Determination of isotopic labeling of proteins by precursor ion scanning liquid chromatography/tandem mass spectrometry of derivatized amino acids applied to nuclear magnetic resonance studies

Article abstract of DOI:10.1002/rcm.6204

RATIONALE A method has been developed for the quantitation of isotopic labeling of proteins using liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the application of protein nuclear magnetic resonance (NMR) studies. NMR relies on specific isotopic nuclei, such as ¹³C and ¹⁵N, for detection and, therefore, isotopic labeling is an important sample preparation step prior to in-depth structural characterization of proteins. The goal of this study was to develop a robust quantitative assay for assessing isotopic labeling in proteins while retaining information on the extent of labeling for individual amino acids. METHODS Complete digestion of proteins by acid hydrolysis was followed by derivatization of free amino acids with 6-aminoquinolyl N-hydroxysuccinimidyl carbamate (AQC) forming derivatives having identical MS/MS fragmentation behavior. Precursor ion scanning on a hybrid quadrupole-linear ion trap platform was used for amino acid analysis and determining isotopic labeling of proteins. RESULTS Using a set of isotope-labeled amino acid standards mixed with their unlabeled counterparts, the method was validated for accurately measuring % isotopic contribution. We then applied the method for determining the ¹³C isotopic content of algal proteins during a feeding study using ¹³C₆-glucose- or ¹³C-bicarbonate-supplemented culture media as well as the level of labeling in mussel byssal threads obtained after feeding with labeled algae. CONCLUSIONS This method is ideally suited for assessing the extent of protein labeling prior to NMR studies, where the isotopic labeling is a determining factor in the quality of resulting protein spectra, and can be applied to a multitude of different biological samples. Copyright

Full text of DOI:10.1002/rcm.6204

Research Article
Received: 11 September 2011
Revised: 12 January 2012
Accepted: 16 February 2012
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2012, 26, 1165–1174
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6204
Determination of isotopic labeling of proteins by precursor ion
scanning liquid chromatography/tandem mass spectrometry of
derivatized amino acids applied to nuclear magnetic resonance
studies
André LeBlanc1, Alexandre A. Arnold1, Bertrand Genard2, Jean-Bruno Nadalini2,
Marc-Olivier Séguin Heine1, Isabelle Marcotte1, Réjean Tremblay2 and Lekha Sleno1
*
1Université du Québec à Montréal, Chemistry Department, Montréal, QC, Canada
2Université du Québec à Rimouski, Institut des sciences de la mer de Rimouski (ISMER), Rimouski, QC, Canada
RATIONALE: A method has been developed for the quantitation of isotopic labeling of proteins using liquid
chromatography/tandem mass spectrometry (LC/MS/MS) for the application of protein nuclear magnetic resonance
(NMR) studies. NMR relies on specific isotopic nuclei, such as 13C and 15N, for detection and, therefore, isotopic labeling
is an important sample preparation step prior to in-depth structural characterization of proteins. The goal of this study
was to develop a robust quantitative assay for assessing isotopic labeling in proteins while retaining information on the
extent of labeling for individual amino acids.
METHODS: Complete digestion of proteins by acid hydrolysis was followed by derivatization of free amino acids with
6-aminoquinolyl N-hydroxysuccinimidyl carbamate (AQC) forming derivatives having identical MS/MS fragmentation
behavior. Precursor ion scanning on a hybrid quadrupole-linear ion trap platform was used for amino acid analysis and
determining isotopic labeling of proteins.
RESULTS: Using a set of isotope-labeled amino acid standards mixed with their unlabeled counterparts, the
method was validated for accurately measuring % isotopic contribution. We then applied the method for deter-
mining the 13C isotopic content of algal proteins during a feeding study using 13C6-glucose- or 13C-bicarbonate-
supplemented culture media as well as the level of labeling in mussel byssal threads obtained after feeding with
labeled algae.
CONCLUSIONS: This method is ideally suited for assessing the extent of protein labeling prior to NMR studies, where
the isotopic labeling is a determining factor in the quality of resulting protein spectra, and can be applied to a multitude
of different biological samples. Copyright © 2012 John Wiley & Sons, Ltd.
Protein fibers such as silk, collagen or mussel byssal threads
are complex biopolymers which have unique mechanical
properties exquisitely tuned to the tasks that they perform.[1,2]
These macroscopic properties are a consequence of the
structure of these proteins at a molecular level. It is thus
desirable to unravel their structure in order to produce bio-
inspired synthetic materials, which can be achieved using
solid-state nuclear magnetic resonance (SS-NMR). The major
drawback of NMR is its lack of sensitivity, a weakness which
can be offset by isotopic labeling. This strategy is routinely
used in the study of large soluble proteins and becomes a
compulsory step in the study of insoluble proteins such as silk
or mussel byssal threads by high-resolution SS-NMR of carbon
or nitrogen nuclei. The natural abundances of the NMR-active
isotopes of these nuclei (13C and 15N) are only 1.1 and 0.37%,
respectively, thus leading to very weak NMR signals. Isotopic
enrichment not only directly increases the signal-to-noise ratio
of the NMR spectrum; it has also a much more marked effect
on the number of NMR acquisitions needed for a similar
quality spectrum. A 10-fold increase in isotopic enrichment,
for example, is thus equivalent to a 100-fold increase in the
number of averaged acquisitions. This reduction in acquisition
time becomes a critical step when performing the two-
dimensional NMR experiments required for the structural
study of proteins. For these types of experiments, a 10-fold
increase in isotopic labeling will reduce the total experi-
mental time to a couple of days and the experiments thus
become feasible.
