Differentiating n-terminal aspartic and isoaspartic acid residues in peptides

Article abstract of DOI:10.1021/ac201223d

Formation of isoaspartic acid (isoAsp) is a common modification of aspartic acid (Asp) or asparagine (Asn) residue in proteins. Differentiation of isoAsp and Asp residues is a challenging task owing to their similar properties and identical molecular mass

Full text of DOI:10.1021/ac201223d

ARTICLE
pubs.acs.org/ac
Differentiating N-Terminal Aspartic and Isoaspartic Acid Residues in
Peptides
Nadezda P. Sargaeva,† Cheng Lin,† and Peter B. O’Connor†,‡,*
†Mass Spectrometry Resource, Department of Biochemistry, Boston University School of Medicine, 670 Albany Street, R504, Boston,
Massachusetts 02118, United States
‡Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, U.K.
S Supporting Information
b
ABSTRACT: Formation of isoaspartic acid (isoAsp) is a
common modification of aspartic acid (Asp) or asparagine
(Asn) residue in proteins. Differentiation of isoAsp and Asp
residues is a challenging task owing to their similar properties
and identical molecular mass. It was recently shown that they
can be differentiated using ionÀelectron or ionÀion interaction
fragmentation methods (ExD) because these methods provide
diagnostic fragments c + 57 and z• À 57 specific to the isoAsp
residue. To date, however, the presence of such fragments has
not been explored on peptides with an N-terminal isoAsp residue. To address this question, several N-terminal isoAsp-containing
peptides were analyzed using ExD methods alone or combined with chromatography. A diagnostic fragment [M + 2H À 74]+• was
observed for the doubly charged precursor ions with N-terminal isoAsp residues. For some peptides, identification of the N-terminal
isoAsp residue was challenging because of the low diagnostic ion peak intensity and the presence of interfering peaks. Supplemental
activation was used to improve diagnostic ion detection. Further, N-terminal acetylation was offered as a means to overcome the
interference problem by shifting the diagnostic fragment peak to [M + 2H À 116]+•.
soaspartic acid (isoAsp) is an isomer of aspartic acid (Asp) that
can be formed by either isomerization of an Asp or deamida-
hand, carboxypeptidase Y cleaves N-terminally to an isoAsp
residue recognizing its R-carboxylic acid as if it is a carboxyl-
terminal amino acid.17
I
tion of an asparagine (Asn) residue. Under physiological condi-
tions, both reactions proceed via formation of a cyclic
succinimide intermediate, followed by rapid hydrolysis to form
a mixture of Asp and isoAsp, typically in a 1:3 ratio in short,
unstructured peptides.1,2 In proteins, the ratio could be different
from 1:3 and influenced by structure.3,4 The succinimide forma-
tion from Asp is ∼10À40 times slower than from Asn at neutral
pH, yet the reaction rate may vary greatly depending on the
adjacent residues, protein conformation, and the proximity of the
Asp/Asn residue to the protein surface.1,5,6
Asn deamidation and Asp isomerization are common in vivo,
particularly in long-lived proteins, which are often associated
with aging,7 eye lens abnormalities,8 and amyloid diseases such as
Alzheimer disease.9,10 These modifications may alter protein
conformation, activity, and stability and trigger protein aggrega-
tion. For example, the 3-dimensional crystal structure of ribonu-
clease U2B revealed that formation of isoAsp32 led to a single
turn unfolding of the R-helix to form a U-shape loop structure,
affecting the hydrolytic activity of the protein.11 Single Asp
isomerization was shown to deactivate the antigen-binding region
of the immunoglobulin gamma (IgG)-2 antibody.12 The presence
of the isomerized Asp may also affect the proteolytic stability of
the protein. For example, Lys-C proteolysis is hindered when the
Lys residue is adjacent to an isoAsp in IgG-1 antibody;13 isoAsp
residues are also resistant to Asp-N proteolysis.14À16 On the other
Although in vivo Asn deamidation is irreversible, the isoAsp
accumulation can be minimized by protein L-isoaspartyl O-methyl-
transferase (PIMT),10 which catalyzes isoAsp conversion to Asp.
