Determination of true ratios of different N-glycan structures in electrospray ionization mass spectrometry
Determination of true ratios of different N-glycan structures in electrospray ionization mass spectrometry
Clemens Grünwald-Gruber 0
Andreas Thader 0
Daniel Maresch 0
Thomas Dalik 0
Friedrich Altmann 0
0 Department of Chemistry, University of Natural Resources and Life Sciences , Vienna, Muthgasse 18, 1190 Vienna , Austria
An ideal method for the analysis of N-glycans would both identify the isomeric structure and deliver a true picture of the relative, if not absolute, amounts of the various structures in one sample. Porous graphitic carbon chromatography coupled with electrospray ionization mass spectrometry (ESI-MS) detection has emerged as a method with a particularly high potential of resolving isomeric oligosaccharides, but little attention has so far been paid to quantitation of the results obtained. In this work, we isolated a range of structures from Man5 to complex type N-glycans with zero to four sialic acids and blended them into an equimolar Bglyco tune mix^. When subjected to liquid chromatography-ESI-MS in positive and negative modes, the glyco tune mix clearly demonstrated the futility of quantitation of N-glycans of different overall composition, different number of sialic acids, and strongly differing size without compensation for their very different molar responses. Relative quantitation of human plasma N-glycans was performed with correction factors deduced from this external glyco tune mix. Addition of just one isotope-coded internal standard with enzymatically added 13C-galactose led to absolute quantification in the same experiment.
N-Glycan; Sialylation; Quantitative glycomics; Mass spectrometry; Electrospray ionization
Structural assignment of N-glycans and O-glycans has been a
major issue in the last few decades, with the clearest advances
having been achieved with mass spectrometry (MS)  and
even more with liquid chromatography (LC)–MS [2, 3]. In
terms of power to define a particular structure, MS surpasses
chromatographic techniques such as hydrophilic interaction
LC (HILIC)–high-performance LC (HPLC) or capillary zone
electrophoresis of fluorescently labeled glycans . These
techniques, however, offer the advantage of an inherently
identical molar response of all N-glycan species in a sample
because of the invariable stoichiometry of the fluorophore [4,
5]. Correct relative quantitation of the components within a
sample therefore requires well-separated peaks as facilitated
by the latest ultraperformance HILIC columns . Even then,
with samples being more complex than antibody or plasma
N-glycans, peak overlap will increasingly become a problem
and requires the higher definition power of MS.
The term Brelative quantitation^ may be understood in two
rather different ways. First, it refers to the comparison of two
or more samples with the aim of finding upregulated or
downregulated glycoforms. Second, it can mean determination of
the correct ratios of different glycan structures within one
The first aim, comparison of different samples, can be
accomplished Blabel-free^ by consecutive analyses [7–9] or it
can be realized by derivatization with isotopically labeled
reagents. Light and heavy forms of 2-aminopyridine ,
anthranilic acid [11, 12], 2-aminobenzoic acid [13, 14], aniline
[15–17], and phenyl-3-methyl-5-pyrazolone [18, 19] have
been used. Other chemistries targeting the reducing end used
carbonyl-reactive tandem mass tags , Girard’s reagent
, TMT-labeling of N-glycosylamines or reducing glycans
[22, 23], or hydrazide based reagents [24, 25]. Two samples
from cell cultures can be compared by metabolic
incorporation of 15N into N-acetylglucosamine (GlcNAc) residues .
Several groups used permethylation to introduce heavy
isotopes [27–29], whereby H/D permethylation introduced a
remarkably strong deuterium effect . Only a few of these
approaches have been applied to biologicals samples such as
glycans from carcinoma cell lines [20, 26], differentiating
murine stem cells , or serum from healthy donors  and
from patients with esophageal diseases [23, 29]. One study
screened ovarian cancer samples from a biorepository and
identified a number of glycans.
However, none of these approaches consider the differing
molar response of glycans of different size, different number
of sialic acids, different general architecture (oligomannosidic
vs complex type), different charge states, and different
tendency to form adducts with sodium or ammonium in electrospray
ionization (ESI) MS . In addition, chemical modification
of glycans is essentially not compatible with the one method
that has the greatest ability to separate structural isomers; that
is, porous graphitic carbon (PGC) chromatography coupled
with ESI-MS [2, 3]. Nevertheless, impressive separation of
permethylated glycans by PGC chromatography has been
reported recently , and allows the introduction of methyl
groups of differing isotopic composition. However, PGC
separation of reduced but otherwise unmodified glycans is still
the standard format .
