Peptide dimethylation: Fragmentation control via distancing the dimethylamino group

Article abstract of DOI:10.1007/s13361-014-0951-7

Direct reductive methylation of peptides is a common method for quantitative proteomics. It is an active derivatization technique; with participation of the dimethylamino group, the derivatized peptides preferentially release intense a₁ ions. The advantageous generation of a₁ ions for quantitative proteomic profiling, however, is not desirable for targeted proteomic quantitation using multiple reaction monitoring mass spectrometry; this mass spectrometric method prefers the derivatizing group to stay with the intact peptide ions and multiple fragments as passive mass tags. This work investigated collisional fragmentation of peptides whose amine groups were derivatized with five linear ω-dimethylamino acids, from 2-(dimethylamino)-acetic acid to 6-(dimethylamino)-hexanoic acid. Tandem mass spectra of the derivatized tryptic peptides revealed different preferential breakdown pathways. Together with energy resolved mass spectrometry, it was found that shutting down the active participation of the terminal dimethylamino group in fragmentation of derivatized peptides is possible. However, it took a separation of five methylene groups between the terminal dimethylamino group and the amide formed upon peptide derivatization. For the first time, the gas-phase fragmentation of peptides derivatized with linear ω-dimethylamino acids of systematically increasing alkyl chain lengths is reported.

Full text of DOI:10.1007/s13361-014-0951-7

B American Society for Mass Spectrometry, 2014
J. Am. Soc. Mass Spectrom. (2014) 25:1694Y1704
DOI: 10.1007/s13361-014-0951-7
RESEARCH ARTICLE
Peptide Dimethylation: Fragmentation Control via Distancing
the Dimethylamino Group
Adam J. McShane, Yuanyuan Shen, Mary Joan Castillo, Xudong Yao
Department of Chemistry, University of Connecticut, Storrs, CT 06269, USA
Abstract. Direct reductive methylation of peptides is a common method for quantita-
tive proteomics. It is an active derivatization technique; with participation of the
dimethylamino group, the derivatized peptides preferentially release intense a1 ions.
The advantageous generation of a1 ions for quantitative proteomic profiling, however,
is not desirable for targeted proteomic quantitation using multiple reaction monitoring
mass spectrometry; this mass spectrometric method prefers the derivatizing group to
stay with the intact peptide ions and multiple fragments as passive mass tags. This
work investigated collisional fragmentation of peptides whose amine groups were
derivatized with five linear ω-dimethylamino acids, from 2-(dimethylamino)-acetic
acid to 6-(dimethylamino)-hexanoic acid. Tandem mass spectra of the derivatized
tryptic peptides revealed different preferential breakdown pathways. Together with energy resolved mass
spectrometry, it was found that shutting down the active participation of the terminal dimethylamino group in
fragmentation of derivatized peptides is possible. However, it took a separation of five methylene groups between
the terminal dimethylamino group and the amide formed upon peptide derivatization. For the first time, the gas-
phase fragmentation of peptides derivatized with linear ω-dimethylamino acids of systematically increasing alkyl
chain lengths is reported.
Key words: Peptide fragmentation, Collisional dissociation mechanism, Energy-resolved mass spectrometry,
Peptide derivatization, Active derivatization, Passive derivatization, Peptide dimethylation, Dimethylamino pep-
tides, Quantitative proteomics, Multiple reaction monitoring mass spectrometry
Received: 21 March 2014/Revised: 13 May 2014/Accepted: 6 June 2014/Published Online: 5 August 2014
caused by all the derivatized peptides sharing common reporter
ions; therefore, peptides with similar masses and elution time
Introduction
he use of peptide derivatization is a staple of proteomic
quantitation [1]. Derivatization-based mass spectrometry
produce elevated background for the peptides in quantitation
[4, 5].
T
(MS) quantitation is characterized by its sample-throughput
capabilities allowing multiple samples to be analyzed simulta-
neously. Different applications are dependent upon fragmenta-
tion control of the derivatized peptide. In applications of iso-
baric mass tagging reagents [1, 2], the derivatized peptides are
isobaric, but after fragmentation produce differentiable reporter
ions in tandem MS (MS/MS) spectra. Peptide derivatization
with these types of reagents are Type I active derivatizations
(Scheme 1), classified by the strong signals directly observed
from the fragmented derivatization group [3]. One significant
pitfall of these reagents is the limited dynamic range, which is
Reductive methylation has been used as both mass-
difference tagging for MS quantitation [6–8] and, recently,
isobaric mass tagging for MS/MS quantitation [9–11].
Dimethylation is a relatively simple reaction only requiring a
reducing agent and formaldehyde. This reaction is expeditious
without any substantial formation of side products. When
stable isotope labels are used in the reducing agent and/or
formaldehyde, peptides can be introduced with a designed
number of stable isotope labels for MS-based quantitative
measurements. This method has been broadly applicable be-
cause of its easy implementation [12].