Asn deamidation and Asp isomerization are known to play a role in
apoptosis18 and have been proposed to serve as a molecular timer of
biological events.6 Furthermore, PIMT-mediated isoAsp-Asp inter-
conversion may regulate synaptic transmission in presynaptic
proteins.18 In vitro Asp isomerization and Asn deamidation can
also occur during protein production and storage, where the PIMT
enzyme is not available and the isoAsp products accumulate with
time. IsoAsp buildup could be detrimental to protein structural
integrity and stability, which affects the shelf life and potency of the
therapeutic monoclonal antibodies (mAb) in the pharmaceutical
industry as well as all other protein-based new drug entities. To
avoid undesirable consequences due to drug degradation, such
modifications have to be carefully monitored.
A number of techniques have been developed to detect iso-
Asp, and they are often used in conjunction. These include the
Edman degradation reaction,19 PIMT-utilizing assays,20 affinity en-
richment of isoAsp-containing proteins,21 use of isoAsp-specific
Received: May 13, 2011
Accepted: July 7, 2011
Published: July 07, 2011
r
2011 American Chemical Society
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Figure 1. ECD spectra of the angiotensin II (a) and its modified variant with isoAsp at the N-terminus (b). Insets show side-chain losses from the
charge-reduced species [M + 2H]+•. IsoAsp specific fragment [M + 2H À 74]+•, produced as a result of the CRÀCβ bond cleavage within the N-terminal
isoAsp residue, is present in the modified variant only, (b) inset.
antibodies,10,22 endoproteinase Asp-N-based approaches,12,14,23 iso-
topic 18O labeling,4,24,25 and various liquid chromatography (LC)-
based techniques: hydrophobic interaction chromatography,26 size-
exclusion high performance liquid chromatography (HPLC),12
cation exchange HPLC,26 reversed-phase (RP) HPLC,27,28 and ultra
HPLC.29 Asp/isoAsp differentiation has been demonstrated by
tandem mass spectrometric approaches, including collisionally acti-
vated dissociation,30,31 fast atom bombardment,32 and postsource
decay fragmentation.33 Recently, isoAsp identification and quantita-
tion by tandem MS methods employing ionÀelectron or ionÀion
interactions (ExD) based on the diagnostic fragment ions c + 57 and
z• À 57 that result from the CRÀCβ bond cleavage unique to isoAsp
residues have been developed34À39 and successfully applied.23,40,41
All the above-mentioned MS methods were accomplished in the
positive ion mode; however, in the negative ion mode, the isoAsp-
containing peptides were not differentiated.42
Despite a large number of existing approaches, isoAsp identi-
fication remains a challenging task. In HPLC, the elution order of
Asp/isoAsp-containing peptides depends on conditions of se-
paration (column type, mobile phase, temperature, etc.), materi-
als, and the instrument used.23,28 In particular, as was previously
shown,29,43 the elution order can be inverted when isoAsp is
locatedat theN-terminus, leading to erroneous assignment. Thus,
unless a standard mixture of the samepeptide pair separated at the
same condition is available, a further analysis would be required
for correct isomer assignment. ExD-based tandem MS seems to
be a fast and accurate approach well suited for this task. However,
to the best of our knowledge, a detailed ExD study of peptides
with N-terminally located isoAsp residues has not been per-
formed, and the N-terminal isoAsp-specific fragments have not
yet been demonstrated. In this work, the potential of ExD for
N-terminal isoAsp residue identification was investigated either
by performing the ExD analysis alone or following RP-HPLC
separation. The role of N-terminal acetylation and the effectof ion
activation in isomer differentiation were also examined.
