Tandem mass spectrometric analysis of a mixture of isobars using the survival yield technique

Article abstract of DOI:10.1007/s13361-011-0195-8

Collision induced dissociation tandem mass spectrometry experiments were performed to unequivocally separate compounds from an isobaric mixture of two products. The Survival Yield curve was obtained and is shown to consist in a linear combination of the curves corresponding to the two components separately. For such a mixture, a plateau appears on the diagram in lieu of the continuous decrease expected allowing for the structural study of the two components separately. The width of the plateau critically relates to the fragmentation parameters of the two molecular ions, which need to be sufficiently different structurally for the plateau to be observed. However, at constant fragmentation parameters, we have observed the width significantly increases at large m/z. This makes the separation more and more efficient as isobars have larger m/z and the technique complementary to those applicable at low m/z only. We have observed that the vertical position of the plateau relates linearly to the relative concentration of the two compounds that may be useful for quantification. Repeatability was estimated at 2% on a quadrupole ion trap. An advantage of using survival yield curves only, is that a priori knowledge of the respective fragmentation patterns of the two isobars becomes unnecessary. Consequently, similar performances are obtained if fragments are isobaric, which is also demonstrated in our study. The critical case of reverse peptides, at low m/z and similar fragmentation parameters, is also presented as a limitation of the method. American Society for Mass Spectrometry, 2011.

Full text of DOI:10.1007/s13361-011-0195-8

B American Society for Mass Spectrometry, 2011
J. Am. Soc. Mass Spectrom. (2011) 22:1744Y1752
DOI: 10.1007/s13361-011-0195-8
RESEARCH ARTICLE
Tandem Mass Spectrometric Analysis of a Mixture
of Isobars Using the Survival Yield Technique
Antony Memboeuf, Laure Jullien, Rémy Lartia, Bernard Brasme, Yves Gimbert
Département de Chimie Moléculaire, UMR 5250-ICMG-FR 2607, CNRS Université Joseph Fourier, BP 53, 38041 Grenoble,
Cedex 9, France
Abstract
Collision induced dissociation tandem mass spectrometry experiments were performed to
unequivocally separate compounds from an isobaric mixture of two products. The Survival Yield
curve was obtained and is shown to consist in a linear combination of the curves corresponding
to the two components separately. For such a mixture, a plateau appears on the diagram in lieu
of the continuous decrease expected allowing for the structural study of the two components
separately. The width of the plateau critically relates to the fragmentation parameters of the two
molecular ions, which need to be sufficiently different structurally for the plateau to be observed.
However, at constant fragmentation parameters, we have observed the width significantly
increases at large m/z. This makes the separation more and more efficient as isobars have
larger m/z and the technique complementary to those applicable at low m/z only. We have
observed that the vertical position of the plateau relates linearly to the relative concentration of
the two compounds that may be useful for quantification. Repeatability was estimated at 2% on
a quadrupole ion trap. An advantage of using survival yield curves only, is that a priori
knowledge of the respective fragmentation patterns of the two isobars becomes unnecessary.
Consequently, similar performances are obtained if fragments are isobaric, which is also
demonstrated in our study. The critical case of reverse peptides, at low m/z and similar
fragmentation parameters, is also presented as a limitation of the method.
Key words: Isobar, Fragmentation, Quantification, Structural analysis, Survival yield technique,
Tandem mass spectrometry, Polyether, Contamination
for mass spectroscopists. The identification and, possibly,
elimination of an isobar becomes essential in order to
Introduction
here is a growing demand on mass spectrometric
techniques for high-throughput analysis, to perform
perform the structural analysis, ultimately to quantify, a
compound of interest. This may require tedious preparative
work, coupling with chromatographic separation techniques
or more recently ion mobility spectrometry, possibly
combined with complex procedures [7, 8]. A simpler and
faster alternative consists of using collision induced disso-
ciation tandem mass spectrometry experiments combined
with the kinetic method. Using this technique, mixtures of
chiral or isomeric peptides may bind to transition metals,
allowing for quantitative analysis [9, 10]. This is, however,
limited to low mass compounds. A number of techniques
utilizing branching ratios of product ions were also devel-
oped [6, 11, 12]. Alternatively, MS/MS spectra obtained at
T
more reliably and provide more information on increasingly
complex samples: the analysis of compounds in biological
samples [1], in the field of petroleomics [2, 3], to study
isomers [4, 5], or simply for separating unresolved peaks due
to technical limitations [6]. With this respect, the identifica-
tion and quantification of isobars remain challenging tasks
Electronic supplementary material The online version of this article
(doi:10.1007/s13361-011-0195-8) contains supplementary material, which
is available to authorized users.