A solution to the problem is the use of internal standards
labeled without alteration of the overall structure, which has
recently been presented in the form of 13C-labeled
N-acetylated glycans . A range of glycan structures
covering the species relevant for the analysis of monoclonal
antibodies was prepared, including a disialylated glycan. Many
g l y c o p r o t e i n s , h o w e v e r, c o nt a i n t r i s i a l y l a t ed a n d
tetrasialylated N-glycans, and in our experience and that of
others these more complex glycans show the strongest
deviations of molar response [32–34]. Preparation of isotopically
labeled glycans of this complexity, however, becomes a highly
demanding task, and hence the resulting standard mixtures
would be too expensive for routine use. Therefore, Mehta
et al.  concentrated on a set of three natural glycans with
zero or two sialic acids for the analysis of permethylated
glycans by nanospray MS and matrix-assisted laser desorption
ionization time-of-flight (MALDI-TOF) MS . Both
strategies yield absolute quantitation and hence also true relative
proportions of the glycans considered. For characterization of
biopharmaceutical glycoproteins, relative quantitation appears
to be the more relevant task, which is particularly
compounded by multiantennary, highly sialylated structures
if it is conducted by MS . A recent approach to this task
therefore involved enzymatic simplification of N-glycans by
sialidase, galactosidase, and fucosidase. Thereby, the original
bias toward high-mannose structures was clearly diminished
. MALDI-TOF MS of permethylated glycans circumvents
the problems arising from carboxyl groups and is held to
reflect truly the molar proportions of the glycans in a sample.
This was convincingly substantiated in a recent study by
comparison of MALDI-TOF MS and HILIC–HPLC data .
In this work we generated an equimolar mixture of
N-glycans with natural isotope patterns covering a wide
range of structures from Man5 to tetrasialylated glycans
(Table 1). This calibration mixture clearly demonstrated
the very different molar responses of different glycan
species and the usefulness of this standard for instrument
tuning and determination of true molar ratios. Absolute
quantitation—if required—can then be achieved with
application of just one or maybe two isotopically labeled
internal standards, which in this work were obtained via
enzymatically prepared UDP-13C6-galactose.
Materials and methods
Preparation of complex N-glycan standards
The nonsialylated diantennary glycan A4A4 (G2) was
prepared from a pepsin digest of bovine fibrin by digestion with
PNGase A and reduction with NaBH4. Final purification was
achieved by chromatography on PGC on a 3 mm x 150 mm
column (Thermo Scientific, Vienna, Austria) with a gradient
from 5 to 30% acetonitrile in 65 mM ammonium formate of pH 3.0.
Fractions were analyzed by capillary PGC-LC–ESI-MS .
4 4 6 (BAAF^; G2F; see
The fucosylated standard A A F
Table 1 for structures and their abbreviations) was isolated
by preparative PGC chromatography from human IgG .
The monosialyated and disialyated glycans Na6-4A4/
A4Na6-4 (G2S1) and Na6-4Na6-4 (BNaNa^; G2S2) were
obt a i n e d b y i n c u b a t i o n o f G 2 ( A 4 A 4 ) w i t h α 2 , 6
sialyltransferase. To this end, a His-tagged version of rat
ST6Gal I devoid of 96 residues at the N-terminus was
expressed in the baculovirus/insect cell system. The G2S1
isomers (Na6-4A4 and A4Na6-4) and G2S2 were isolated by
The trisialylated standard Na6-4[Na6-4 Na6-4] (G3S3) was
isolated from the glycans released from bovine fetuin .
The tetrasialylated [Na3-4 Na3-4][Na3-4 Na3-4]F6 (G4FS4)
was extracted by preparative PGC chromatography from
erythropoietin, which was obtained as a by-product of a
feasibility study for biosimilar production.
Man5 was prepared from Aspergillus oryzae amylase by
PGC chromatography. Man9 was isolated from the N-glycan
pool of white beans by HILIC on a TSKgel Amide-80 column
(Tosoh Bioscience, Griesheim, Germany) .
The reference compounds were dried several times to
remove any ammonium acetate or formate, taken up in water,
and subjected to amino sugar analysis with consideration of
the molar content of amino sugar .