’ EXPERIMENTAL SECTION
Peptides and Reagents. Angiotensin II (Ang II) peptide
variant iDRVYIHPF (hereinafter iD represents isoAsp in peptide
sequences), synthetic peptides AcÀDGVGDVGGVHÀNH2
and AcÀiDGVGiDVGGVHÀNH2, and amyloid beta 1À10
peptide variant NAEFRHNSGY with Asn residues at positions
1 and 7 (hereafter Aβ1À10 (N1N7)) were custom synthesized
by Peptide 2.0 (Chantilly, VA, USA). Ang II (DRVYIHPF) and
Aβ1À10 (DAEFRHDSGY) were ordered from AnaSpec (San
Jose, CA, USA). Formic acid (FA) was purchased from Thermo
Scientific (Rockford, IL, USA). HPLC grade acetonitrile (ACN)
and methanol (MeOH) were obtained from Fisher Scientific (Fair
Lawn, NJ, USA). Deionized water was purified by a Millipore
Milli-Q Gradient system (R = 18.2 MΩ cm and TOC = 9À
12 ppb) (Billerica, MA, USA). Ammonium bicarbonate (ABC)
was ordered from Sigma-Aldrich (St. Louis, MO, USA).
Deamidation. The Aβ1À10 peptide variant (NAEFRHN-
SGY) was incubated at 0.44 mM concentration in 0.1 mM ABC,
at pH ∼7.7 and 37 °C for 4 days. The resulting peptide mixture was
separated on a reversed-phase C18 column prior to MS/MS analysis.
Chromatography. RP-HPLC peptide separations were per-
formed on an Agilent 1200 Series system (Agilent Technologies,
Wilmington, DE, USA) using a reversed-phase C18 column
Vydac 218TP5215 (150 Â 2.1 mm, 3 μm particles, 300 Å pore
size). Four nanomoles of peptide mixture (in 20 μL) was injected
directly into the column and eluted using a linear gradient of
acetonitrile with 0.1% FA (0 min,1% B; 20 min, 11% B) at 0.7 mL/
min flow rate at 60 °C. The chromatograms were measured using
UV detection at 214 nm. Fractions were collected and refrigerated
at 4 °C prior to MS/MS analysis.
Mass Spectrometry. ExD analyses were performed on the
following:
1. A solariX FTICR instrument (Bruker Daltonics, Billerica,
MA, USA) with a 12 T actively shielded magnet: electron
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Figure 2. Chromatogram of the peptide mixture, generated by partial deamidation of the Aβ1À10 (N1N7) peptide variant (top panel). Each
chromatographic peak was labeled based on ECD results. The four ECD spectra shown correspond to the VIIIÀXI fractions with the isoAsp specific
fragments indicated in the insets. Additional spectra for fractions IÀVII are available in Supporting Information Figure S1.
capture dissociation (ECD), hotECD, and electron transfer
dissociation (ETD). Peptides were nanosprayed at 0.5À
2 μM concentration in 50:50 ACN/H2O with 0.1% FA
following HPLC separation with off-line fraction collection or
in 50:50 MeOH/H2O with 0.1% FA when infused directly.
Doubly charged molecular ions were isolated and irradiated
with low- (cathode bias 0.4À0.8 V) or high- (cathode bias 4.5
V (hotECD)) energy electrons for 0.05À0.5 ms to produce
fragments. Each ExD spectrum was the result of 100 scans.
2. An LTQ-Orbitrap XL with ETD capability (Thermo
Scientific, San Jose, CA, USA): ETD with or without
supplemental activation (SA). Peptides were nanosprayed
in solutions as stated above at ∼0.5 μM using a robotic
Nanomate source (Advion, Ithaca, NY, USA). Activation
energy parameter “En” of 5 or 15 was applied when SA was
on (typical En values range from 0À20).
Fluoranthene was used as the anion radical reagent in all ETD
experiments with 150À250 ms reagent accumulation, and
50À150 ms reaction time was used to produce fragments. Data
acquired on solariX and amaZon were analyzed using Bruker’s
ESI Compass DataAnalysis 4.0 software. The Orbitrap data were
analyzed using Thermo’s Xcalibur 2.0.7 software.