Correspondence to: Antony Memboeuf; e-mail: a.memboeuf@laposte.net
Received: 14 April 2011
Revised: 8 June 2011
Accepted: 12 June 2011
Published online: 19 July 2011

A. Memboeuf, et al.: Tandem MS of Isobars Using SY
1745
“low” and at “high” collision energies may be used to make
this structural analysis. The latter procedures rely on the fact
that fragment ions of the two isobars are formed at different
collision energies according to differences in the structures
of the isobaric components. More precisely, different
fragmentation parameters (activation energy and entropy)
may be associated with the formation of fragment ions due
to differences in the nature of the fragmentation or
dissociation processes. However, when monitoring branching
ratios, one needs to have an a priori knowledge of MS/MS
spectra for both isobars. Moreover, in the case fragment ions
are also isobaric, tandem MS technique does not allow even for
the qualitative identification of the presence of two compo-
nents. In this paper, we present a simple MS/MS based method,
using the survival yield technique, to overcome these issues.
This allows for qualitative identification of the mixture, even
when fragments are also isobars, without a priori knowledge of
fragment ions. MS/MS spectra for two isobaric homopolymers
obtained at relatively large polymerization degree are presented
both separately and in mixtures. The main features observed
are discussed in detail, in particular which structural informa-
tion can be obtained for both components from this procedure.
The possibility for quantitative analysis is presented, as well as
the performance of the method and how it is affected by mass
to charge ratio. Finally, a critical case consisting of two reverse
peptides is also discussed in detail.
reverse 1 was separated from small amount of β-branched
peptide resulting from aspartimide formation using the follow-
ing gradient: 10% of eluent B for 2 min, followed by 10% to
40% in 15 min. Reverse 1 peptide was isolated at 10.94 min and
base-to-base resolved from its main impurity (tR=11.30 min).
Peptide reverse 2 was purified using the following gradient: 0%
of eluent B for 2 min, followed by 0% to 100% in 15 min.
Peptide was isolated at 8.60 min. Fractions were gathered and
lyophilized to afford white powder in ca. 60% yield.
ESI Quadrupole Ion Trap MS/MS Experiments
The experiments were performed on a Bruker Esquire
3000plus ion trap mass spectrometer (Bruker Daltonics,
Bremen, Germany). Samples of synthetic polymers were
dissolved in pure MeOH at 5.10–5 M, LiCl was added at 10–
5
M. Peptides were dissolved in water/acetonitrile 1:1 with
0.1% formic acid to generate protonated signal. The settings
of the instrument were the following: capillary voltage 2 kV;
nitrogen nebulizer pressure 11 psi; nitrogen dry gas flow
5 L/min; dry gas temperature 300°C; He collision gas was
used at an average pressure in the ion trap of 1.25×10–
5
mbar (uncorrected gauge reading). The number of trapped
ions was approximately 10,000 and was kept constant by
using the ion charge control (ICC) option throughout all
measurements. Isolation was performed using a 0.8 Th
window and followed immediately by a 50 ms cooling
period (fragmentation delay). Molecular ions were frag-
mented by applying an excitation voltage during 100 ms
(fragmentation time) over a 10 Th mass to charge window
(fragmentation width). SmartFrag option was turned off and
the 27% default mass cut-off was applied. Recorded mass
spectra were analyzed using Data Analysis 3.3 software
(Bruker Daltonics, Bremen, Germany). During tandem MS
(MS/MS) experiments, 1 min acquisition was performed at
each excitation voltage. For an improved statistic and
repeatability, data are further summed up over the last 30 s
of the acquisition stage in order to generate a MS/MS
spectrum from which peaks intensities can be further
extracted. For accurate quantitative analysis the duration of
acquisition may be increased and data summed up over
1 min or more in order to reduce error bars.
Experimental
Chemicals
Samples of synthetic polymers poly(lactic acid) –PLA, HO-
(C3H4O2)n-H–, and poly(tetramethylene glycols) –PTMEG,
Terathane or PTHF for poly(tetrahydrofuran), HO-(C4H8O)n-
H– both with number-average molecular weight 1000 g/mol
were used. PTMEG was purchased from Sigma-Aldrich
(Schnelldorf, Germany) and PLA was kindly provided by J.