The molecular basis for MS/MS quantitation of peptides
with direct dimethylation is the facile collisional cleavage of
the first N-terminal amino acid residues. Strong signals for
derivatized a1 ions (stable quaternary amine ions) are charac-
teristic in MS/MS spectra of the derivatized peptides [13–15].
Dimethylation of peptides is considered a Type II active
Electronic supplementary material The online version of this article
(doi:10.1007/s13361-014-0951-7) contains supplementary material, which is
available to authorized users.
Correspondence to: Xudong Yao; e-mail: x.yao@uconn.edu

A. J. McShane et al.: Fragmentation of Dimethylamino Peptides
1695
Scheme 1. Categorization of peptidyl chemical derivatizations
derivatization, where the derivatizing group promotes the
cleavage of the first amide bond of peptides (Scheme 1). Col-
lisional fragmentation of directly dimethylated peptides has
been studied in detail [14, 15]. When the methyl groups carry
differential stable isotope labels, the derivatized peptides can,
in principle, be quantified based on the correspondingly labeled
a1 ions. This quantitation using MS/MS measurement could be
advantageous. These a1 ions are peptide-specific and, thus,
lessen the interference from co-eluting peptides experienced
for peptides derivatized with isobaric mass tagging reagents. In
order to observe such interference, derivatized peptides would
need to have similar elution times and masses as well as the
same first N-terminal amino acid residue; a rare occurrence.
However, authentic quadrupole sampling of dimethylated pep-
tide ions with different numbers of isotope labels as precursors
can be problematic. Efforts have been made to develop an
isobaric mass tagging capability using peptide dimethylation.
One method derivatizes peptides at both N- and C-termini, but
with complementary numbers of stable isotope labels. In this
method, triplex isobaric peptide termini labeling with
dimethylation, peptide precursors are isobaric and fragment
ions carry differentiable stable isotope labels and thus the ion
intensity of the fragments can be used for quantitation [9].
In line with recent advancements in the utilization of ultra-
high resolving MS for proteomics, differences in mass defects
of carbon, hydrogen, oxygen, and nitrogen isotopes have been
exploited [10, 11, 16–19]. The scope of isobaric mass tagging
is, thus, being expanded, opening exciting new opportunities in
proteome quantitation. In principle, all of the currently prac-
ticed, derivatization-based quantitative proteomic methods can
be adapted to better utilize contemporary mass spectrometers.
Two recent reports use peptide dimethylation. Peptides in
comparison carry derivatizing groups with different
isotopologue labels (i.e., two atoms of 13C versus 2H). There-
fore, during the low-resolution selection of precursor ions, the
differentially-labeled peptides are co-selected authentically.
However, when MS/MS are recorded with ultra-high resolving
power, fragment ions can be baseline-separated for quantita-
tion. Both works use Lys-C for protein digestion so that each
resulting peptide carries two derivatizing groups. It is interest-
ing to note that there are more derivatized y ions than b ions
available to be used for quantitation [10, 11]; in other words the
ε-dimethylamino group on the lysine side chain at the peptide
C-terminus is more stable than the α-dimethylamino group at
the N-terminus.
Multiple reaction monitoring (MRM) MS is at the forefront
of targeted, quantitative MS because of its high selectivity,
sensitivity, and method robustness [20]. Stable isotope labeled
references are commonly used to assemble methods for stable
isotope dilution (SID) MRM MS. The generation of quantita-
tion reference peptides can be achieved by derivatizing pep-
tides with stable isotope labeled chemicals for mass-difference
tagging [21, 22]. Although the quantitation utility of the
derivatized reference peptides can be comparable to those
produced by metabolic labeling techniques [23], these reagents
have yet to be broadly adopted. The limited adoption of the
commercial reagents for MRM MS exemplifies a common
problem for using active derivatizations of peptides for
MRM-based MS quantitation: the derivatizing groups are not
efficiently held as intact mass tags for the precursor ions of the
derivatized peptides or for their fragments.
MRM-based methods require the detection of strong signals
for both precursor ions and fragment ions to obtain low quan-
titation limits. Furthermore, multiple distinguishable fragment
ions are preferred to secure the method’s specificity. If the
derivatizing group is cleaved from the fragment ions, these
fragments are no longer distinguishable among peptides with
differential derivatizations. Therefore, MRM-based quantita-
tion can benefit from passive derivatization of peptides
(Scheme 1) [3]: during MRM analysis, derivatized peptides
preserve the derivatizing groups on the intact peptide precursor
ions in the ionization region of the instrument and keep these
derivatizing groups on multiple fragment ions during fragmen-
tation of the derivatized peptides in the collision cell. We
hypothesize that if the dimethylamino group on derivatized
peptides is disengaged from the adjacent amide bond, a passive
derivatization will be achieved for MRM-based peptide analy-
sis. This derivatization will keep the increased gas-phase ba-
sicity of dimethylamino peptides from the tertiary amine of the
dimethylamino group and, accordingly, the enhanced MS sig-
nals [24], but not activate particular peptide bonds in a biased
manner. Herein, we present a systematic study of how

1696
A. J. McShane et al.: Fragmentation of Dimethylamino Peptides
diversely a dimethylamino group participates in collisional
fragmentation of peptides with the increased distance from
the first N-terminal amino acid residue. In addition, the change
of role for the dimethylamino group from an active participant
in fragmentation to a passive mass tagging one is reported.