’ RESULTS AND DISCUSSION
Diagnostic Fragments for the N-Terminal Asp/isoAsp. As
originally proposed, ECD of peptides with isoAsp located at the
nth position may generate a unique complementary ion pair cnÀ1
•
+ 57 and zmÀn+1 À 57 as a result of the CRÀCβ bond cleavage,
where m is the total number of amino acid residues and 57, or
•
more accurately, 56.9976 corresponds to the mass of a C2HO2
group. For an isoAsp residue located at the N-terminus, n = 1,
•
3. An amaZon ion trap instrument (Bruker Daltonics, Bill-
erica, MA, USA): ETD with or without smart decomposi-
tion (SD). Peptides were electrosprayed (2 μL/min, glass
capillary temperature 220 °C) at 1 μM concentration in
50:50 MeOH/H2O with 0.1À1% formic acid using an
Apollo II ion source.
and the diagnostic fragments become c0 + 57 and zm À 57. The
c0 fragment is essentially an ammonia molecule that is neither
•
informative nor observed in ECD spectra, and the zm ion
contains no sequence information and is usually assigned as a
loss of ammonia from the charge-reduced species [M + 2H À
NH3]+•. Thus, when an N-terminal isoAsp residue is present, the
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Figure 3. ECD spectra of the synthetic peptide variants with Asp (a) and isoAsp residues (b) amidated at the C-terminus and acetylated at the
N-terminus. IsoAsp1 and isoAsp5 specific fragments, [M + 2H À 116]+• and z6 À 57, are observed only for the isoAsp-peptide variant. Asp side-chain
•
losses are also indicated, (a) inset.
C-terminal diagnostic fragment would be [M + 2H À (NH3 +
residues on the basis of the diagnostic fragments observed; that
is, [M + 2H À 74]+• for the isoAsp1 and c6 + 57 for the isoAsp7,
as shown in Figure 2. Nondeamidated Asn residues were
distinguished on the basis of the 0.984 mass difference of the
precursor ions and their fragments containing Asn, as shown in
C2HO2 )]+• or [M + 2H À74.0242]+• (hereinafter indicated as
•
[M + 2H À 74]+•), and the N-terminal diagnostic fragment
would be C2H5NO2+• (m/z = 75.032). Although the m/z = 75
peak is usually below the low mass cutoff, it is still possible to look
for the corresponding neutral loss [M + 2H À 74]+•.
•
the example of z6 fragments (Figure S1 of the Supporting
To see if the isoAsp can be differentiated from the Asp when
located at the N-terminus, ECD spectra of the two synthetic
isomers of Ang II were directly compared (Figure 1). The two
spectra were very similar with only a few exceptions. Careful
examination revealed the presence of the specific fragment
Information, fractions I and V). Fraction VI contained both
peptides DAEFRHNSGY and NAEFRHiDSGY, which are iso-
mers and thus indistinguishable at the MS level, yet could be
easily identified by ECD because some fragment ions exhibited
two components, separated by 0.984 Da (Figure S1 of the
Supporting Information, fraction VI, inset). On the basis of the
“z8 ” À 57 or [M + 2H À 74]+• only in the ECD spectrum of
•
•
the isoAsp-containing Ang II peptide. In contrast, the loss of
60.0211 (C2H4O2) and a double CO2 loss were observed only
for the Asp peptide. Indeed, the loss of 60 is characteristic of the
Asp side chain44 and cannot be formed from the isoAsp. The loss
of two CO2 molecules can be generated by a combined loss from
the C-terminus and from the Asp side chain. Although the CO2
loss can also come from an isoAsp residue, it is normally of a
much lower abundance37,41 or undetectable.38 For example, the
relative abundance of the two components within the z6 isotopic
cluster, the DAEFRHNSGY peptide comprises only a small
fraction of fraction VI. This is consistent with the fact that the
DAEFRHNSGY peptide has a lower probability of formation
compared with the NAEFRHiDSGY peptide (from original
peptide NAEFRHNSGY) because the Asn in the NA sequence
has a slower deamidation rate than Asn in the NS sequence.45,46
Although the separation of this closely related mixture of
peptides was challenging and for some peaks there was no
baseline separation, all peptides were successfully identified by
ECD, highlighting the utility of ECD in isoaspartomic research.