Table 1 N-Glycan structures used in this work with their names and masses used. The IgG style abbreviations give the number of galactoses (G) and
sialic acids (S) and presence of fucose (F)
Preparation of high-mannose N-glycan standards
Fungal amylase was purified by passage over Sepharose S100
with 50 mM ammonium acetate of pH 6.0. A pepsin digest of
the enzyme was passed over Sephadex G50m with 1% acetic
acid as the solvent. The glycopeptide fraction was then treated
with peptide N-glycosidase A. The digest was passed through
a C18 silica cartridge (HyperSep C18, 25 mg; Thermo
Scientific, Waltham MA, USA) and the flow-through was
applied to a PGC cartridge (HyperSep Hypercarb, 25 mg;
Thermo Scientific). N-Glycans were eluted with 50%
acetonitrile in ammonium formate buffer of pH 3.0. As this
material contained about 60% Man6, it was digested with
recombinant α-1,2-specific mannosidase MNS1 from
Arabidopsis thaliana . The digest was again subjected to
the purification steps described above. The purity of the final
product was verified by PGC-LC–ESI-MS .
Preparation of 13C6-labeled complex N-glycans
UDP-13C6-galactose was prepared by incubation of
13C6-galactose (Cambridge Isotope Laboratories, Tewksbury,
MA, USA) with galactokinase (Sigma-Aldrich, Vienna,
Austria). The galactose 1-phosphate was converted to the
nucleotide sugar in the presence of UPD-glucose by human
galactose 1-phosphate uridylyltransferase, which was
recombinantly expressed in Escherichia coli BL21 and
purified via its His6-tag (a yeast enzyme with this activity is
now commercially available from Sigma-Aldrich ). The
UDP-13C6-galactose was finally purified by PGC
chromatography with use of a slightly alkaline buffer as described in
This UDP-13C6-galactose was then used to regalactosylate
fibrin glycans previously degalactosylated by Aspergillus oryzae
β-galactosidase . Bovine β-1,4-galactosyltransferase
(Sigma-Aldrich, Vienna, Austria) was used in 50 mM
tris(hydroxymethyl)aminomethane–HCl buffer for transfer of
13C6-galactose. The thus produced C13A A
4 4 (C13G2) was
sialylated with α-2,6-sialyltransferase (see earlier) to arrive at
singly and doubly sialylated (C13G2S2) standards with a 12-Da
increment compared with the natural versions.
A trisialylated standard was prepared by partial
degalactosylation of fetuin asialo N-glycans, isolation of the
G2 form, followed by incorporation of just one 13C6-galactose
Quantification of individual N-glycans
The isolated standard compounds, both natural and heavy
isotopologues, were subjected to hydrolysis with 4 M HCl
acid for 4 h at 100 °C followed by reduction with NaBH4
and amino sugar analysis . Analyses were repeated at least
four times. Relative standard deviations of better than 5.5%
were obtained. Molar concentrations were calculated on the
basis of the respective glycan structure under the assumption
of near to complete recovery of GlcNAc residues as
Quantitative N-glycan analysis of serotransferrin, human
plasma, and human serum albumin
Transferrin, human serum albumin (HSA), and human plasma
(obtained from buffy coat preparations purchased from the
University Clinic for Blood Group Serology and Transfusion
Medicine, Graz, Austria) were digested with PNGase F as
described in . In short, the sample was denatured in 2%
sodium dodecyl sulfate at 60 °C for 10 min. On dilution with
100 mM ammonium bicarbonate buffer containing 2% Igepal
CA-630 (Sigma-Aldrich, Vienna, Austria) PNGase F (0.4 mU
per 6 μL plasma; Roche, Mannheim, Germany) was added.
The mixture was incubated for 16 h at 37 °C, whereupon 151
pmol of the isotope-coded C13G2S2 standard per microliter of
plasma was added to the sample. Released glycans were
purified with PGC solid-phase extraction cartridges (HyperSep
C18, 25 mg; Thermo Scientific, Waltham, MA, USA). Elution
was done with 55% acetonitrile in ammonium formate buffer.
Finally, the glycans were reconstituted in high-quality water.
All experiments were done in triplicate.
MS measurement and data interpretation
All samples were measured in positive and negative mode
with two different instruments, a quadrupole time-of-flight
(Q-TOF) instrument (maXis 4G; Bruker, Bremen, Germany)
and an ion trap instrument (amaZon speed ETD; Bruker,
Bremen, Germany). Standard source settings (capillary
voltage 4.5 kV, nebulizer gas pressure 0.5 bar, drying gas 5 L/min,
200 °C) were used. Instrument tuning was optimized for a mid
mass range (500–3000-Da molecules).