resolution MS (AccuTOF DART; JEOL, Peabody, MA, USA).
Detailed syntheses and characterizations can be found in
Supplementary Information (SI) 1–5.
Peptide Derivatization
The dimethylamino acid (1.0 equivalent), NHS (1.2 equiva-
lents), and DIC (1.0 equivalent) were first dissolved in DMSO,
and then diluted with anhydrous DCM. The solution was incu-
bated overnight at room temperature. A solution containing five
peptides, NSILTETLHR, LSLVPDSEQGEAILPR,
LSEPAELTDAVK, YGGFLR, and SVILLGR dissolved in
10% DIEA/DMF, was added to the activated dimethylamino
acid. The solution was incubated overnight at room temperature.
The coupling was then quenched with 20% formic acid/H2O on
ice. The solution was first dried with a speed-vac (Savant
SC100; Thermo Fisher), then lyophilized (Labconco FreeZone
Plus, Kansas City, MO, USA). After drying, the sample was
desalted via an empty spin column (Thermo Fisher) packed with
hydrophilic-lipophilic-balanced (HLB, Oasis Waters, Milford,
MA, USA) reversed-phase sorbent. The sample was then dried
and reconstituted with FA/H2O for liquid chromatography (LC)-
MS analysis (Shimadzu HPLC pumps/controller, Kyoto, KYT,
Japan and HTC PAL autosampler, Carrboro, NC, USA).
Experimental
Chemicals
2-Aminoacetic acid (999%), 3-aminopropanoic acid (99%), 4-
aminobutanoic acid (999%), 5-aminopentanoic acid (997%),
6-aminohexanoic acid (999%), methanol (999.9%), diethyl
ether (999%), glacial acetic acid (999.7%), N,N-
diisopropylethylamine (99.5%, DIEA), N-hydroxysuccinimide
(98%, NHS), dimethyl sulfoxide (999.7%, DMSO), anhydrous
N,N-dimethylformamide (999.8%, DMF), 4-(4,6-dimethoxy-
1, 3, 5-triazin-2-yl)-4-methylmorpholinium chloride (≥96.0%,
DMTMM), N- (3-dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride (≥98.0%, EDC), and anhydrous dichlorometh-
ane (999.5%, DCM) were purchased from Sigma-Aldrich (St.
Louis, MO, USA). Formic acid (88% and 99.5%),
trifluoroacetic acid (97%, TFA), acetonitrile (99.9%, ACN),
and formaldehyde (37%) were purchased from Fisher (Hano-
ver Park, IL, USA). Concentrated hydrogen chloride acid
(36.5%–38%) was purchased from J. T. Baker (Center Valley,
PA, USA). N,N'-diisopropylcarbodiimide (99%, DIC) was pur-
chased from Alfa Aesar (Ward Hill, MA, USA). The ultrapure
water was obtained from a Direct-Q 3 UV water purification
system (EMD Millipore, Billerica, MA, USA). The peptides
LSLVPDSEQGEAILPR (95%), LSEPAELTDAVK (99%),
YGGFLR (98%), and SVILLGR (99%) were purchased from
Peptide 2.0 (Chantilly, VA, USA). The peptide NSILTETLHR
(95%) was purchased from Anaspec (Fremont, CA, USA). The
isotopic peptide SVIL[L-13C615N]GR (99%) was purchased
from Thermo Fisher (Rockford, IL, USA).
LC-MS and LC-MS/MS Studies
A 10 cm, 1.0 mm i.d. Hypersil Gold HPLC column (Thermo
Fisher Scientific) with, 175 Å, 3 μm C18 resin was used.
Solvent A was composed of 98.8% H2O, 1.0% ACN, and
0.2% FA and solvent B was 98.8% ACN, 1.0% H2O, and
0.2% FA. The LC gradient was 5% to 35% solvent B for
45 min. The column temperature was 60°C. The hybrid mass
spectrometer used was a QSTAR Elite (AB SCIEX, Framing-
ham, MA, USA). Positive electrospray ionization (ESI) was
used, with parameters of 5.5 kV for the spray voltage and
source temperature of 300°C. The initial collision energies
(CE) for the doubly- and triply charged peptides were obtained
from Equations 1 and 2, respectively [25].