Peak Interference. Caution should be taken if the peptide of
interest contains methionine (Met) or isoleucine (Ile) residues
because they may produce side-chain loss fragment ions with a
m/z close to that of the diagnostic fragment ion: the Met side-
chain loss (C3H6S) results in a peak at [M + 2H À 74.01902]+•,
and the Ile side-chain loss with an additional ammonia loss
(C4H8 + NH3) at [M + 2H À 73.0891]+•. Although these
interfering peaks could lead to erroneous identification of N-
terminal isoAsp, they do not usually pose a problem if the analysis
•
a6 À NH2 À CO2 peak was very abundant in the ECD spectrum
of the Ang II peptide, but was not detected in that of its isoAsp
variant. Thus, in Figure 1a, the CO2 loss from the charge-reduced
species was probably generated from the C-terminus.
Analysis of the Deamidation Products of Aβ1À10 (N1N7)
by Off-Line RP-HPLC, Followed by ECD. An Aβ1À10 peptide
variant with Asn residues at positions 1 and 7 was deamidated to
artificially introduce isoAsp residues into the sequence. The
resulting mixture of peptides was separated on a C18 reversed-
phase column. Eleven peaks were detected in the chromatogram
(Figure 2, top panel), collected into fractions, and further
analyzed using ECD. It was possible to differentiate and to define
the elution order of the isomeric peptides containing isoAsp
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Figure 4. N-terminal isoAsp residue diagnostic fragment peak [M + 2H À 74]+• detection for the Aβ1À10 peptide variant (iDAEFRHiDSGY, fraction
XI in Figure 3, top panel) by (a) ETD on LTQ-Orbitrap, with supplemental activation energy parameter 15; and (b) ECD on SolariX. Insets show
diagnostic peaks and their S/N ratios.
is performed on an instrument with sufficient resolving power.
For example, differentiating the singly charged N-terminal iso-
Asp signature fragment ion at m/z = 1000 from the interfering
Met side-chain loss ion would require a mass resolving power
of >190 000, which is easily achievable on an FTICR mass
spectrometer such as the one used here.
are no such losses observed in the isoAsp-peptide ECD spectrum,
•
except for the z6 À CO2. This ion was produced as a result of a
•
secondary reaction following the z6 fragment ion formation via
hydrogen rearrangement that occurs only when the CR radical
formed is adjacent to the isoAsp side chain. Similarly, a peak at
m/z = 832 also has the CO2 loss, which could have been formed as
a result of a secondary reaction upon the loss of an acetamide
(C2H5NO).
N-Terminal Acetylation. For experiments conducted on
instruments with limited mass resolving power, such as ETD
analysis done on an ion trap instrument, the peak interference
problem could be circumvented by shifting the diagnostic peak
out of the small molecule and side-chain loss region. This can be
done by, for example, N-terminal acetylation. In this case, the
C-terminal diagnosticfragmentwould be [M + 2H À (C2H5NO+
It should be noted that if the N-terminally acetylated peptide
contains a tryptophan (Trp) residue, the Trp side-chain loss of
C8H6N• (116.05002) would interfere with the isoAsp diagnostic
fragment peak [M + 2H À 116.0348]+• (to correctly identify
such fragments at m/z ∼ 1000, the minimum required resolving
power is 65 000). In addition, no N-terminal isoAsp diagnostic
fragment ion was observed in the N-terminally acetylated pep-
tide, likely because of its small size and lack of a charge carrier.