De Winter (Mass Spectrometry Research Group of UMONS,
Mons, Belgium). Peptides with inverted sequences H-
(SDGRGAF)-NH2 (reverse 1) and H-(FAGRGDS)-NH2
(reverse 2) were synthesized in our lab (Nanobio Platform).
They were assembled on an ABI433 peptides synthesizer
following Fmoc strategy. The following side-chain protected
amino acids were used: Ser(tBu); Arg(pbf); and Asp(tBu). Rink
amide resin, loaded to 0.79 mmol/g, was used. Synthesis were Results and Discussions
performed on a 0.1 mmol scale using 10 equivalents amino acids
MS/MS Spectra of Lithium Cationized PLA
dissolved in 2 mL NMP, 9.5 equivalents HBTU and 20
and PTMEG and of Their Mixture
equivalents DIEA. Deprotection was performed by a NMP/
piperidine (4:1) solution and monitored by UV. Final depro-
tection was achieved by shaking resin in 50 mL TFA/H2O/TIS
(95:2.5:2.5) for 2 h. Deprotection solution was evaporated to
dryness and the resulting oily residue was precipitated in Et2O
(40 mL) and centrifuged. White precipitate was washed by Et2O
(3×30 mL). Crude mixture was purified by RP-HPLC on a
Gilson GX-281 apparatus. Eluent A is a 0.1% TFA solution and
eluent B is 0.1% TFA solution in MeCN/H2O (9:1). Peptide
We have performed tandem mass spectrometric analysis of
lithium cationized PLA and PTMEG homopolymers, both
separately and in mixtures. These homopolymers have
different structures but monomers are essentially isobaric.
Our analysis was performed with degree of polymerization
varying from 14 up to 23. The corresponding m/z range goes
approximately from m/z 1000 up to m/z 1700, and has been
selected to avoid overlapping of multiply charged ion

1746
A. Memboeuf, et al.: Tandem MS of Isobars Using SY
distributions, which would complicate the picture, and to
assess the technique on large mass to charge ratio. In
Figure 1, tandem mass spectra were obtained at two
excitation voltages: 0.8 V (spectra d, e, and f) and 1.3 V
(spectra a, b, and c). They were obtained for pure
homopolymers, [PLA23 + Li]+ (spectra a and d) and
[PTMEG23 + Li]+ (spectra c and f), and for a 5:1 mixture
of PLA/PTMEG (spectra b and e). A 5:1 ratio was found
more suitable for illustrative purposes; a detailed analysis of
this aspect is presented in the following section discussing
quantitative information. For convenience, and without loss
of information, these spectra are presented on the same mass
range going from m/z 1350 up to m/z 1700; spectrum for
[PLA23 + Li]+ is flat outside this m/z range and the same
series of peaks for [PTMEG23 + Li]+ extend down to
approximately m/z 1000 from which fragment ions are
minor. Spectra a and c clearly exhibit similarities; indeed,
the same series of peaks regularly spaced by 72 Th from
molecular ions peak is observed, although peak intensities
from [PLA23 + Li]+ tend to decrease at low m/z. One also
notices an additional series present in the MS/MS spectrum
of [PTMEG23 + Li]+ that is actually missing for [PLA23 + Li]+
and is shifted from the aforementioned series by −18 Th (for
detailed discussions of fragmentation mechanisms and MS/MS
spectra interpretations, one may refer to recent publications
[13, 14]). However, in the case of a mixture of PTMEG
samples with PLA, which we may refer to as contamination
throughout the discussion, visual inspection is not sufficient to
distinguish between fragment ions (spectrum b in Figure 1).
Clearly, in spite of their structural differences, tandem mass
spectra of this mixture of homopolymers do not allow even for
a qualitative identification of contamination. At this point, it is
important to note the large difference in the degree of
fragmentation achieved between the two homopolymers at
the same excitation voltage: 0.8 V almost the entire population
of [PLA23 + Li]+ molecular ions have already fragmented
(spectrum d), when, on the contrary, tandem mass spectrum f
of [PTMEG23 + Li]+ does not exhibit a single fragment ion.