Dimethylamino Acid Synthesis
Each amino acid (8 mmol) was dissolved in formic acid (5 mL,
88%) and formaldehyde (1 mL). The synthesis of 3-
(dimethylamino)-propanoic acid (denoted as dim-3), 4-
(dimethylamino)-butanoic acid (denoted as dim-4), 5-
(dimethylamino)-pentanoic acid (denoted as dim-5), and 6-
(dimethylamino)-hexanoic acid (denoted as dim-6) was carried
out in a microwave reactor (CEM Discover-S; Matthews, NC,
USA), with the following conditions: 100 W, 110°C, 20 to
120 min. 2-(dimethylamino)-acetic acid (denoted as dim-2)
was conventionally refluxed at 100°C for 1 h. Concentrated
HCl (1 mL) was added after heating. The solvent was dried in
vacuo. After drying, a white precipitate was obtained and
washed with glacial acetic acid (5 mL) three times. The
dimethylamino acid was then recrystallized with methanol
and diethyl ether. After purification, the reagents were charac-
terized with nuclear magnetic resonance (NMR) spectroscopy
(Avance III 400 MHz; Bruker, Billerica, MA, USA) and high-
CE ¼ ð0:057 Â m=zÞ − 4:265
ð1Þ
CE ¼ ð0:031 Â m=zÞ þ 7:082
ð2Þ
These equations were empirically adjusted for the non-
derivatized peptides (Supplementary Table S1), and for dim-2-
peptides (Supplementary Table S2). The new equations from the
dim-2-peptides were applied to the dim-3- through dim-6-peptides.
Energy Resolved (ER)-MS Studies
An AB SCIEX 4000 QTRAP triple quadruple mass spectrom-
eter with direct syringe infusion of the sample solutions was
used for the ER-MS studies. The ESI was in positive mode with

A. J. McShane et al.: Fragmentation of Dimethylamino Peptides
1697
Scheme 2. General reaction for derivatizing peptidyl amines with dimethylated amino acids
parameters of 5.5 kV for the ion spray and a source temperature
of 200°C. Each peptide (500 fmol/μL) was individually infused
with a flow rate at 5 μL/min. The DP was optimized before the
collision studies were started. The CE was ramped from 5 to
40 V in 0.5 V/s increments. The resolution was set to unit for
Q1 and high for Q3. The dwell time was 200 ms. Three
replicates were taken for each peptide measured (non-
derivatized YGGFLR through dim-6-YGGFLR).
low and varying yields for the derivatization products. A mixed
solvent of DMSO and DCM served well for activating the
dimethylamino acids using DIC/NHS. This solvent system
was miscible with the mixture of DIEA and DMF, which was
used as the solvent for preparing peptide solutions for
derivatization.
MS Signal Enhancement of Dimethylamino Peptides
Results and Discussion
Microwave-Assisted Synthesis of Dimethylamino
Acids
The percentage of chemical conversion (PCC) and signal yield
for mass spectrometry (SYMS) were quantitatively measured
for peptides dim-2-SVILLGR through dim-6-SVILLGR. After
quenching the derivatization reaction, an equal amount of
isotopically labeled SVIL[L-13C615N]GR was added as the
quantitation reference, although GSVIL[L-13C615N]GR would
be a closer reference. The MS signal for the residual unreacted
SVILLGR, compared with that for the isotopic quantitation
reference, allowed for calculation of the PCC value for each
derivatization. The average PCC for all five derivatizations of
SVILLGR was 85% (Supplementary Equation S1). Further
optimization of derivatization reactions was not performed in
this work because these reactions were used to generate suffi-
cient amounts of dimethylated peptides for mechanistic inves-
tigations not to be directly applied for proteome quantitation.
The SYMS value for a derivatization was calculated based on
the MS signal of the derivatized peptide (e.g., dim-2-
SVILLGR) and that of the isotopic quantitation reference.
The average SYMS for all five derivatized peptides was
170% (Supplemental Equation S2). This average SYMS was
further corrected for the derivatization completeness (or aver-
age PCC), giving an adjusted SYMS of 200% (Supplementary
Equation S3), for the addition of dimethylamino acids to the N-
terminus of SVILLGR.
The established Eschweiler-Clarke reaction [26, 27] was used
to dimethylate the primary amines of five amino acids: 2-
aminoacetic acid, 3-aminopropanoic acid, 4-aminobutanoic
acid, 5-aminopentanoic acid, and 6-aminohexanoic acid. They
have increasing numbers of methylene groups, one through
five (dim-2 through dim-6, respectively), distancing the amino
group from the carboxylic acid. Full conversion of 3-
aminopropanoic acid to dim-3 required 8 h of conventional
refluxing. Other amino acids were even less reactive towards
amine dimethylation. In an attempt to expedite the
dimethylation of 3-aminopropanoic acid to 6-aminohexanoic
acid, microwave irradiation was utilized. All of the initial five
substrates were converted to the corresponding products within
20 to 120 min of irradiation (SI 1–5).