Therefore, other types of N-terminal elongation could be used
for a more confident identification of the N-terminal isoAsp.
Presumably, the use of larger tags may help to avoid undesired
interference from the side-chain losses and for the detection of
the N-terminal diagnostic ion.
Analysis of the N-Terminal isoAsp by ETD. Peptides with
N-terminal isoAsp were also analyzed using ETD. The N-term-
inal isoAsp diagnostic fragment peak [M + 2H À 74]+• was
observed for all peptides, although in some cases, diagnostic peak
detection was challenging because it exhibited low intensity. In
particular, in ETD (LTQ-Orbitrap) of isoAsp-containing
Aβ1À10 peptide, the peak corresponding to the diagnostic
fragment ion was barely observable above the noise threshold
C2HO2 )]+• = [M + 2H À 116.0348]+• (hereinafter indicated as
•
[M + 2H À 116]+•), and the N-terminal diagnostic fragment
+•
would be C4H7NO3 with m/z of 117.042. The effect of
N-terminal acetylation was examined on the synthetic peptide
Ac-DGVGDVGGVH-NH2 and its isoAsp-variant AcÀiDGVGiD-
VGGVHÀNH2 using ECD. Specific diagnostic fragment peaks
were observed for both isomers (Figure 3, insets): [M + 2H À
116]+• was presentexclusively inthe isoAsp-peptidespectrum, and
the neutral lossof 60 (C2H4O2 = 60.0211) was observed only from
the charge-reduced species of the Asp-peptide variant, indicated as
[M + 2H À 60]+•. The peak at m/z = 894.4195 in the isoAsp-
peptide spectrum (Figure 3, lower panel inset) is not related and
corresponds to the neutral loss of 59 (C2H5NO = 59.0371) from
the acetylated N-terminus. In addition, many of the fragments
containing the Asp residue of the Asp-peptide also exhibit an
accompanyingloss of CO2 (Figure3, upperpanel); however, there
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Table 1. The Effect of Additional Energy Input on the Diagnostic Fragment Formationa
peptide
angiotensin II (iDRVYIHPF)
[M + 2H À 74]+•
973.5259
amyloid β (iDAEFRHiDSGY)
[M + 2H À 74]+•
1123.4807
synthetic (Ac-iDGVGiDVGGVH-NH2)
[M + 2H À 116]+•
signature ion
m/z
837.4218
method
S/N
Rel. Int.
S/N
Rel. Int.
S/N
Rel. Int
ETD Orbitrap no SA
ETD Orbitrap SA, En=5
ETD Orbitrap SA, En=15
ECD solariX
8.2
40.5
0.04
6.6
6.4
0.0022
0.0022
0.0014
0.0095
0.0096
0.0044
0.007
316.3
313.6
293.9
120.5
291.3
98.4
0.0793
0.0803
0.0706
0.0549
0.0524
0.0828
0.0786
0.0818
0.053
0.032
0.029
0.025
0.059
0.057
0.052
32.99
104.6
128.9
145.8
17.7
7.6
35.0
28.0
4.0
hotECD solariX 4.5 V
ETD solariX
ETD amaZon no SD
8.2
160.7
ETD amaZon SD
92.2
17.3
0.0036
162.3
a Relative intensity was calculated as the absolute intensity of the diagnostic fragment divided by the sum of the absolute intensities of all c and z
fragments.