In the case of silks or byssal threads, animals are fed a
labeled diet to incorporate stable isotopes into the protein
fibers. In order to optimize the fiber labeling, the efficiency
and kinetics of the transfer between labeled food to the
secreted fibers must first be determined. Experimentally,
these two parameters (efficiency and kinetics) appear to
* Correspondence to: L. Sleno, Laboratory for Bioanalytical
Mass Spectrometry, Chemistry Department, Université du
Québec à Montréal, C.P. 8888 Succ. Centre-Ville, Montréal,
Québec, Canada, H3C 3P8.
E-mail: sleno.lekha@uqam.ca
Rapid Commun. Mass Spectrom. 2012, 26, 1165–1174
Copyright © 2012 John Wiley & Sons, Ltd.

A. LeBlanc et al.
vary with the organism species, type of food and environ-
mental conditions.[3,4] We have seen that, in the case of
mussels, the most efficient and less costly strategy consists
in producing labeled microalgae which will subsequently
be fed to the mussels (unpublished data). Consequently,
a method for determining the isotopic labeling of total
protein mass is required. To be useful for protein NMR
studies, the method should be amenable to the
measurement of 13C or 15N enrichment and allow labeling
levels to be distinguished between different amino acids as
well as be able to discriminate between partial and total
labeling. Indeed, partially labeled amino acids can yield
ambiguous NMR patterns which can lead to wrong
assignments and thus render an accurate NMR analysis
impossible.
EXPERIMENTAL
Materials
Alanine, arginine, aspartic acid, asparagine, glutamic acid,
cysteine, glutamine, glycine, histidine, isoleucine, lysine (HCl
salt), methionine, phenylalanine, proline, serine, threonine, tryp-
tophan, tyrosine and valine, 13C915N-tyrosine, 13C6(ring-labeled)-
phenylalanine, 13C515N-methionine, 13C515N2-glutamine, glucose,
13C6-glucose, di(N-succinimidyl) carbonate and formic acid
(ultrapure) were all obtained from Sigma-Aldrich (Oakville,
ON, Canada). Sodium [13C]-bicarbonate was purchased from
Cambridge Isotope Laboratories (Cambridge, MA, USA).
Concentrated hydrochloric acid was supplied by Fisher
Scientific (Ottawa, ON, Canada). Sodium tetraborate decahy-
drate and 6-aminoquinoline were purchased from Alfa Aesar
(Ward Hill, MA, USA). HPLC grade methanol and acetonitrile
were obtained from Caledon Laboratories Ltd. (Georgetown,
ON, Canada). Ultrapure water was supplied by a Synergy
purification system (Millipore, Billerica, Massachusetts, USA).
Several techniques exist that can measure isotopic enrich-
ment of molecules, including isotope ratio mass spectrometry
(IRMS), gas chromatography/mass spectrometry (GC/MS)
and, most recently, liquid chromatography/mass spectrometry
(LC/MS). The choice of analytical method for measuring
isotope enrichment is highly dependent on the needs of the
experiment. In this study, in order to obtain an accurate
estimate of isotopic labeling of amino acids from algal and
byssal protein extracts, the main priorities for the analytical
method were speed, cost effectiveness and the flexibility to
determine either 13C or 15N enrichment. IRMS experiments
yield very accurate results for isotope measurements; however,
they do not allow any structural information to be extracted
from the data since all molecules are transformed into
combustion products prior to entering the ion source, also
necessitating highly specialized instrumentation,[5] and,
furthermore, would not be easily amenable for simultaneous
determination of 15N enrichment. GC/MS is a common techni-
que used for isotope analysis and has often been applied to the
study of amino acid isotopic enrichment.[6–9] Most GC/MS
analyses use time-consuming derivatization steps in order to
transform analytes into volatile species and resulting electron
ionization (EI) spectra are often very difficult to interpret when
many isotopomers are present at once.[10] LC/MS for isotope
analysis of amino acids has previously been reported,
demonstrating the power and applicability of this
technique.[11–14] LC/MS is also amenable to the evaluation of
the extent of labeling within a given amino acid.[15] Previous
methods have relied on full scan or single ion monitoring
detection. In this study, we have developed an LC/MS/
MS-based method for determining isotopic labeling of proteins
following acid hydrolysis and rapid amino acid derivatization.
A related method has been described by Hess et al.[4] for the
determination of isotopic enrichment in silk proteins by
derivatization of hydrolyzed amino acids with Na-(2,4-
dinitro-5-fluorophenyl)-L-alaninamide (FDAA) and single
quadrupole MS analysis, with results being shown for four
amino acids (Ala, Gly, Pro, Glu). The present study, using a
selective precursor ion scan for detecting 6-aminoquinolyl
N-hydroxysuccinimidyl carbamate (AQC)-derivatized amino
acids, presents data from a majority of protein amino
acids and is shown for determining isotopic enrichment
of both algal proteins and mussel byssus thread samples.
The method allows the evaluation of feeding protocols of
both the microalgae and mussels, for further structural
investigation of suitably labeled mussel byssal threads
by NMR.