The purified samples were loaded on a PGC column
(100 mm x 0.32 mm, 5 μm; Thermo Scientific, Waltham,
MA, USA) with use of 65 mM ammonium formate buffer of
pH 3.0 (positive mode) or 10 mM ammonium carbonate
(negative mode) as the aqueous solvent. A gradient from 1%
solvent B (80% acetonitrile plus 20% solvent A) to 68% solvent
B in 40 min was applied, at a flow rate of 6 μL/min. Detection
was performed with the Q-TOF or ion trap mass spectrometer
equipped with the standard ESI source in data-dependent
acquisition mode (switching to MS/MS mode for eluted peaks),
directly linked to the Thermo Ultimate 3000 UPLC system.
MS scans were recorded (range 150–2200 Da for the Q-TOF
instrument and 400–1600 Da for the ion trap instrument) and
the four highest peaks were selected for fragmentation.
Instrument calibration was performed with an ESI calibration
Data interpretation was done with DataAnalysis 4.0
(Bruker, Bremen, Ger many). Theoretical isotopic
distributions were calculated with Compass isotope pattern
calculator (Bruker, Bremen, Germany). For quantification
with the Ball peaks^ strategy, the four (Man5) to seven
(G4S4F) highest isotopic peaks (resulting in at least 96.5%
of the overall theoretical peak areas) in the extracted ion
chromatogram were integrated (detailed in Table S1). All charge
states and adduct ions were considered if their top peak
reached at least 4% of the base peak of the particular spectrum.
MS spectra of glycans in positive and negative mode
When preparing the glycans for the equimolar reference
mixture (Table 1), we stumbled on the startling complexity of MS
results for glycans. First, this arises from the splitting of peaks
into isotope patterns, which differ depending on the size of the
glycans (Fig. 1). Very different results are thus obtained when
the monoisotopic peak, the most abundant peak, or Ball^ peaks
(covering 96.5% of the theoretical isotope pattern; all charge
states and adduct peaks) were considered. In addition, most
N-glycans tend to occur in two charge states in ESI and form
adduct peaks of different relative height (Fig. 1). The most
relevant adduct peaks in PGC LC–ESI-MS in positive mode
correspond to ammonium-containing ions as the eluents
usually used contain either ammonium formate (for positive
mode analysis [32, 33]) or ammonium carbonate (for negative
mode analysis ). In negative mode, carbonate adducts were
generated, whereas formate adducts were found if the eluent
contained ammonium formate (not shown). Some
complication arises from the oligomannosidic glycans’ tendency to
undergo in-source fragmentation, which is almost absent for
Man9 but increases from Man8 to Man5.
A Bglyco tune mix^ for correction of ion abundances
and its application to the plasma glycome
Ion abundances (intensities) can be obtained from either the
monoisotopic peaks or the entire footprint of the analyte. In
the absence of an appropriate standard, a makeshift solution
toward relative quantification could be to consider all isotopic
peaks (more exactly, those making up at least 96.5% of the
entire theoretical isotope cluster as detailed in Table S1), charge
states, and adducts of an analyte. This approach, however,
resulted in ion abundance values highly discrepant with the actual
stoichiometry (Fig. 1). The most serious deviations were
observed between neutral and highly sialylated species, and here
again with the ion trap instrument in positive mode (blue bars in
Fig. 1), where the apparent ratio between G4FS4 and neutral
G2 was 1:9.3 despite their being present in equal molar
amounts. The smallest divergence was seen again with the
ion trap instrument in negative mode, with a spread of 1:4.5
Fig. 1 Peak fine structures and
ion abundancies of mass
spectrometry (MS) peaks as
determined with the equimolar
Bglyco tune mix.^ The mixture
was subjected to porous graphitic
carbon liquid chromatography
(PGC-LC) with MS detection in
either positive mode or negative
mode with a maXis 4G
(QTOF) instrument or an amaZon
ion trap instrument (both Bruker).
The top panels show the isotope
and adduct footprints of a smaller,
neutral and a large tetrasialylated
N-glycan. The lower panels
compare the ion abundances as
determined from extracted ion
chromatograms for either only the
monoisotopic peak (Bsingle
peak^) or Ball^ peaks (see
BMaterials and methods^). The
numbers above the bars are the
correction factors for the Bsingle
peak^ approach relative to G2.
Technical error was below 3%
(red bars in Fig. 1) between G4FS4 and the disialylated G2S2.