Figure 5. N-terminal isoAsp residue diagnostic fragment peak [M + 2H À 74]+• detection using ETD without (a) or with (b) SA for the modified Ang II
peptide variant (iDRVYIHPF). Both spectra were acquired on Orbitrap. Insets show the S/N ratio of the [M + 2H À 74]+• peak and the appearance of
additional side-chain losses upon SA (His and Tyr).
level, even with the supplemental activation (Figure 4a, inset). In
addition, some low-abundance peaks were barely detectable or
ion activation (via SA, hotECD, and SD) on the diagnostic fragment
peak intensity was investigated for all isoAsp-peptides studied. The
S/N ratios and the relative intensities (Rel. Int.) of the diagnostic
ion peaks are presented in Table 1. At first, the Rel. Int. was
calculated, but it did not reflect the true picture for some peptides
because the absolute intensity of all fragments could change
significantly with ion activation. Absolute S/N ratio seemed to be
a better indicator than relative intensity. For example, with the
Ang II ETD spectrum acquired on the amaZon instrument,
application of SD resulted in a 5-fold increase in the S/N ratio of
•
•
absent in the ETD spectrum, e.g. a5 , and z7 À CO2. ECD
provided a 4.5 times larger S/N ratio for the diagnostic fragment
peak (Figure 4b, inset), possibly owing to the higher energy
available in the ECD process than in the ETD process. Thus, the
effect of additional energy input on diagnostic ion generation was
further explored by means of supplemental activation.
The Effect of Ion Activation. Supplemental activation can
improve fragment detection.39,47,48 Thus, the effect of supplemental
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the diagnostic fragment peak, while the Rel. Int. value did not
change much (Table 1).
ChemistryDepartmentandWarwickCentre for AnalyticalScience
(EPSRC funded EP/F034210/1) are gratefully acknowledged.
A direct comparison of the results acquired on different
instruments is not possible because of the difference in multiple
operating parameters, energetics of the reactions, and the mass
analyzers. When comparing spectra obtained using the same
instrument, the results suggest that supplemental activation gen-
erally leads to higher S/N ratios for diagnostic ions. The effect of
additional energy input on fragmentation depends on the peptide
sequence, particularly on the presence of basic residues and side-
chain interactions. An example is shown on the ETD data acquired
on an Orbitrap instrument with or without SA for the modified
Ang II peptide (Figure 5). Similar to the ETD results acquired
on an amaZon instrument mentioned earlier, the S/N ratio of
the diagnostic ion became 4 times greater when SA was applied
(Figure 5b).
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’ CONCLUSIONS
The N-terminal isoAsp residue was differentiated from Asp
using ECD, hotECD, and ETD. On the basis of the detection
of the specific fragments [M + 2H À 74]+• (isoAsp1), c6 + 57
(isoAsp7), and [M + 2H À 60]+• (Asp1, Asp7) it was possible to
differentiate a mixture of nine peptides containing isoAsp, Asp,
and Asn residues, produced upon partial deamidation of the
Aβ1À10 (N1N7) peptide and separated by a RP-HPLC system.
This result illustrates that HPLC separation followed by ExD
analysis can be a powerful method for isoAsp-containing peptide
mixture differentiation. Although the detection of the N-terminal
diagnostic fragment peak can be challenging, it is possible to
substantially improve its detection by application of supplemen-
tal ion activation. In some cases, the S/N ratio of the signature
fragment ion peak was increased by upto6times. Severalinterfering
peaks may further complicate the analysis when Met or Ile amino
acids are present. This can be overcome by the N-terminal
elongation, with, for example, acetylation, which introduces a mass
shift of the diagnostic peak to the smaller m/z region, where fewer
side-chain losses are available. However, if a Trp residue is present in
the sequence, other types of elongation would be preferred. In
addition, with a larger tag attachedto theN-terminus, anN-terminal
diagnostic peak may become detectable.
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Supporting Information. ECD spectra of chromato-
b
graphic peaks I, IVÀVII from the partially deamidated Aβ1À10
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Corresponding Author
*Phone: +44 024 7615 1008. Fax: +44 7615 1009. E-mail: p.
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’ ACKNOWLEDGMENT
The authors acknowledge Eugene Moskovets and Alexander
Cherkassky for helpful discussions. This work was supported by
the NIH/NCRR-P41 RR10888, NIH/NIGMS-R01GM078293,
and NIH/NCRR-S10 RR025082 grants. The Warwick University
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