Preparation of Dimethylamino Peptides
Five synthetic peptides were used as models to investigate
collisional fragmentation mechanisms of dimethylated pep-
tides: NSILTETLHR, LSLVPDSEQGEAILPR,
LSEPAELTDAVK, YGGFLR, and SVILLGR. They possess
a wide variety of properties (Supplementary Table S3). Peptide
LSEPAELTDAVK was chosen as a model peptide for lysine-
containing peptides, which have two primary amines. Tandem
mass spectra for these non-derivatized peptides are shown in
Supplementary Figures S1a–f.
The enhanced MS signal for the derivatized peptides can be
attributed to the increased gas-phase basicity, compared with
the underivatized SVILLGR. These derivatizations convert a
primary N-terminal amino group to a tertiary amine, which is
more basic and results in more favorable protonation in the gas
phase. Signal enhancement of 130% to 240% was also reported
Peptidyl amino groups on the model peptides were
derivatized with dimethylamino acids whose carboxylic groups
were activated in situ (Scheme 2). The activation of carboxylic
groups was mediated by DIC and NHS. Solvent played a
dominating role in this peptide derivatization. The
dimethylamino acids were soluble in aqueous solution. How-
ever, derivatization of the peptides using water-soluble media-
tion reagents, including EDC/NHS and DMTMM, resulted in
Scheme 3. Nomenclature of fragment ions as represented by
dim-3-YGGFLR

1698
A. J. McShane et al.: Fragmentation of Dimethylamino Peptides
(a)
(b)
Scheme 4. Mechanism of f1 production from dim-2 derivatized peptides
for peptides with dimethylated lysine side chains, compared
with their non-dimethylated counterparts [24]. The increased
basicity of peptides carrying the dimethylamino group is likely
also attributing to the charge-state shift from a mixture of
singly- and doubly charged ions for SVILLGR and YGGFLR
to the mostly doubly charged for their derivatized counterparts,
and predominantly doubly charged LSLVPDSEQGEAILPR to
a doubly- and triply charged mixture (Supplementary
Figure S7a–c). The coalescence to doubly charged SVILLGR
f1
f1
58.06
58.06
100
100
DIM-2-YGGFLR
[M+2H]2+ 399.22
DIM-3-YGGFLR
[M+2H]2+ 406.23
90
90
80
70
60
80
70
60
ab2
50
50
y1
175.10
y1
*a1
[MH-f1]+
263.10
175.10
40
30
20
10
0
M
221.12
40
712.31
*a2
754.36
ab4
ab3
320.12
278.11
ab1
206.07
30
y5
549.29
a4
467.21
*b1
249.11
f2
20
10
0
397.16
86.09
[M+2H]2+
aa4
y5
549.32
406.23
439.18
100
200
300
400
500
600
700
800
100
200
300
400
500
600
700
800
m/z
m/z
Figure 1. MS/MS spectrum of dim-2-YGGFLR
Figure 2. MS/MS spectrum of dim-3-YGGFLR

A. J. McShane et al.: Fragmentation of Dimethylamino Peptides
1699
Scheme 5. Mechanism of f1 production from dim-3 derivatized peptides
from the singly- and doubly charged mixture could also attri-
bute to the increase in SYMS.
the dimethylamino group are denoted as f1, (4) b and a ions
carrying an acetyl group (the complementary products from the
α-β cleavage) are denoted as ab and aa, respectively, and (5) the
ions from the amide bond cleavage between the derivatizing
group and the original N-terminus of the peptide are denoted as
f2, and the corresponding production of the singly charged ion
as the original peptide is denoted as oM. A general trend of the
increased coverage of b and derivatized b ions was observed for
the derivatized peptides compared with their underivatized
counterparts (Supplementary Figures S1–S6) [6].
Dim-2 derivatization results in peptides with the same
chemical structure as peptides with direct reductive
dimethylation; collisional fragmentation mechanism for the
directly methylated peptides has been studied in detail
(Scheme 4a) [14, 15]. For instance, dim-2-YGGFLR (MS/
MS spectrum shown in Figure 1) is the same as dimethylated
GYGGFLR. Dimethylated peptides are known to produce
enhanced signals for *a1 ions [13–15]. In the nomenclature
system used in this paper, the f1 ion was observed for dim-2-
YGGFLR (or *a1 for dimethylated GYGGFLR) at m/z 58.06
(Figure 1; Scheme 4). Generation of f2 ions (or *b1 for
dimethylated GYGGFLR) at m/z 86.20 can be accounted for
by the oxozolium mechanism [28] or the aziridin-2-one path-
way (Scheme 4b), which further produces the f1 ion [29].