So, in addition to the enormous differences in molar response,
even the relative order of the glycans differed between
polarities and instruments. The values actually measured are highly
dependent on the tuning of the instrument and thus cannot be
transferred from one instrument to another as was already
obvious from the again rather different relative responses
previously found with a Waters Q-TOF instrument in negative mode
. The inferior performance of the ion trap instrument for
highly sialylated glycans may reflect inadequate tuning, but it
also reminds us of the poor sensitivity found for sialylated
glycopeptides when the same type of trap was used .
Uncorrected quantification via only the monoisotopic
peak of the most relevant ion type yielded even more
distorted results, with a spread of 20 and 150 for the
Q-TOF instrument and the ion trap instrument respectively.
However, use of the results obtained with the equimolar
standard allows the calculation of correction factors, which
would compensate for different responses. Then again, one
has the choice of using the analyte’s entire footprint
(isotope peaks, charges states, adducts) or just the
monoisotopic peak of the most abundant charge state. The Ball peaks^
strategy—though preferable if no correction is applied—has
three obvious disadvantages: (1) it requires more work to
the set up the extracted ion chromatogram parameters, (2) it
may require more time for extraction of the respective
traces, and, most relevant, (3) it maximizes the risk of
collecting peaks from contaminants. Although this risk also
exists if only the monoisotopic peak is considered, it can
largely be avoided—or at least noticed—by verification of
the isotopic distribution of this one ion type. Although in
this study we used monoisotopic masses throughout for the
Bsingle peak^ strategy, choosing the second isotope peak
could be a worthwhile choice for larger glycans. Notably,
the ion abundance values for smaller glycans such as Man5
are unduly low because of their tendency to fragment.
Plasma contains many more structures than just the seven
contained in the standard mixture [8, 43]. Some of these
additional glycans are just isomers with, for example, different
sialic linkages or branching (Fig. 2), and it may well be
assumed that these have similar response factors within the
boundaries of measuring accuracy (typically around 5%)
(Table 2). Other structures differ, for example, by the
presence/absence of fucose, galactose, or a bisecting
GlcNAc, and the deviations may be larger, but correction with
factors for the respective Bnearest neighbor^ will yield
reasonably good approximations of true values (Table 2). Large
differences in retention time and hence solvent composition may
Fig. 2 PGC-LC separation of triantennary, trisialylated N-glycans in the
presence of the isotope-coded standard C13Na6-4[Na6-4Na6-4]. Extracted
ion chromatograms for m/z = 961.68 (solid lines) and m/z = 963.68
(dashed lines) are shown. Peak annotations of serum and fetuin glycans
are based on previous structural elucidation  together with the
empirical rule that α-2,3-linked sialic acids cause increased retention.
Sialic acids (diamonds) pointing upward are α-2,6-linked, those
pointing downward α-2,3-linked. EIC extracted ion chromatogram,
EPO erythropoietin, Fuc fucose, Gal galactose, GlcNAc
Nacetylglucosamine, Man mannose, Neu5Ac N-acetylneuraminic acid
reduce the gain in correction. As an example, the acetonitrile
content changes by a factor of 1.25 during the elution of the
trisialoglycans (Fig. 2) and thus may lead to deviations that,
however, appear small compared with the huge original error.
The equimolar Bglyco tune mix,^ whose concentration is
known, could be applied for absolute quantitation via external
calibration, and this strategy has just recently been realized by
others . For work with complex samples such as plasma or
tissues, which require extensive processing steps before the
actual analysis, it appears advisable to take into account losses
during workup. Hence, we decided to generate
stableisotope-labeled standards, as detailed in the following section.
Preparation of 13C6-galactose-labeled oligosaccharides
We used the Leloir pathway for the generation of
UDP-13C-galactose . 13C6-galactose is phosphorylated
and—in the same pot—transferred to UDP in exchange for
unlabeled glucose (Fig. 3). Usually, more than half of the
UDP-glucose is converted to UDP-galactose. Attempts at
optimizing the yield were not made . The desired product can be
easily discriminated by mass from unlabeled UDP-glucose. The
two nucleotide sugars in the reaction mixture were separated by
PGC chromatography (Fig. 3) even though a perfect purification
is not required as neither bovine β-1,4-galactosyltransferase nor
human β-1,3-galactosyltransferase used UDP-glucose (data not
GnGn (i.e., a desialylated and degalactosylated diantennary
N-glycan) was incubated with UDP-13C6-galactose and
β-1,4-galactosyltransferase. The fully galactosylated product
C13A4A4 (or C13G2) was separated from the partially
galactosylated isomers C13A4Gn and C13GnA4 by PGC-HPLC
on a column with an inner diameter of 3 mm. The C13G2 fraction
was further treated with ST6Gal to arrive at singly sialylated
(C13G2S1; actually a mixture of Na6-4A4 and A4Na6-4) and
doubly sialylated (C13G2S2 or exactly icNa6-4Na6-4, ic standing for
isotope coded) glycans, which were isolated by PGC-LC and
thus the charged species C13G2 and C13G2S2 (and C13G2S1)
were available in pure form and could be individually quantitated
via amino sugar analysis.