Production of 13C-labeled microalgae
Glucose diet
Enrichment of microalgae with isotope-labeled glucose was
performed using Amphora sp. obtained from the Centre for
Culture of Marine phytoplankton (CCMP) at Bigelow
Laboratory for Ocean Sciences (West Boothbay Harbor, ME,
USA). Algae were cultured in seawater enriched with f/2
medium[16] at pH 8 and temperature 20.6ꢀ 0.4ꢁC, under
lighting supplied by fluorescent grow lights (intensity of
191.6 ꢀ 9.9 mE/m2s). Seawater was filtered and sterilized prior
to use. A 10 mL volume of the microalgae solution was used
to inoculate a 250 mL Erlenmeyer flask filled with f/2 culture
media supplemented with 5 g/L glucose (50% mixture of
unlabeled and 13C6 labeled glucose). Triplicate cultures were
maintained for 19 days and cell concentrations were measured
each day with a Z2 Coulter counter fitted with a 70-mm orifice
tube (Beckman Coulter Canada, Mississauga, ON, Canada).
Stationary phase growth was achieved at day 19 and the triplicate
cultures had a mean concentration of 1.38 ꢀ 0.24 ꢂ 106 cells/mL.
Sample aliquots were removed every 2 days from day 1 to
day 19, lyophilized and stored (at 4ꢁC) prior to analysis.
Sodium bicarbonate diet
Microalgae (Nannochloropsisocculata) were batch-cultured at
20ꢁC and under continuous illumination during 10 days in
f/2 seawater medium[16] supplemented with 25 mM sodium
[13C]-bicarbonate. f/2 medium was transferred into four glass
bottles (250 mL) then autoclaved with specific seawater setting
to avoid salt precipitation. After the addition of labeled sodium
bicarbonate, the culture system was sealed and the medium
was purged with nitrogen gas to eliminate atmospheric CO2.
The culture system was built to ensure the elimination of
oxygen produced by algae and avoid gas exchange with the
ambient atmosphere. Culture bottles were inoculated with
10 mL of a growing culture (ꢀ20 cells/mL) using sterile
syringes. Sample aliquots were taken after the 10-day culture
period, lyophilized and stored (at 4ꢁC) prior to analysis.
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Amino acid isotope labeling analysis by LC/MS/MS
Production of 13C-labeled mussel byssus
were derivatized in a rapid reaction with 6-aminoquinolyl
N-hydroxy-succinimidylcarbamate (AQC) (see Fig. 1). AQC
was synthesized according to the literature[18] in a one-step
procedure combining di(N-succinimidyl) carbonate and
6-aminoquinoline. Amino acids were derivatized by adding
10 mL of a saturated solution of AQC (approximately 3 mg/mL)
dissolved in anhydrous acetonitrile to a mixture of 20 mL amino
acid standard or protein hydrolysate and 70 mL of borate buffer
(25 mM borate, pH 8.8).
Mytilus edulis were fed with 13C-labeled Nannochloropsisocculata
microalgae (from bicarbonate-labeling experiment) for 2 days
in a 40 L tank filled with UV-treated oxygenated seawater.
Water temperature was maintained at 18ꢁC with a salinity ratio
of 26. Every 12 h, 250 mL of 13C-labeled microalgae culture was
added to the seawater. Subsequently, mussels were placed on a
vertical polyvinyl chloride (PVC) rod using cyanoacrylate glue
suspended 10 mm above a PVC plate for byssus production.
Mussels were submerged in an experimental flume[17] and
submitted to a current velocity of 12 cm/s to stimulate byssus
production. Byssi were sampled every second day for analysis.
All samples (1 mL injections) were separated on a
Zorbax Eclipse Plus C18 column (2.1 ꢂ100 mm, 1.8 mm)
using a Shimadzu Prominence HPLC system (including
two LC20AD-XR pumps with in-line degasser, a SIL20AC
autosampler, and a CTO20A column compartment) with
the following gradient: 5% B with a 2 min hold, up to
70% B at 15 min, 95% B at 15.5 min held until 20 min
before re-equilibration, where mobile phases A and B were
water and methanol (both containing 0.1% formic acid),
respectively, at a flow rate of 0.2 mL/min and a column
temperature of 50ꢁC. Mass spectrometric analysis was per-
formed on a QTRAP 5500 hybrid triple quadrupole-linear
ion trap (AB Sciex, Concord, ON, Canada) in positive ion
mode. The TurboIonSprayW source was operated with an
ionspray voltage of 5000 V at 450ꢁC. The curtain gas was
maintained at 35 psi and GS1 and GS2 gases were held
at 55 and 60 psi, respectively. The declustering potential
(DP) and exit potential (EP) were set to 120 and 10 V,
respectively. A precursor ion scan of the fragment ion at
m/z 171.1 was performed with collision energy (CE) of
30V and at a scan rate of 200 Da/s with a 0.1 Da step size
between m/z 235 and 500. The collision cell exit potential
(CXP) was maintained at 13 V. The offset values in
the resolution tables for both quadrupoles were adjusted
to obtain baseline resolution between consecutive 13C
and/or 15N isotope peaks, reflecting slightly higher than
unit resolution. All raw data was processed with AnalystW
software (version 1.5.1) and MultiQuant™ (version 2.0.2)
software for signal integration.
