Val‐Gly‐OCH3 (7), yielded product
ion spectra that are distinctly different from one another
(Table 1 and also Supporting Information). Similarly to 1
and 3, the CID mass spectra of the [M + H]+ ions (m/z 411)
of 5 and 7 show product ions at m/z 379 ([M + H–CH3OH]+),
233 ([M + H–Fme]+), 215 (loss of (9H‐fluoren‐9‐yl)methanol),
189 ([M + H–Fmoc + H]+) and 179 (C14H11+). Isomer 5 shows
additional peaks at m/z 351 ([M + H–CH3OH–CO]+), 201
The MS/MS CID mass spectra of [M + H]+ ions (m/z 439) of
a pair of positional isomers, Fmoc‐Ala‐Leu‐OCH3 (17) and
Fmoc‐Leu‐Ala‐OCH3 (19), exhibit product ions that are
distinctly different from one another (Fig. 3). Similarly to
previous isomers, the CID mass spectra of [M + H]+ ions (m/z
439) of 17 and 19 show product ions at m/z 407 ([M + H–
CH3OH]+), 379 ([M + H–CH3OH–CO]+), 261 ([M + H–Fme]+),
243 (loss of (9H‐fluoren‐9‐yl)methanol), 229 ([M + H–Fme–
CH3OH]+), 217 ([M + H–Fmoc + H]+), 185 ([M + H–Fmoc + H–
CH3OH]+) and 179 (C14H11+). Isomer 17 shows additional
peaks at m/z 294 (b1+), 201([M + H–Fme–CH3OH–CO]+) and
146 (y1+), whereas isomer 19 shows product ions at m/z 336
(b1+), 158 (b1+–Fme) and 130 (b1+–Fme–CO). These fragmen-
tation pathways have been confirmed by MSn experiments.
Thus, the positional isomers 17 and 19 can be readily
distinguished from one another by means of their character-
istic product ions.
([M + H–Fme–CH3OH]+), 173 ([M + H–Fme–CH3OH–CO]+),
157 ([M + H–Fmoc + H–CH3OH]+), 132 (y1
)
and 129
+
([M + H–Fmoc+ H–CH3OH–CO]+) which are absent for isomer
7, whereas isomer 7 shows product ions at m/z 322 (b1++),
161 ([M + H–Fmoc + H–CO]+), 144 (b1+–Fme) and 116 (b1
–
Fme–CO). These fragmentation pathways have been
confirmed by MSn experiments. Similarly to 1, absence
+
of the b1 ion for isomer 5 containing glycine at the
N‐terminus can be attributed to the unstable aziridinone
structure.[46,47] Thus, the positional isomers 5 and 7 can
be readily distinguished from one another by means of
their characteristic product ions.
Further, we have examined the MS3 CID of m/z 189 ion
([M + H–Fmoc + H]+) of 5 and 7, which were formed via
sequential losses of Fme and CO2 from the [M + H]+
precursor ions. Both the isomers show (Table 1 and also
Supporting Information) an ion at m/z 157 ([M + H–Fmoc +
H–CH3OH]+). Besides, the former shows product ions at m/z
171 ([M + H–Fmoc + H–H2O]+), 132 (y1+) and 129 ([M + H–
Further, MS3 CID of m/z 217 ([M + H‐Fmoc + H]+) of 17 and
19 gives (Fig. 4) low abundance ions at m/z 199 ([M + H–
Fmoc + H–H2O]+), 185 ([M + H–Fmoc + H–CH3OH]+) and 157
([M + H–Fmoc + H–CH3OH–CO]+). Besides, the former shows
an abundant ion at m/z 146 (y1+) and the latter gives low
abundance ions at m/z 189 ([M + H–Fmoc + H–CO]+), 104 (y1
)
+
Fmoc + H–CH3OH–CO]+) and the latter shows
a low
abundance ion at m/z 161 ([M + H‐Fmoc + H‐CO]+) and an
abundant ion at m/z 72 corresponding to the immonium ion
and an abundant ion at m/z 86 corresponding to the
immonium ion (a1+), a characteristic ion for the presence
Table 1. Partial CID of [M + H]+ ions of peptides 5, 7, 9, 11, 13, 15, 21 and 23: m/z values with relative abundances (%) in
parentheses
[M + H−
[M + H−
MS3 of [M + H−
Compound [M + H]+
Fme]+ Fmoc + H ]+
Other ions
Fmoc + H ]+
5
411
411
425
425
459
459
473
473
233(32)
233(18)
247(38)
247(35)
281(30)
281(12)
295(12)
295 (10)
189(60)
189(41)
203(79)
203(98)
379 (100), 351 (1), 215 (1), 201 (10), 179 (3),