Fragmentation Dependence of Dimethylamino
Peptides on Alkyl Chain Length
Tandem MS was performed on a QqTOF mass spectrometer
for all of the derivatized peptides. The derivatized YGGFLR
peptides are used as the exemplary peptides for discussion,
with all others in the SI (Supplementary Figures S1–S6). The
CE was empirically adjusted using Equations 1 and 2 to ensure
proper fragment ion production. The dim-2-peptides were used
for the empirical adjustment, and the CE was increased by 1.1
to 1.5 times, compared with the suggested values from the
equations (Supplementary Table S3). The CE was adjusted
until an approximate G10% precursor ion was observed after
collision-induced dissociation (CID). Equations 1 and 2 were
then corrected accordingly for other dimethylamino peptides.
As shown in Scheme 3, fragment ions are denoted with typical
nomenclature except for the following: (1) b and a ions carry-
ing the derivatizing group are denoted as *b and *a respective-
ly, (2) b and a ions carrying a cyclization product of the
c
c
derivatizing group are denoted as b and a respectively, (3)
ions from the bond cleavage between the α and β carbon from
Scheme 6. Imido lactone formation at the N-terminus of dim-4-, dim-5-, and dim-6-YGGFLR

1700
A. J. McShane et al.: Fragmentation of Dimethylamino Peptides
cyc2+
Generation of f2 ions suggests the favorable protonation of the
dimethylamino group and a facile intramolecular proton trans-
fer to activate the adjacent amide group in the derivatized
peptides (Scheme 4b). In other words, there is an active partic-
ipation of the derivatizing group in the peptide fragmentation.
Dim-3 derivatization extends the distance by one additional
methylene group between the dimethylamino group and the
first amide group on the derivatized peptides, which is formed
upon the attachment of a dimethylamino propionic acid. Two
intense ions at m/z 58.06 (f1) and m/z 754.36 [MH-f1]+ were
observed for dim-3-YGGFLR, together with significant signals
for acetyl b ions (ab1, ab2, ab3, and ab4), shown in Figure 2. The
sum of m/z 58.06 and m/z 754.36 made up the mass balance for
dim-3-YGGFLR, suggesting the existence of a labile bond.
Generation of these two ions can be readily explained by the
McLafferty-type rearrangement (Scheme 5) [30]. It should be
noted that although the ion structure for f1 ions for dim-2-
peptides and dim-3-peptides are the same, mechanisms for
the ion generation are different (Schemes 4 and 5). In general,
the f1 ion was observed for all dim-3 peptides (Figure 2 and
Supplementary Figure S3). On the other hand, not all of the
corresponding [MH-f1]+ ions were observed (Supplementary
Figure S3), which could be due to further breakage of the
primary [MH-f1]+ fragment ions under experimental condi-
tions. According to the categorization of peptide derivatization
shown in Scheme 1, the dim-3 reaction would be classified as a
Type I active derivatization, where there is preferential cleav-
age within the derivatizing group.
397.73
100
90
80
70
60
50
40
30
20
10
0
DIM-5-YGGFLR
[M+2H]2+ 420.24
[M+2H]2+
420.26
y1
175.12
f2
*b4
552.31
cb4
128.11
*a4
524.30
ca1
218.12
y5
549.34
*b3
507.26
M
ca4
712.38
405.24
y4
479.27
492.31
100
200
300
400
500
600
700
800
m/z
Figure 4. MS/MS spectrum of dim-5-YGGFLR
lactone (i.e., cb and ca ions) were observed for dim-4- and dim-
5-YGGFLR. Formation of these ions can be explained by
nucleophilic substitution of the amide oxygen to the α-
methylene carbon of the dimethylamino group (Scheme 6).
Similar mechanisms were reported in a fragmentation study
of lysylglycine [31] and used for explaining the charge mobi-
lization of peptides derivatized with a quaternary amine [32]
and loss of dimethylamine from Nε-dimethyllysine [33]. Dim-
6-YGGFLR produced a similar cyclization product at m/z
404.72 (cyc2+, Figure 5), but at a much reduced intensity and
A common, neutral loss of dimethylamine was observed for
doubly charged ions of dim-4-, dim-5-, and dim-6-YGGFLR,
producing corresponding doubly charged fragments
(Scheme 6). Dim-4 derivatization produced a very strong frag-
ment ion at m/z 390.75 (cyc2+, Figure 3). Dim-5-YGGFLR
gave a doubly charged ion at m/z 397.73 (cyc2+) as the most
intense fragment (Figure 4). These two ions are proposed as
YGGFLR carrying an imido lactone at the N-terminus
(Scheme 6). A complement of b and a ions carrying the imido
without observable complementary ca and b ions. The inten-
sity differences could be related to the stability of imido lactone
rings.