Protein hydrolysis
Lyophilized microalgae (1–1.5 mg) or mussel byssal thread
(0.4–0.6 mg) samples were accurately weighed and dissolved
in 1.0 mL of 6 M HCl. The samples were hydrolyzed for 24 h
at 110ꢁC, using a VWR Digital Heatblock (VWR International,
Mississauga, ON, Canada). Sample tubes were opened and
the acid was evaporated, rinsed with 1 mL water, and further
evaporated using a SpeedVac concentrator (Fisher Scientific,
Ottawa, ON, Canada) before reconstituting the sample in a
final volume of 500 mL of water.
Standard preparation
Standard mixtures of 0, 1, 2, 3, 4, 5, 10, 20, 40, 50, 60, 80, 90, 95,
96, 97, 98, 99 and 100% (labeled/unlabeled concentrations) of
glutamine, methionine, phenylalanine and tyrosine were
prepared at a total (labeled + unlabeled) concentration of
1 mM for each amino acid in water.
Derivatization and LC/MS analysis
The reconstituted algal and byssal hydrolysates were centri-
fuged at 13000 rpm for 30 s to sediment particulates prior to
derivatization and diluted 10-fold in water. Amino acids
Figure 1. AQC derivatization reaction of amino acids (a) and example of extracted precur-
sor ions in the case of a 13C-labeling experiment for monitoring alanine (b).
Rapid Commun. Mass Spectrom. 2012, 26, 1165–1174
Copyright © 2012 John Wiley & Sons, Ltd.
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A. LeBlanc et al.
Data analysis
precursor ion scan selectively detects AQC-derivatized
free amine-containing molecules. Furthermore, the AQC-
derivatization reaction is a one-step process that can be
performed directly in the injection vial and is complete
within a few minutes. In short, AQC derivatization is ideal
for isotopomer analysis of amino acids since it is a very fast
and easy reaction that allows for separation of amino acids
by reversed-phase HPLC and the use of a selective and
sensitive precursor ion scanning method that does not
include any isotope contribution from the derivatizing agent.
The remaining concern in the development of a method
for accurate and reproducible measurement of amino acid
isotope enrichment was cross-isotope spectral interference.
Quadrupoles are most commonly operated at unit resolution
which does not provide baseline separation of spectral peaks
separated by a difference of 1 Da. For our method, both filtering
quadrupoles (Q1 and Q3) were tuned for increased resolution in
order to obtain baseline separation of neighboring isotopomers
separated by 1 Da, as well as to assure that there would be no
isotope pattern skewing due to a contribution from non-
monoisotopic (AQC) fragment ions. Setting the instrument
parameters for increased resolution in both quadrupoles does
decrease its sensitivity; however, this is generally not a problem
for these types of applications since, typically, a few milligrams
of dried protein extract are easily available which represents a
large excess considering the sensitivity of modern day mass
spectrometry. To further insure that there would be no interfer-
ence when calculating the relative areas of isotope peaks of
each amino acid, the mass window used for each EIC was care-
fully chosen to avoid any contribution from neighboring
isotopomers. By choosing mass windows of ꢀ0.4 m/z relative
to the theoretical m/z value for the isotopomer of interest, we
cover nearly the entire spectral width of each peak while
avoiding any contribution from its neighbors.
Extracted ion chromatograms (EICs) were integrated from pre-
cursor ion scan data using a mass selection window of ꢀ0.4 m/z
relative to the theoretical m/z value for each isotopomer
precursor ion. The area measured for each isotopomer was
divided by the sum of areas for all possible isotopomers (up
to fully labeled) of a given amino acid. The resulting ratio was
expressed as a percentage in order to obtain a measure of the
relative contribution from each isotopomer to the total amount
of amino acid, as shown in the following equation:
!
AM
n
% contribution of isotopomer ðCÞ ¼
ꢂ 100% (1)
P
N
AM
n
n¼0
where A represents the area of the EIC peak, Mn is the
isotopomer from amino acid ’M’ with ’n’ labeled atoms, and
N is the maximum number of labeled atoms for amino acid ’M’.
From this data, the total labeling percentage was calculated
by weighting each isotopomer according to the number of
labeled atoms they contain, then dividing by the maximum
number of labeled atoms, which corresponds to the atom
percent (AP) labeled, as shown below:
P
N
n¼1
ðCM Þn
n
labeled AP ðof amino acid MÞ ¼
(2)
N
For example, in a 13C-labeling experiment, the isotopomer
masses of AQC-derivatized alanine are shown in Fig. 1. If the
extracted precursor ion at m/z 260 (n= 0) were twice the size
of the peak area for fully labeled alanine (n= 3) at m/z 263,
and no other isotopomer peak was detected, then the total
label atom percent (AP) would be calculated as 33%, since
exactly one-third of the total pool of alanine carbons would be
13C-labeled.
The calculation of the percentage of isotope label for a given
amino acid is based on the ratio of the EIC peak area of each
isotopomer relative to the sum of all isotopomer signals.
The quantitative information is therefore extracted from a
chromatographic peak rather than from a spectral peak,
removing any possibility of skewed results due to chromato-
graphic shifts from isotopically labeled ions. In order to
calculate the total isotopic contribution, the areas corresponding
to each isotopomer are weighted according to the number of
labeled atoms they contain, resulting in an absolute percentage
of labeling, or atom percent (AP). The data can then be treated
according to the needs of the experiment, for example a correc-
tion factor can be applied to account for natural abundance,
thus yielding a % enrichment value. Isotopomers can also be
analyzed individually, since the data from each is available, pre-
serving the opportunity for determining the distribution of
labels incorporated in any amino acid for a given experiment.