This striking difference suggests the fragmentation parameters
(activation energy and entropy) are significantly different and
that these homopolymers may be separated according to this
criterion.
as a function of the excitation voltage: this leads to the SY
curve. This technique has already been used in fundamental
studies [15–18], but also to discriminate molecular ions
according to their structures [19–21]. When doing so in the
same experimental conditions for both homopolymers
separately and for a mixture of 5:1 (PLA/PTMEG), one
obtains the curves displayed in Figure 2. In the case of a
pure homopolymer, the SY curve has the shape of a sigmoid
starting from 1 at low excitation voltages (no fragment ions
observed) decreasing continuously to 0 at higher excitation
voltages (no molecular ions remain). An important point is
that at the same molecular ion mass, the SY curve obtained
for lithium cationized PTMEG is shifted to much higher
excitation voltages compared with the one obtained for PLA.
This is in agreement with our recent observation that
polyethers do fragment at significantly higher excitation
voltages compared with polyesters [19]. On the contrary, the
SY curve obtained for a mixture of the two samples exhibits
a plateau in place of the continuous decrease with the
excitation voltage. After the [PLA23 + Li]+ component has
fully fragmented to form fragment ions (at approximately
0.8 V according to the curve obtained for pure [PLA23 + Li]+),
only lithium cationized PTMEG molecular ions remain and do
not fragment until a 1 V excitation voltage is applied.
Consequently, from 0.8 up to 1 V, the excitation process does
not lead to the formation of additional fragments, and the ratio
expressed in Eq. (1) remains constant on that range of
excitation voltages. Starting from 1 V, the SY ratio starts to
decrease again as [PTMEG23 + Li]+ is fragmenting, to finally
reach 0 value at high excitation voltages (complete fragmenta-
tion of both cationized homopolymers).
Following this reasoning, three regimes can be identified:
a “low excitation regime” with spectra obtained at excitation
voltages lower than 0.8 V, an “intermediate excitation
regime” that consists in a plateau (from 0.8 up to 1 V)
and, a “high excitation regime” corresponding to the second
decrease of the SY curve from 1 up to 1.4 V. Combining this
nomenclature with previous qualitative arguments, the
content of structural information of tandem MS spectra
obtained at each regime may now be discussed in more
detail. Fragment ions that are formed at the low and at the
intermediate excitation regimes are obviously produced by
the first component only, that is [PLA23 + Li]+. Hence, MS/
MS spectra are characteristic of this isobar only and, by
doing mass-selection of fragment ions obtained at voltages
lower than 1 V, multistage mass spectrometry can be
performed on this component only, avoiding isobaric
contamination and ambiguous structural analysis. Comple-
mentarily, molecular ions surviving the excitation process at
the intermediate and at the high excitation regimes are
clearly of the second type, that is [PTMEG23 + Li]+. This is
obviously the case as the entire population of molecular ions of
the first component has been discarded from the MS/MS
spectrum through fragmentation. Similarly, a second isolation
stage of molecular ions may then be performed to study the
structure of the second component as if there were no isobaric
Structural Analysis of Isobaric Compounds
in a Mixture Using the Survival Yield Technique
The survival yield technique uses survival yield (SY) ratio,
which is a convenient quantitative measure of the fragmentation
degree that is defined according to:
IM
P
SY ¼
ð1Þ
IM
þ
IF
where IF stands for the intensities of fragment ions peaks and
IM for the molecular ions peak intensity. Using this
definition, one may record and plot the value of this ratio

A. Memboeuf, et al.: Tandem MS of Isobars Using SY
1747
Figure 1. MS/MS spectra for [PLA23 + Li]+ (spectra a and d), [PTMEG23 + Li]+ (spectra c and f) and a 5:1 mixture (spectra b and e),
obtained at 0.8 V (spectra d, e, and f) and 1.3 V (spectra a, b, and c) excitation voltages
Figure 2. Survival Yield curves for [PLA23 + Li]+ (filled squares with dotted line) and [PTMEG23 + Li]+ (filled triangles with dotted
line) and a 5:1 mixture of [PLA23 + Li]+/ [PTMEG23 + Li]+ (filled stars with dot dashed line)

1748
A. Memboeuf, et al.: Tandem MS of Isobars Using SY
contaminant. Clearly, using this technique, separation and
structural analysis of isobars can be performed using multistage
mass spectrometric experiments only. Results are clearly
unequivocal and do not depend on the a priori knowledge of
the SY curves of pure analytes. However, the presented
strategies critically rely on the identification of a plateau that
enables clearly distinguishing between excitation regimes.
ratio can be linearly related with the ratio of the two
components (inset of Figure 3) with very good approximation.