173 (1), 157 (8), 132 (1), 129 (2)
189 → 171(1), 157(100),
132(2)b, 129(12),
7
379 (22), 322(30)a, 215 (5), 201 (1), 179
(100), 161(1), 144 (50), 116 (9)
189 → 161(5), 157(2),
72(100)c,
9
393 (100), 365 (2), 229 (1), 215 (12), 187 (2),
179 (3), 171 (8), 146 (2), 143 (4)
203 → 185(1), 171(100),
146(5)b, 143(17)
11
13
15
21
23
393 (36), 381 (1), 336 (25)a, 229 (7), 215 (1),
179 (100), 158 (60), 130 (27)
203 → 185(1), 175(4), 171
(3), 86(100)c
237(100) 427 (45), 399 (1), 263 (1), 249 (6), 221 (1),
205 (5), 180 (8), 177 (3)
237 → 219(1), 205(100),
180(32)b, 177(47)
237(100) 427 (15), 415 (1), 370 (6)a, 263 (5), 221 (1),
192 (43), 179 (20), 164 (51), 148 (2), 146 (3)
237 → 219(1), 209(1), 205
(30), 177(1), 120(100)c
251 → 233(10), 219(22),
191(6), 180(100)b
251(72)
441(100), 413 (4), 294 (1)a, 277 (1), 263 (2), 235
(1), 219 (1), 191 (1), 180 (28), 179 (1), 163 (1)
441(100), 413 (1), 370 (5)a, 277 (3), 263 (1), 219 (1),
192(33), 191(1), 179(20), 164(46), 148(2), 146(4)
251(67)
251 →233(1), 219(12), 191
(1), 120(100)c, 104(1)b
ab1 ion; y1 ion; a1 ion.
+
b
+
c
+
Rapid Commun. Mass Spectrom. 2011, 25, 1949–1958 Copyright © 2011 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm

M. Ramesh et al.
+
of leucine at the N‐terminus. The formation of y1+ and/or a1
the MS3 spectra of 17 and 19 are also useful in establishing
whether alanine or leucine is located at the N‐terminus.
Similarly, another pair of isomeric peptides, 21/23 (Fmoc‐
Ala‐Phe‐OCH3/Fmoc‐Phe‐Ala‐OCH3), can be clearly dis-
tinguished from each other from their characteristic frag-
mentation in both MS2 and MS3 (Table 1 and also Supporting
Information).
The positional isomeric pairs of corresponding acids, 2/4,
6/8, 10/12, 14/16, 18/20 and 22/24, can be clearly distinguished
from one another, except that the masses of the C‐terminal
ions are decreased by 14 Da.
ions from [M+ H–Fmoc + H]+ of 17 and 19 is in agreement
with earlier reports of unprotected dipeptides.[29] Thus,
x5
x5
407
100
80
60
40
20
0
(a)
179
261
217
229
b
146
185
201
294
439
450
379
243
250
150
200
300
m/z
350
400
Negative ion CID of isomeric peptide acids
Peptides containing a free terminal carboxylic acid are known
to readily form negative ions; hence, we examined the
possibility of differentiating these peptide acids (Scheme 1)
under negative ion ESI conditions. The negative ion ESI mass
spectra of all the dipeptide acids show [M–H]–, [M‐H‐9H‐
fluoren‐9‐yl) methanol]– and [M–H–Fmoc + H]– ions. The
direct formation of [M–H–Fmoc + H]– ions and absence of
[M–H–Fme]– ions under negative ion conditions can be
explained by a plausible mechanism that may involve a 1,5‐H
migration from the fluorenyl group to the ‐NH‐ leading to the
concomitant loss of CO2 and Fme (Scheme 5).
x2
x5
407
100
80
60
40
20
0
(b)
179
217
261
b
439
158
336
130
243
229
185
200
379
150
250
300
m/z
350
400
450
Figure 3. ESI‐MS/MS spectra of [M + H]+ ions (m/z 439) of
(a) 17 and (b) 19 at 23 eV.
145
100
185
146
100
(a)
(a)
80
80
+
y1
60
40
60
40
367
20
0
20
0
157
217
171
199
200
150
200
250
300
350
60
80
100
120
140
m/z
160
180
220
m/z
145
86
100
80
60
40
20
0
100
80
60
40
20
0
(b)
(b)
+
a1
185
+
y1
104
367
217
189
171
157
160
199
200
80
100
120
140
m/z
180
220
150
200
250
300
350
m/z
Figure 4. ESI‐MS3 spectra of [M + H‐Fmoc + H]+ ions (m/z
217) of (a) 17 and (b) 19 at 27 eV.
Figure 5. ESI‐MS/MS spectra of [M–H]– ions (m/z 367) of (a)
2 and (b) 4 at 15 eV.