RESULTS AND DISCUSSION
Calculation of % isotope labeling
Amino acid separation by reversed-phase chromatography
normally requires derivatization due to the polar nature of the
analytes. This poses an interesting problem for amino acid
isotopomer analysis by reversed-phase LC/MS since the
resulting derivatized amino acids will have an altered isotope
pattern compared to the free amino acids, thus complicating
data analysis. Our derivatization method using AQC, a
common reagent used for reversed-phase amino acid analysis
and designed for fluorescence detection,[18] permits the use of
a selective precursor ion scanning method that addresses this
problem. The fragmentation of AQC-derivatized amino acids
yields a prominent, and thus highly sensitive, product ion at
m/z 171 that results from the loss of the AQC moiety.[19] Since
the ion at m/z 171 is the loss of the entire monoisotopic deriva-
tization agent, the isotopic pattern detected from the precursor
ion scan corresponding to the AQC-derivatized amino acids is
dictated exclusively by the free amino acids. Moreover, the pre-
cursor ion scan adds a level of selectivity that non-MS/MS
methods, such as single ion monitoring, do not provide. The
possibility of mass interference during the analysis of all
isotopomers for multiple amino acids from a relatively complex
biological sample is a legitimate concern. Fortunately, the
Method validation using isotope-labeled standards
In order to test our method of measuring isotopic enrichment,
amino acid standards prepared at varying unlabeled to labeled
ratios were tested. The standards were prepared by mixing four
unlabeled amino acids (phenylalanine, methionine, glutamine
and tyrosine) with their corresponding fully 13C- and
15N-labeled counterparts (with the exception of phenylalanine,
which was 13C6 only) in precise ratios. It is difficult to prepare
standards that will perfectly reflect biological samples, which
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Amino acid isotope labeling analysis by LC/MS/MS
majority (89.5%) of the samples being within ꢀ10% of the
expected value. In order to obtain these standard curves, a final
concentration of 200 nM derivatized amino acid standard
representing between 29 and 36 pg of each pre-derivatized
amino acid (from the four standard tested) were injected onto
the column. The performance and sensitivity of the method is
clearly suitable for the application described since we would
always be starting with an initial protein sample of at least
0.1 mg prior to hydrolysis.
Amino acid analysis and protein hydrolysis
In order to obtain a good measurement of the degree of isotopic
enrichment in proteins, we have opted for one of the simplest
and most commonly used methods: acid hydrolysis using
concentrated hydrochloric acid. Like most other hydrolysis
methods, this one does not permit the analysis of all amino
acids. By combining different hydrolysis methods, it is possible
to recover every amino acid and it is important to note that
our derivatization and LC/MS methods can be adapted to be
compatible with any hydrolysis method.
There are six amino acids that we cannot directly measure
using our combination of acid hydrolysis and LC/MS method,
as shown in Table 2. Cysteine and tryptophan are known to be
destroyed by acid hydrolysis and asparagine and glutamine are
deamidated to their carboxylic acid counterparts, aspartic acid
and glutamic acid, respectively.[20] Even though we cannot
directly analyze asparagine or glutamine, all of their 13C-labeling
information is incorporated into the data obtained from
aspartic acid and glutamic acid. This complicates the individual
analysis of these four amino acids and renders the measure-
ment of side-chain 15N-labeling for asparagine and glutamine
impossible when using acid hydrolysis. It is best to use other
hydrolysis methods[16] if the precise labeling information for
these amino acids is required. Two more amino acids, histidine
and arginine, are not sensitive enough to be analyzed using
our method, mainly due to the highly polar nature of their
AQC derivatives, and therefore very early elution of these
derivatives using an acidic mobile phase. If these amino acids
are specifically of interest, simply using a more basic mobile
phase, such as ammonium acetate, would increase their
retention on a C18 column, thus increasing their sensitivity.
The formic acid mobile phase, however, was chosen as the best
choice since, overall, we obtain very good resolution and
sensitivity of a majority of the amino acid derivatives.
Representative chromatograms of overlaid monoisotopic
EICs from a mixture of all 20 standard amino acids (without
hydrolysis), each at a concentration of 0.5 mM, and a
hydrolyzed algal protein sample (from an initial amount of
1 mg lyophilized algae) are illustrated in Fig. 4. Using the
precursor ion scanning method from m/z 235–500, we did not
observe peaks in the standard mix for cysteine, arginine and
histidine, since most of the ion intensities for these amino acid
AQC derivatives are doubly charged and therefore below the
mass range detected (see Table 2). However, if cysteine is
protected to remove the free thiol group prior to protein
hydrolysis, the corresponding protected AQC derivative will
be measurable with the same precursor ion scanning method
as described here. Basic amino acids, such as lysine, arginine
and histidine, since they are doubly charged will not have
complete resolution between isotopomers, and therefore
will not be taken into account in the final assay for protein
Figure 2. Mass spectra (from m/z 335 to 346) from different
standard mixtures of ring-labeled (13C6) phenylalanine.
will contain various degrees of isotope labeling, since
commercially available standards are manufactured to be
isotopically uniform. These standards suit our purpose of
validating the method by testing our ability to correctly
determine isotope ratios and to calculate a percentage of
isotope enrichment from these ratios. As an example of the
results obtained with these standards, Fig. 2 shows the mass
spectra from different standard mixtures of phenylalanine.