The correlation coefficient R2 was found to be 0.983 if the
curve was enforced to go through (0; 1) and R2=0.992 with a
standard deviation of 0.041 when the intercept was allowed to
vary (in such case the curve goes through y=0.94 in place of 1).
The value of the intercept corresponds to the position of the SY
curve in the absence of the first contaminant (that is for a
sample of pure PTMEG), it is thus a good sign as it goes
smoothly through this value. The same linearity would be
obtained by plotting the SY ratio as a function of PTMEG/PLA
ratio (in place of PLA/PTMEG), since the position of the
plateau (brutto value) corresponds to the amount of the second
component (PTMEG) relative to the amount of the first
component (PLA). One may also note there is not a one to
one correspondence between the SY ratio at the plateau and the
PLA/PTMEG quantities introduced in the mass spectrometer.
For example, when a 1:1 mixture of PLA/PTMEG was
analyzed, the SY ratio determined was close to 0.92. The
slope of the linear fit with intercept (0; 1) is 0.164, when one
would appreciate to have unity for straight quantitative
analysis. Obviously differences in the lithium cation affinity,
in the ionization or isolation efficiency or in the excitation
efficiency (theoretically, there is 0.2 Th difference between
these two isobars) may be responsible for this discrepancy.
Moreover, controlling the initial concentration for single
oligomers is not feasible as the concentration reflects only the
number-average molecular weight of the polymer mass
distribution not the one of a single oligomer. In spite of all
these complications, we still observe a linear tendency that
is very encouraging, and allows for using this methodology
Quantification of Contaminant and Tandem Mass
Spectra Correction
An important feature is that at the intermediate regime, one
simultaneously observes fragment ions of the first component
and molecular ions of the second. Therefore, after an excitation
voltage corresponding to this regime has been applied, between
0.8 and 1 V in our case, the two isobars are completely
separated. By reckoning the ratio of molecular ions peak
intensity over the sum of fragment ions peaks intensities (more
rigorously, the value of the SY ratio normalized to its
maximum value, which is simply unity), one would thus
expect the relative proportion of the two components to be
unambiguously determined. To assess this anticipation, mix-
tures of PLA/PTMEG with ratio 1:1, 3:1, 5:1, 7:1, 8:1, 9:1, and
11:1 were prepared, and the corresponding SY curves
measured to be compared with SY curves for pure oligomers
(ratio 1:0 and 0:1). Results are presented in Figure 3. Clearly,
increasing the quantity of PLA relative to PTMEG leads to a
shift of the plateau to lower values of the SY ratio, which
feature does agree with our previous understanding of the SY
curve behavior. Furthermore, this qualitative finding can be
supplemented by quantitative estimations: the inverse of SY
Figure 3. SY curves for PLA/PTMEG mixtures at relative concentrations 0:1, 1:1, 5:1, 8:1, and 1:0, in the same experimental
conditions. The inset shows the correlation between the inverse of the SY ratio at the plateau (R) and the ratio PLA/PTMEG
from sample preparation 0:1, 1:1, 5:1, 7:1, 8:1, 9:1, and 11:1

A. Memboeuf, et al.: Tandem MS of Isobars Using SY
1749
for quantitative purposes using, e.g., the standard addition
method or a priori calibration. In order for these observa-
tions to be more practical, one may use the following
strategy: at first, the compound of interest must be studied
by means of drawing SY curve for the existence of a
plateau to be evidenced. This demonstrates the isobaric
contamination. Then, MS/MS spectra must be acquired at a
single voltage corresponding to the intermediate regime (at
the plateau) for different concentrations of the analyte or of
the contaminant. The monitoring of the SY ratio at this
unique excitation voltage at different concentrations, allows
for drawing a titration curve as presented in the inset of
Figure 3. This way, the drawing of multiple SY curves,
which is rather time-consuming, is avoided and quantification
can be quickly obtained following an experimental sequence
similar to the standard addition method.
For quantitative purposes, it is important to have an
evaluation of the performance of the proposed methodology.
To this end, we have performed stability as well as a
repeatability analysis of tandem MS spectra acquired for the
mixture at an excitation voltage of 0.9 V (at the middle of
the plateau). The same spectrum has been recorded
separately 10 times, successively, in the same experimental
conditions and following the same methodology (acquisition
and data treatment methods) on three different days. From
the combination of these stability and repeatability analyses,
we obtained at most 2% deviation from the average value.