O
R1
O
R1
H
H
N
H
O
C
N
H
O
NH
- Fme+CO2
O
H2N
R2
O
R2
[M-H-Fmoc+H]-
O
O
[M-H]-
Scheme 5. Proposed mechanism for the concomitant loss of Fme and CO2.
wileyonlinelibrary.com/journal/rcm
Copyright © 2011 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2011, 25, 1949–1958

Study of Nα‐Fmoc‐protected dipeptide isomers by ESI‐MSn
Negative ion ESI CID of the [M–H]– ion (m/z 367) of isomeric
acids 2 and 4 yields (Fig. 5) a low abundance ion at m/z 171
(loss of (9H‐fluoren‐9‐yl)methanol) and an abundant ion at m/z
145 ([M–H–Fmoc + H]–). To differentiate the isomeric pair of 2
and 4, we have examined the MS3 of [M–H–Fmoc+ H]– ions.
Figure 6 shows the MS3 spectra of [M–H–Fmoc + H]– ions
(m/z 145) of 2 and 4 which are significantly different from
one another. The CID spectra of both the isomers display ions at
m/z 127 ([M–H–Fmoc + H–H2O]–) and 101 ([M–H–Fmoc +
H–CO2]–). In addition, 2 shows an abundant ion at m/z 88 (y1 )
–
–
and a low abundance ion at m/z 73 (c1 ), whereas the latter
‐
shows an abundant ion at m/z 74 (y1 ) and low abundance ions at
x10
m/z 87 (c1 ),84 ([M–H–Fmoc+ H–CO2–NH3]–) and 57 (z1 ). The
formation of c1– and z1– ions can be explained by the mechanism
shown in Scheme 6.[48] It is noteworthy that N‐terminal b1– ions
are absent, instead c1– ions are significant which are normally
reported to form under electron‐capture dissociation.[49] Thus,
the positional isomers 2 and 4 can be readily distinguished
from one another by means of different product ions.
–
–
88
100
(a)
80
-
y1
60
-
c1
40
20
0
73
145
101
Similarly to 2 and 4, negative ion ESI CID of the [M–H]– ion
(m/z 395) of another pair of isomeric acids 6 and 8 yields a low
abundance ion at m/z 199 (loss of (9H‐fluoren‐9‐yl)methanol)
and an abundant ion at m/z 173 ([M‐H‐Fmoc + H]‐) (Support-
ing Information). The MS3 spectra of [M–H–Fmoc + H]– ions
(m/z 173) of these isomers are distinctly different from one
another (Table 2 and also Supporting Information). The
spectra display ions at m/z 155 ([M–H–Fmoc + H–H2O]–) and
129 ([M–H–Fmoc + H–CO2]–). Besides, 6 shows an abundant
127
60
80
100
m/z
120
140
x10
x10
101
100
80
60
40
20
0
(b)
74
-
57
y1
-
z1
–
–
145
ion at m/z 116 (y1 ) and low abundance ions at m/z 99 (z1 ) and
84
-
c1
–
73 (c1 ), whereas the latter shows an abundant ion at m/z 74
87
127
‐
–
(y1 ) and low abundance ion at m/z 57 (z1 ). Thus, the
positional isomers 6 and 8 can be readily distinguished from
one another by means of different product ions.
60
80
100
m/z
120
140
Figure 6. ESI‐MS3 spectra of [M–H–Fmoc + H]– ions (m/z 145)
of (a) 2 and (b) 4 at 34 eV.
Another four pairs of isomeric acids, 10/12, 14/16, 18/20
and 22/24, can also be clearly distinguished from each other
R1
O
H
N
R1
O
H2N
O
H
N
O
H2N
O
R
O
R1
O
R1
O
H
N
H2N
OH
NH
O
H2N
O
R
O
R1
R1
O
O
NH
NH
H2N
OH
O
H2N
CH
O
-
O
z1- (m/ z 57)
c1
R
R1
O
NH
O
H2N
CH
O
R
z1-
c1-
–
–
Scheme 6. Proposed mechanism for the formation of c1 and z1 ions.
Rapid Commun. Mass Spectrom. 2011, 25, 1949–1958 Copyright © 2011 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm

M. Ramesh et al.
Table 2. Partial CID of [M–H]– ions of peptide acids 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24: m/z values with relative abundances (%)
in parentheses
Compound
[M–H]–
[M–H–A*]–
[M–H–Fmoc + H]–
MS3 of [M–H–Fmoc + H]–
6
395
395
409
409
443
443
199(1)
199(6)
213(1)
213(4)
247(3)
247(15)
173(100)
173(100)
187(100)
187(100)
221(100)
221(100)
173 → 155 (1), 129 (85), 116 (100)d, 99 (1)f, 73 (5)e
173 → 155 (5), 129 (100), 74 (68)d, 57 (4)f
8
10
12
14
16
187 → 169(2), 143 (38), 130 (100)d, 113 (1)f, 73 (16)e
187 → 169(1), 143 (100), 126 (3), 74 (64)d, 57 (2)f
221 → 177 (5), 164 (100)d, 147 (74)f, 73 (25)e
221 → 204 (45), 177 (5), 163 (1)e, 160 (30), , 129 (1)a,
111 (1), 91 (5)b, 85 (90)c, 74 (100)d
18
20
22
24
423
423
457
457
227(4)
227(4)
261(12)
261(14)
201(100)
201(100)
235(100)
235(100)
201 → 183 (3), 157 (100), 140 (1), 130 (46)d, 113 (1)f, 87 (6)e
201 → 183 (2), 157 (40), 140 (1), 129 (2)e, 88 (100)d, 71 (1)f
235 → 191 (10), 164 (39)d, 147 (100)f, 87 (38)e
235 → 218 (22), 191 (3), 174 (7), 163(1)e, 143 (1)a,
99 (20)c, 91(1)b,88 (100)d
*A= (9H‐fluoren‐9‐yl)methanol
aLoss of toluene; C7H7+; loss of CO2 from m/z 129/m/z 143 (16 and 24); dy1 ion; c1 ion; z1 ion.
b
c
–
e
‐
f
–
by their characteristic fragmentation in MS3 spectra (Table 2
and also Supporting Information).
[3] K. A. Ahrendt, J. A. Olesen, M. Wakao, J. Trias, J. A. Ellman.
Identification of potent and broad‐spectrum antibiotics
from SAR studies of a synthetic vancomycin analogue.
Bio. Org. Med. Chem. Lett. 2003, 13, 1683.
[4] I. Wagner, H. Musso. New naturally occurring amino acids.
Angew. Chem. Int. Ed. Engl. 1983, 95, 827.
CONCLUSIONS
[5] (a) L. A. Carpino, G. Y. Han. The 9‐fluorenylmethoxycar-
bonyl function, a new base‐sensitive amino‐protecting
group. J. Am. Chem. Soc. 1970, 92, 5748; (b) L. A. Carpino.
The 9‐fluorenylmethyloxycarbonyl family of base‐sensitive
amino‐protecting groups. Acc. Chem. Res. 1987, 20, 401–407.
[6] B. Paizs, S. Suhai. Fragmentation pathways of protonated
peptides. Mass Spectrom. Rev. 2005, 24, 508, and references
cited therein.
[7] A. G. Harrison, I. G. Csizmadia, T. H. Tang , Y. P. Tu. Reaction
competition in the fragmentation of protonated dipeptides.
J. Mass Spectrom. 2000, 35, 683.
[8] B. Paizs, S. Suhai. Theoretical study of the main fragmenta-
tion pathways for protonated glycylglycine. Rapid Commun.
Mass Spectrom. 2001, 15, 651.
Positive and negative ion ESI tandem mass spectrometry has
been shown to be very useful for the structural characteriza-
tion and differentiation of six pairs of Nα‐Fmoc‐protected
dipeptide positional isomers. While MS/MS of the proton-
ated dipeptides containing Ala‐, Val‐, Leu‐ and Phe‐ at the N‐
terminus gives rise to intense b1 ions, MS3 of [M + H–
+
+
+
Fmoc + H]+ yields y1 and/or a1 ions. Further, negative ion
MS3 CID has also been found to be useful for differentiating
these isomeric peptide acids. In contrast to positive ions, b1
ions are absent, instead significant c1 , z1 and y1 ions are
–
–
–
–
observed in the negative ion MSn spectra.
[9] M. M. Cordero, C. Wesdemiotis. Tandem mass spectro-
metry of peptides: sequence information based on neutral
losses. I. Isomeric dipeptides. Org. Mass Spectrom. 1993,
28, 1041.
[10] E. Mammoliti, G. Sindona, N. Uccella. Characterization of
Z‐blocked isomeric dipeptides by fast atom bombardment
tandem mass spectrometry and kinetic energy release
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[11] M. J. Polce, D. Ren, C. Wesdemiotis. Dissociation of the
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2000, 35, 1391.
[12] M. Karas, F. Hillenkamp. Laser desorption ionization of
proteins with molecular masses exceeding 10 000 daltons.
Anal. Chem. 1988, 60, 2299; (b) T. Yoshida, K. Tanaka, Y. Ido,
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SUPPORTING INFORMATION
Additional supporting information may be found in the
online version of this article.
Acknowledgements
The authors thank Dr. J. S. Yadav, Director, IICT, Hyderabad,
for facilities and Dr. M. Vairamani for cooperation. M. R. is
thankful to CSIR, New Delhi, for the award of Senior
Research Fellowship, B. R. is thankful to DST, New Delhi, for
the award of a Junior Research Fellowship and T.M.V and H.
P.H. thank for CSIR, New Delhi, for financial assistance.
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