The spectra clearly show the typical baseline resolution
obtained between isotopomers using our method. In the case
of the standard mixtures, the total percentage of labeled atoms
was corrected for the natural isotopic abundance for each
amino acid by adjusting the targeted percentage based on the
theoretical isotope distributions of the standard mixtures and
the isotopic purity of the labeled amino acids. The results show
excellent linearity and consistent results for all standard amino
acids (Fig. 3 and Table 1). The precision of the method is
excellent, with all standard samples injected having
a
coefficient of variance (CV) within 20%. The accuracy of the
method is also very good, with all standard samples having a
calculated AP within ꢀ2.5% and a percent accuracy within
ꢀ20% of the expected theoretical value, including the vast
Figure 3. Calibration curves of standard amino acids (Gln,
Phe, Tyr, Met) at different labeling percentage mixtures.
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A. LeBlanc et al.
Table 1. Atom percent labeling for mixtures of unlabeled and labeled amino acid standards
Gln Phe Met
Tyr
%
labeled Calc%
Calc%
(Theo)
Calc%
(Theo)
Calc%
(Theo)
std
(Theo)
%Δ
0.9
%CV
%Δ
4.1
%CV
%Δ
%CV
%Δ
%CV
0
1.1 (1.1)
19.7
11.4
13.4
7.5
18.0
6.9
3.2
1.9 (1.8)
2.8 (2.8)
1.9
8.5
8.2
8.5
3.5
10.7
2.1
12.4
4.2
1.8
9.4
0.3
0.9
0.4
0.3
0.4
0.1
0.0
2.7 (2.6)
3.7 (3.6)
5.0
5.0
8.7
11.9
0.4
11.5
14.6
6.2
1.1 (1.1)
2.2 (2.1)
ꢃ0.6
5.9
ꢃ9.4
ꢃ0.1
ꢃ8.4
2.6
14.0
ꢃ5.4
3.5
3.9
6.8
1
1.7 (2.0) ꢃ14.8
0.3
2
2.4 (3.0) ꢃ19.0
3.3 (3.8) ꢃ10.9
4.9 (4.5)
2.7 (3.0)
8.2
3
3.7 (3.9)
5.8 (4.9)
ꢃ5.6
18.8
ꢃ9.7
16.0
7.6
4.4 (4.7)
5.4 (5.7)
6.8 (6.7)
ꢃ5.9
ꢃ5.6
1.8
6.1 (5.4)
16.3
18.6
13.6
4.8
4.0 (4.0)
19.9
19.3
17.2
14.5
6.4
4
6.4 (6.4)
4.5 (4.9)
5
5.3 (5.9)
6.9 (7.3)
ꢃ5.0
12.4
ꢃ7.2
1.8
6.0 (5.9)
10
20
40
50
60
80
90
95
96
97
98
99
100
12.4 (10.7)
21.8 (20.3)
41.4 (39.6)
10.9 (11.5) ꢃ5.0
20.3 (21.2) ꢃ4.4
37.9 (40.7) ꢃ6.8
50.3 (50.5) ꢃ0.4
58.2 (60.3) ꢃ3.4
79.7 (80.0) ꢃ0.3
88.9 (89.8) ꢃ1.0
13.5 (12.0)
19.9 (21.5)
41.3 (40.6)
48.2 (50.2)
61.5 (60.0)
81.9 (79.7)
88.2 (89.6)
94.6 (94.7)
96.4 (95.7)
97.3 (96.7)
98.2 (97.7)
99.2 (98.7)
12.1 (10.7)
19.1 (20.2)
40.8 (39.5)
48.6 (49.1)
56.8 (58.9)
80.2 (78.4)
88.0 (88.3)
93.9 (93.2)
94.7 (94.2)
95.6 (95.2)
97.5 (96.2)
98.3 (97.2)
99.3 (98.1)
15.9
5.0
5.8
4.5
0.6
8.5
46.2 (49.3) ꢃ6.3
3.6
ꢃ4.1
4.5
ꢃ1.1
ꢃ3.5
2.3
4.5
62.4 (59.0)
80.4 (78.6)
89.1 (88.4)
93.6 (93.3)
95.1 (94.3)
96.0 (95.3)
97.6 (96.2)
98.5 (97.2)
99.2 (98.2)
5.6
2.3
0.8
0.4
0.9
0.8
1.4
1.3
1.0
1.3
2.5
2.6
2.9
4.1
2.8
3.5
1.8
0.6
ꢃ1.6
1.5
ꢃ0.3
0.7
3.1
95.1 (94.8)
96.2 (95.7)
96.7 (96.7)
98.2 (97.7)
99.0 (98.7)
0.4
0.4
0.0
0.5
0.3
0.6
0.0
1.7
0.7
1.1
1.3
0.8
1.2
0.6
0.6
0.5
0.6
0.3
0.4
0.8
0.3
0.6
0.2
1.4
0.4
0.3
0.5
0.3
1.2
0.1
0.1 100.3 (99.7)
0.2 100.2 (99.7)
0.5
0.1
1.2
0.0
%Δ: Percent difference between the calculated AP labeling and the theoretical value.
%CV: Standard deviation as a percentage from triplicate injections.