Using this quantitative estimate, one may try recovering
“pure” MS/MS spectra for both homopolymers at any
excitation voltages using information obtained at the
intermediate excitation regime. We have observed that by
applying simple spectral subtraction using Eqs. S-1 or S-2
according to the excitation regime (see Supplementary
Material), corrected SY curves compare very well to SY
curves for pure analytes. These results must be combined
with the fact that the position of the plateau scales linearly
with the relative quantities of the two components (see
above). By doing so, SY curve of the mixture can be
considered as a linear combination of the curves of the two
analytes along the y-axis, while it is not sensitive to
contamination along the x-axis. Simply, the nature and
quantity of molecular ions do not affect the fragmentation
energetic behavior of the other isobaric compounds and, at
constant experimental conditions, the shape and position of
the SY curve characterize the compound. However, details
of MS/MS spectra, more precisely peaks relative intensities,
are significantly different. The latter results demonstrate that
consecutive fragmentations constitute an important part of
the fragmentation processes in our experimental conditions.
Details of this part of the study, containing graphs and
formulas, are presented in the Supplementary Material.
associated with the fragmentation processes observed should
have sufficiently different values to observe a plateau
(intermediate regime). We have recently demonstrated [15]
the collision energy necessary to achieve a certain degree of
fragmentation increases linearly with the mass to a very good
approximation (R290.996). This analysis was performed on
another polyether, namely poly(ethylene glycols), and tested
on a wide variety of conditions (different instruments, data
statistical treatment, experimental conditions …). Moreover,
when structurally different compounds are studied, the same
linearity is observed but with different rate of increase [19].
Combining these two observations suggests the shift of the SY
curves observed for isobars would increase with their mass
providing the fragmentation parameters remain independent of
the molecular ion mass. Consequently, the width of the
intermediate regime observed in Figure 2 must increase with
masses of the isobars due to molecular ions masses (as a
consequence of the degrees of freedom effect and invariability
of fragmentation parameters [15, 22]). To assess this predic-
tion, we have reproduced the SY curves as presented in
Figure 2 for lithium cationized PLA and PTMEG with
polymerization degree varying from 14 up to 23 (from
approximately m/z 1000 up to m/z 1700) under the same
experimental conditions (see Figure 4). The widths of the
plateaus, which may be called the isobaric resolving power,
have been estimated using SY curves for pure homopolymers,
considering voltages necessary to achieve more than 99%
fragmentation of [PLA23 + Li]+ and less than 1% fragmentation
of [PTMEG23 + Li]+. Error bars were overestimated, consid-
ering 0.02 V uncertainties, when determining the latter
voltages. The linear fit is in agreement with our expectation
and demonstrates that the resolving power of the technique can
increase significantly with molecular ion mass (the width is
doubled between m/z 1000 and m/z 1700). Considering the
fragmentation parameters remain constant with respect to the
polymerization degree, this technique allows for an efficient
Figure 4. Width of the plateau (intermediate regime) as a
function of molecular ions mass for a mixture of [PLA23 + Li]+/
[PTMEG23 + Li]+. Details on calculations and estimation of
error bars are presented in the text
Size Effect on the Separation Power
The technique presented requires that isobars fragment at
different excitation voltages. In other terms, parameters

1750
A. Memboeuf, et al.: Tandem MS of Isobars Using SY
separation of isobars and may enable refined structural
characterization according to the fragmentation parameters at
large m/z. If the linearity observed can be explained as a
consequence of the degrees of freedom effect, the negative
intercept suggests that PLA/PTMEG mixtures with masses
below m/z 500 cannot be resolved. The negative values are
associated with the fact that [PTMEGn + Li]+ would simply start
fragmenting before [PLAn + Li]+ has fully decomposed.