Doubly charged dim-6-YGGFLR, compared with dim-4-
and dim-5-YGGFLR, produced full series of *a and *b ions
carrying the derivatization group, together with y ions
(Figures 3, 4, and 5). This suggests that in the dim-6-peptide
the preferential cleavage via the mechanism in Scheme 5
c
[M+2H]2+
f2
114.10
427.24
100
100
DIM-6-YGGFLR
[M+2H]2+ 427.25
DIM-4-YGGFLR
[M+2H]2+ 413.24
90
90
80
80
y1
70
cyc2+
390.75
70
175.10
60
60
y1
175.13
50
50
*b3
*a4
ca1
419.21
538.30
40
30
20
10
0
40
y5
549.31
204.11
f2
*b4
*a3
391.22
2+
30
142.12
566.29
[M+2H]
*a1
413.26
ca4
465.25
cyc2+
277.19
20
*b5
679.33
cb4
*a5
M
ca2
M
y5
404.72
712.34
712.38
*b1
10
0
549.36
493.24
651.37
261.16
305.18
100
200
300
400
500
600
700
800
100
200
300
400
500
600
700
800
m/z
m/z
Figure 3. MS/MS spectrum of dim-4-YGGFLR
Figure 5. MS/MS spectrum of dim-6-YGGFLR

A. J. McShane et al.: Fragmentation of Dimethylamino Peptides
1701
Scheme 7. N,N-dimethyl lactam ion loss from dim-4-, dim-5-, and dim-6-YGGFLR
becomes much less competitive. In other words, the dim-6
derivatization is passive (Scheme 1). Similar results were also
observed for other dim-6 derivatized peptides (Supplementary
Figure S6). It is also interesting to note that the intensity for the
residual doubly charged precursor was high (Figure 5), using
the collision energy obtained from the same calculation as
those for other derivatized peptides (Supplementary Table S2),
dim-2-YGGFLR through dim-5-YGGFLR.
Double derivatization of LSEPAELTDAVK was obtained
at both the N-terminus and the lysine side chain. For example,
dim-4-LSEPAELTDAVK (Supplementary Figure S4d) pro-
duced fragments that corresponded to the cyclization occurring
at both sites. The first cyclization product (m/z 727.45) was
80% relative intensity and the second (m/z 704.90) was 30%
relative intensity. In comparison, one cyclization product (m/z
741.43) was observed for dim-5-LSEPAELTDAVK at 15%
relative intensity (Supplementary Figure S5e), which reports
the bond cleavage at the N-terminus.
Replicate 1
Replicate 2
Replicate 3
Sigmoidal Fitting
100
80
60
40
20
0
Another Preferential Cleavage Pathway
of Dim-4-, Dim-5-, and Dim-6-Peptides
CE50
Two ions related to cleavage of the derivatizing group from the
doubly charged dim-4-YGGFLR precursor were observed; one
dominantly at m/z 114.10 (f2) and the other strong ion at m/z
712.38 (oM, Figure 3). Dim-5-YGGFLR and dim-6-YGGFLR,
cleaving at the same amide bond, also produced ion pairs at m/z
128.15 and 712.38 and at m/z 142.12 and 712.34, respectively.
However, the relative ion intensities for the corresponding f2
ions, although strong, were not dominantly high for dim-5- and
dim-6-YGGFLR (Figures 4 and 5). The favorable cleavage of
the amide bond can be accounted for by the mechanism pro-
posed in Scheme 7. The intensity differences among these ions
are on the same order of the facility for intramolecular proton
transfer, requiring the formation of a pseudo ring of 7, 8, or 9
atoms, with the increasing entropy penalty. Although dim-2-
and dim-3-peptides have a more facile intramolecular proton
10
20
30
40
50
CE (V)
Figure 6. Survival curve of dim-6-YGGFLR. The precursor was
selected in Q1 and Q3 with the CE being ramped from 5 to 35 V
in 0.5 V/s increments. Three replicates were obtained for each
YGGFLR peptide, and the data was normalized to the maxi-
mum intensity. After sigmoidal dose-response fitting the CE50
was obtained

1702
A. J. McShane et al.: Fragmentation of Dimethylamino Peptides
transfer through a pseudo 5- or 6-membered ring (Schemes 4b
and 5), the nucleophilic attack of the deprotonated
dimethylamine to the carbon of protonated amide would expe-
rience high ring constraints; the generation of similar f2 ions
would require the formation of a ring with three or four atoms
following the mechanism in Scheme 7. Protonated aziridin-2-
ones, however, are viable intermediates for a1 ions of peptides
upon collisional dissociation (Scheme 4b) [29].
preferentially produce f1 and/or f2 ions and their complemen-
tary fragment ions (Schemes 4, 5, 6, and 7), this linear corre-
lation suggests that these different mechanisms result in mini-
mal differential effects on CE required for the breakage of
doubly charged precursor ions.