Table 2. Amino acid characteristics following protein hydrolysis, AQC derivatization and LC/MS analysis
Amino
acid
# C
atoms
# N
atoms
MW
(g/mol)
m/z (AQC
derivative)
RT
(min)
Comment
Hisa
Asn
Arga
Gln
6
4
6
5
3
2
4
5
4
3
5
6
9
3
5
5
11
6
9
3
2
4
2
1
1
1
1
1
1
1
2
1
1
1
1
2
1
1
155.1
132.1
174.1
146.1
105.0
75.0
133.0
147.1
119.1
89.0
115.1
146.1
181.1
121.0
149.1
117.1
204.1
131.1
165.1
163.6, 326.1
303.1
3.3
4.9
Low sensitivity (charge/early elution)
Acid hydrolysed to aspartic acid
Low sensitivity (charge/fragmentation)
Acid hydrolysed to glutamic acid
173.1, 345.2
317.1
5.3
6.4
Ser
276.1
6.5
Gly
246.1
6.8
Asp
Glu
304.1
7.4
318.1
7.8
Thr
290.1
8.2
Ala
260.1
8.7
Pro
286.1
9.2
Lysab
Tyr
244.1, 487.1
352.1
10.6
11.0
11.1
11.3
11.8
13.4
13.5
13.6
Doubly derivatized and doubly charged
Degraded during acid hydrolysis
Cysab
Met
Val
231.6, 462.1
320.1
288.1
Trp
Ile/Leu
Phe
375.1
Degraded during acid hydrolysis
Coelution of isobaric species
302.1
336.1
aDoubly charged species as base peak.
bDoubly derivatized amino acid as major species.
hydrolysates. One alternative for measuring the isotopic
contributions for these two amino acids, as well as lysine, is to
use a mobile phase that is less acidic and therefore would more
likely form the 1+ charge state. The acidic mobile phase is much
more sensitive for the detection of AQC derivatives of amino
acids and therefore was chosen for this study.
One advantage of this approach is that baseline resolution is
not needed for all amino acids; however, it is crucial that all
amino acids with the potential to share isobaric isotopomers
are well resolved chromatographically. For hydrolyzed protein
samples, our method therefore has the ability to measure 13C
isotope enrichment with data originating from 15 of the 20
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Amino acid isotope labeling analysis by LC/MS/MS
Figure 4. Extracted ion chromatograms of monoisotopic
peaks for AQC-derivatized amino acid standard mix (at
0.5 mM amino acid concentration) (a) and protein hydrolysate
from representative algal sample (b).
amino acids, removing the doubly charged derivatives (Lys,
His, Arg) and those that are destroyed by acid hydrolysis
(Cys, Trp), providing enough coverage to obtain a reliable
estimate of the total 13C enrichment of the protein sample.
Analysis of 13C enrichment in proteins from glucose-fed
algae
A cost-effective strategy to obtain labeled byssal threads is to
first label algae which will subsequently be used to feed the
mussels. In the current study, we have tested two possibilities
for labeling of algae: feeding with labeled glucose, as well as
growing algae in the presence of labeled salts. Our method
was first tested on protein extracts from algae that were fed
with isotopically labeled 13C6-glucose (50%) for a period of
19 days. Algae were taken from the growth medium
every second day for analysis and the total percentage of
13C-labeled carbon atoms was calculated (see Table 3). The
labeling kinetics is thus established and the optimal time
for algae harvesting prior to mussel feeding determined.
The results do not include data for methionine, since it is both
one of the lesser sensitive of the amino acids and one of the least
abundant in proteins; therefore, the resulting signal obtained
from our samples was not sufficient for reliable data to be
extracted, resulting in data quantified from 14 amino acids.
In order to compare the mass spectra originating from a
standard algal sample with those from the labeled samples,
threonine is used as an example in Fig. 5. The mass spectrum
of threonine obtained from an algal sample taken on the first
day of labeling is, as expected, very similar to that observed
from the standard. A difference is clearly seen, however, in
the mass spectra between the first (day 1) and last day
(day 19) of the feeding experiment. The relative areas of the
labeled threonine isotopomers are visibly larger compared to
the unlabeled threonine in the day 19 sample, as we can start
to see a fully labeled threonine peak starting to emerge. Since
the algae were continuously fed 13C-labeled glucose during
the experiment, we expect protein extracts to have increasing
13C content throughout the 19 days of sampling. When the
average 13C-labeling percentage of amino acids was calculated,
we indeed observed
a continuous increase in labeling
throughout the sampling period (Fig. 6), reaching a plateau of
labeling when the algae entered the stationary phase of
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A. LeBlanc et al.
While the glucose-feeding experiment does serve as an
excellent example of the applicability of our method, the
overall percentage of labeling of the algae is not sufficient to
obtain a satisfactory labeling of byssal threads for NMR
studies; therefore, we assessed a second method of algal
production using labeled inorganic salts as carbon source.
Analysis of 13C enrichment in protein from sodium
bicarbonate-fed algae and mussel byssus
Sodium bicarbonate is a common carbon source for microalgae
and therefore it was expected that a 13C-labeled bicarbonate
diet would allow a more efficient incorporation of 13C into algal
proteins. In order to maximize labeling efficiency through the
labeled salt diet, a culture system was designed to ensure the
elimination of oxygen produced by algae and avoid gas
exchange with the ambient atmosphere. Indeed, this experi-
mental approach yields algae with significantly higher 13C
labeling than with the previous glucose-feeding experiment,
with a 13C labeling of 84.9% (AP). Figure 7 shows the extracted
ion chromatograms (EICs) corresponding to each of the 13C
isotopomers of alanine, comparing the bicarbonate-fed labeled
algae to unlabeled (control) algae. The areas of each of these
peaks represent the raw data used to calculate the 13C atom
percent labeled (AP). The efficiency of this feeding experiment
is clearly illustrated since the predominant isotopomer
peak from the labeled algae corresponds to the fully labeled
(13C3-alanine at m/z 263) alanine peak.