Obviously, experimental conditions may modify these features;
the mass-limit separation may be shifted to higher or lower
masses, and the resolving power increased at the same molecular
ion mass.
shape that is expected for pure analytes. For an unknown
mixture of isobars with a similar behavior, visual inspection
of tandem mass spectra does not help identifying the
contamination; neither does the analysis of spectra at low
and at high excitation voltages. MS/MS spectra are
contaminated at all excitation voltages and, consequently,
do not provide structural information on one compound only
but rather give mixtures of fragments from both isobars. In
the absence of such a plateau, identifying contamination,
even qualitatively only, is not possible. This also illustrates
the limit of the conventional comparison between tandem
mass spectra obtained at high and at low energies for
structural investigation of isobars. In this respect it is,
however, worth noting that the curve for the mixture is
located in a position that is intermediate to the two curves
obtained for pure compounds. Modification of the SY curve
shape or location seems to be a possible qualitative indicator
of isobaric contamination, but this requires a priori knowl-
edge of the curve for at least one of the two pure
components. Indeed, at constant experimental conditions,
the SY curve for a given analyte has a shape and position
that depend on the molecular mass as well as on the
parameters associated to the fragmentation processes
observed. These processes in turn depend on the details of
the structure of the analyte, making the SY curve a kind of
fingerprint of the compound. Thus, any deviation from the
determined curve, in the same experimental conditions,
demonstrates the presence of an isobaric contaminant.
Limits of the Method
For a mixture of compounds with very similar fragmentation
parameters and low m/z, one would expect no intermediate
regime and a SY curve with a modified shape but without
any plateau. Trying to satisfy those two conditions, we have
studied two protonated peptides with inverted sequences, H-
(SDGRGAF)-NH2 (reverse 1) and H-(FAGRGDS)-NH2
(reverse 2), separately and in a 1:1 mixture. Due to little
structural differences, fragmentation parameters have a good
chance to be also very close. Moreover, those peptides are
exact isobars with m/z 708.3, which is very close to the
resolving limit identified for PLA/PTMEG mixtures (for
which, in addition, fragmentation parameters may be
significantly more different). Results are presented in
Figure 5. As the first component has just started to fragment,
the second component is seemingly fragmenting as well.
Thus, concerning the equimolar mixture, the SY ratio as a
function of excitation voltage consists of a curve with a
continuous and smooth decrease but without any plateau, a
With the SY curves of the two components at hands, we
have tried to estimate the degree of contamination using a
simple weighted combination. This way we could easily
achieve at most 5% deviation from measured values. Such
deviations, however, do not compare favorably to the shift
Figure 5. Survival Yield curves for two protonated peptides with inverted sequences at m/z 708.3: H-(SDGRGAF)-NH2 (reverse
1) and H-(FAGRGDS)-NH2 (reverse 2). SY curve for a 1:1 mixture is also presented

A. Memboeuf, et al.: Tandem MS of Isobars Using SY
1751
of SY curves as they represent 10% to 40% of the
corresponding distances between SY curves for pure proto-
nated peptides. Although not very accurate at this stage, the
method seems nonetheless promising for quantitative eval-
uation of isobars even in the absence of a plateau. The
combination of low m/z and small differences in the
fragmentation parameters leads to a negative value for the
resolving power and thus, necessitates suspicion of isobaric
contamination and a priori knowledge of the structure of one
of the two components to unequivocally decide on the
contamination.
Survival Yield technique only, without necessarily using
complex and tedious sample preparation or cleaning.
Acknowledgments
Département de Chimie Moléculaire (DCM), Université
Joseph Fourier is gratefully acknowledged for financial
support. The authors thank Julien De Winter (UMONS,
Mass Spectrometry Research Group, Mons, Belgium) for
kindly providing them with samples of poly(lactic acid), and
Rodolphe Guéret for technical assistance. A.M. also thanks
Professor K. Vekey and Dr. L. Drahos for support and useful
suggestions and Professor J. C. Tabet for careful reading of
the manuscript.
Conclusions
References
Results presented in this work show the Survival Yield
curve of an isobaric mixture consists in a linear combination
of survival yield curves for the two compounds separately.
Moreover, the survival yield curve for a mixture was shown
to exhibit a plateau. Combining the latter two observations
led to the following conclusions: (1) the existence of a
plateau demonstrates that the sample is made up of a mixture
of compounds and allows for studying the structure of the
two components separately and unambiguously, and (2) the
vertical position of the plateau can be used for quantifying
the two components of the mixture, and there is no need for
a priori knowledge of the respective fragmentation patterns.
These results are, however, limited to compounds with
sufficiently different fragmentation parameters, where “suf-
ficiently” is not well-defined and is a priori not restrictive, as
it may partly be compensated by the size effect. This is well-
demonstrated by the increase in the width of the plateau as
m/z becomes larger.
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