The underivatized YGGFLR showed positive deviation of
1.81 V from the linear correlation (Figure 7). This deviation
signifies the peptide as a mechanistic indicator, reporting that
there is no special bond in the peptide, which is particularly
labile to gas-phase collision. The same is true for the positive
deviation of 2.59 V for dim-6-YGGFLR (Figure 7). Although
dim-4- to dim-6-YGGFLR share a common fragmentation
pathway releasing the derivatizing groups as N,N-dimethyl
lactams (Scheme 7), the increased distance between the
dimethylamino group and the amide bond formed upon the
peptide derivatization leads to a discrete increase in the stability
of intact precursors from dim-5- to dim-6-YGGFLR, shown as
the positive deviation (Figure 7). This agrees with the obser-
vation that the ε-dimethylamino group on the lysine side chain
is a passive mass tag (Supplementary Figure S2e), stable to
collisional fragmentation in the gas phase [10, 11].
ER-MS to Categorize Peptides into Active
and Passive Derivatization Groups
ER-MS is an excellent tool to evaluate the energy requirements
for the generation of fragment ions and elucidation of fragmen-
tation patterns and mechanisms [28]. ER-MS was performed
for YGGFLR and dim-2- through dim-6-YGGFLR. The CE
profiles, in the laboratory frame, for the breakdown of the
derivatized peptides were recorded on a triple quadrupole
instrument. After normalization to the initial precursor ion
intensity, triplicate measurements were combined as a single
data set for sigmoidal dose-response fitting to produce the
survival curve (Figure 6), giving a CE value at 50% (CE50)
of residual precursor ions. Fitting plots for all of the six
YGGLFR peptides are shown in Supplementary Figure S8
and the formulae and the goodness of fitting are reported in
Supplementary Table S4. CE50 values for these peptides were
plotted against m/z values of doubly charged precursor ions
(Figure 7). Dim-2-, dim-3-, dim-4-, and dim-5-YGGFLR cor-
related linearly with a slope of 0.153 and adjusted R2 of 0.9994.
Together with the fact that different pathways operate to
Two Routes for Selective Activation
of Dimethylamino Peptides
There are two general mechanisms for a derivatizing group
actively participating in preferential fragmentations around the
N-termini of derivatized peptides. Both start from the prefer-
ential protonation of the dimethylamino group. This proton
then either (1) transfers to the adjacent amide group acting as
30
YGGFLR
DIM-2(to 5)-YGGFLR
DIM-6-YGGFLR
Linear Fit of DIM-2(to 5)-YGGFLR
25
20
15
y = 0.153x - 40.7
R 2 = 0.9994
360
380
400
420
440
m/z
Figure 7. CE50 and m/z correlation for dimethylamino YGGFLR peptides

A. J. McShane et al.: Fragmentation of Dimethylamino Peptides
1703
an acid catalyst to facilitate fragmentation reactions (the
mobile-proton regime) as in Schemes 4b, 5, and 7, or (2)
directly polarizes the α-methylene carbon of the protonated
dimethylamino group for the nucleophilic substitution by the
adjacent amide oxygen (the charge-directed regime) as in
Scheme 6. Proton migration from basic amino acids, including
histidine, lysine, and arginine, has been shown to promote
preferential neighboring cleavages [34–36]. The first general
mechanism also operates in the preferential fragmentation of
peptides derivatized with isothiocyanates [37, 38]. The impor-
tance of the intramolecular proton transfer is evident in this
study. When the intramolecular proton transfer becomes less
favorable for dim-6-YGGFLR, the preferential activation of the
adjacent amide bond diminishes and so do the subsequent
fragmentation products. In comparison, proton transfer from
non-methylated ε-amine at the peptide N-terminus faces com-
peting paths because of the complex intramolecular solvation
of the protonated amine group [39]; therefore activation of the
first amide group via acid catalysis confronts similar
competitions.
For the second general mechanism, although activa-
tion of the α-methylene carbon of the protonated
dimethylamino group stays the same more or less, the
direct nucleophilic substitution by the amide oxygen
becomes less entropically favorable with the distancing
of the protonated dimethylamino group from the neigh-
boring amide. Succinctly, a quantized change happens
from dim-5-YGGFLR to dim-6-YGGFLR or from active
derivatization to passive derivatization (Scheme 1).
with NMR spectroscopy and the Leadbeater Lab for use of
their scientific microwave.
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Acknowledgments
The authors greatly appreciate financial support from the Cys-
tic Fibrosis Foundation (YAO07XX0) and the NCI/NIH
(1R21CA155536-01). They thank Song Li for his assistance

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