Figure 5. Mass spectra (from m/z 288 to 296) and integration
windows from AQC-derivatized threonine from standard
(a), algal sample on day 1 (b), and algal sample day 19 (c).
The amount of algae labeling provided by this feeding tech-
nique is sufficient in order to move on to the next step and assess
the labeling incorporation into mussel byssus. This algal culture
was used to study the efficiency of the mussel feeding and
byssus production protocols. Normally, byssus production is
stimulated by transferring the mussels in a tank designed to pro-
duce a current which forces the mussels to produce byssal thread
as a means to immobilize themselves on a solid support. Due to
the volume of this tank and its configuration having relatively
high currents, the mussels were pre-fed with labeled algae and
then transferred into the tank for stimulated byssus production.
Every second day, the byssal thread was removed. The optimal
sampling day was then determined by our LC/MS/MS method
in order to maximize the labeling of the byssus sample. From
our data, the optimal labeling was determined to be at day 5,
and the mass spectra in the window of the 13C isotopomers of
valine from these 13C-labeled byssus threads are shown in Fig. 8.
By comparing the labeled byssal thread sample to unlabeled
and labeled algae samples, it is clearly seen that some 13C labels
have been incorporated into the byssus, notably with the
appearance of the fully labeled valine isotopomer peak at
m/z 293. While the byssus that was sampled on day 5 proved
to have the highest 13C-isotopic enrichment, it is still relatively
low at 8.6% (AP), illustrated by the fact that unlabeled valine
still remains the major isotopomer peak. Considering that the
major isotopomer peak for all amino acids in the bicarbonate-
fed algal sample corresponds to the fully labeled amino acid
(as shown for valine), the feeding and sampling methods can
still be improved significantly if feeding is continued during
byssus production. This modest amount of label incorporation,
however, is still enough to reduce the acquisition time ~60-fold
compared to unlabeled byssal thread, allowing for preliminary
NMR structural studies to begin while the mussel feeding and
byssus production protocols are further optimized.
Figure 6. Average percentage of 13C labeling in algal protein
hydrolysates from time-course 13C6-glucose feeding experiment,
with error bars showing analytical reproducibility (n = 3) (left)
and biological variability (n = 3) (right).
growth at day 19. We can clearly see a nice trend in the overall
data even if the percentage change is subtle between samples
from consecutive sampling days, demonstrating that the
method can reliably differentiate samples which differ by about
2% 13C-labeling (AP). The precision of the method for the
analysis of algal samples is clearly consistent with the data from
the standard curves. Three samples were taken at each time
point (biological replicates) and each one of these samples
was injected three times (analytical replicates). The %CV of
the analytical replicates was again excellent: all samples were
under 20%, including over 90% of samples with %CV under
10% (see Table 3). The standard deviations between different
biological replicates suggest that the accuracy of our method
also reflects the results of our standard curves since 91.8% of
the samples are within 2% AP.
An important aspect of this method is that the data pertaining
to all isotopes are available, allowing for deeper analysis of the
data. We can obtain relevant biological information from this
type of analysis, since we can monitor the degree of labeling
for each amino acid and the relative abundance of each
isotopomer, as opposed to only having access to a total amount
of 13C labeling. As previously mentioned, it is very useful
for subsequent NMR studies to be able to discriminate partial
and total labeling of a given amino acid.
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Rapid Commun. Mass Spectrom. 2012, 26, 1165–1174

Amino acid isotope labeling analysis by LC/MS/MS
Figure 7. Extracted ion chromatograms from precursor ion scanning experiment
for alanine 13C isotopomers in unlabeled and 13C-bicarbonate-fed algal samples.
to distinguish between samples that differ by 2% (AP) with a
very modest amounts of amino acid (≤36 pg of pre-derivatized
amino acid) injected onto the column. In short, the performance
of the method, its sensitivity and its accessibility render it an
ideal approach for assessing protein labeling. Within the con-
text of protein structural studies by NMR, this is especially true
since the method is adaptable for the measurement of either 13C
or 15N enrichment and allows one to evaluate labeling levels for
individual amino acids, while also being able to discriminate
between partial and total labeling of a given amino acid.
This new method was used for the measurement of 13C
labeling in algal protein and mussel byssus thread proteins,
demonstrating that we were able to obtain reliable 13C-labeling
data originating from 14 amino acids using the combination of
a simple acid hydrolysis method and our analytical approach
for two different types of biological samples. The method has
allowed us to evaluate two different feeding protocols for
microalgae and subsequently monitor the optimization of
mussel feeding protocols for selecting suitable mussel byssal
threads for further structural investigation.
Figure 8. Mass spectra (from m/z 285 to 295) showing valine
isotopomers from 13C-bicarbonate-fed algae and labeled
mussel byssus sample.
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Rapid Commun. Mass Spectrom. 2012, 26, 1165–1174