Preparation of structures with more antennae and more
sialic acids was undertaken by the stripping off of sialic acids
and—partially—galactose from fetuin glycans. The fraction
with two galactose residues was isolated by HPLC and
furnished with one 13C6-galactose to arrive at C13G3S3
(exactly C13Na6-4[Na6-4Na6-4] in ProGlycAn nomenclature
(http://www.proglycan.com). Complete regalactosylation
with 13C6-galactose would have shifted the mass into a
Table 2 Abundance of N-glycans in human serum. Relative molar abundances were calculated from the peak areas of the monoisotopic peaks and
normalized to the earliest eluted G2S2 (Na6-4Na6-4) variant by the respective correction factors given in Fig. 1
Corrected relative abundance (%)
Absolute quantification was performed with C13 G2S2 as an internal standard with the quadrupole time-of-flight (Q-TOF) instrument
ND not determined
a Not included in equimolar mix. Correction was performed with the Bnearest neighbor^ rule; for example, G1F values were corrected with the factor for G1.
region already occupied by various adduct ions (Fig. 1), hence
the choice of only one 13C6-galactose. Though successful, this
route all too obviously was not suitable for a routine,
largescale preparation. Therefore, the synthesis of triantennary and
tetraantennary glycans was postponed in exchange for the
combination of a broad-range nonlabeled standard set with
just one or two isotope-coded standards for absolute
quantification. However, for the interpretation of elution patterns of
plasma, fetuin, or erythropoietin glycans the isotope-coded
trisialoglycan proved useful (Fig. 2).
Fig. 3 Preparation and isolation by PGC-LC of UDP-13C6-galactose. Isolated peaks were subjected to ion trap MS. Stars symbolize 13C atoms in the
chemical formulas. Gal galactose, Gal-1-P galactose 1-phosphate, Glc glucose
The isotope-coded standards C13G2 and C13G2S2
fortunately occupy parts of the mass spectrum where no adduct
ions interfere (Fig. 4). A small complication occurs when the
isotope-labeled standards are used with the one-peak method
as the isotope pattern of the labeled glycan is not only shifted
by 12 Da but is also altered because of the imperfect isotopic
purity of the 13C-labeled galactose (Fig. 4). The supplier stated
1% 12C, but inspection of the UDP-galactose suggested only
0.85% impurity. This impurity results in a theoretical error of
8.21%; that is, the area of the monoisotopic peak of C13G2
(C13A4A4) should be multiplied by 1.089 to allow a
theoretically correct comparison with natural G2 (A4A4).
Absolute quantif ication of human plasma glycans
and the mysterious glycosylation of bovine serum albumin
The validity of the approach was tested with human
transferrin. With two relevant N-glycosylation sites mainly occupied
by G2S2 (i.e., Na6-4Na6-4) , 70 μg (about 1 nmol) of
transferrin is expected to contain about 1.7 nmol G2S2. The
experimental result gave a content of 1.23 nmol per nanomole
(data not shown). The difference may in part arise from the
moisture content of the glycoprotein.
Applying the same method to human plasma resulted in
absolute concentrations of plasma N-glycans (Table 2).
Whereas immunglobulins are a very abundant class of
glycoproteins in serum, their Fc glycans (G0F, G1F, G2F, and
G2FS1) do not dominate the glycan profile.
A curious case emerged recently when several (glyco)
proteins were analyzed by NMR spectroscopy. HSA gave
clear signals for disialo diantennary N-glycans (G2S2) .
As HSA is generally not considered to be N-glycosylated, this
surprise may come from rare alleles or from impurities
[48, 49]. To determine this, we quantitated the amount of a
possible glycoprotein. We found 67 μg (1 nmol) HSA contained
0.15 nmol G2S2, mainly in the form of Na6-4Na6-4. Thus, a
glycosylated albumin allele would amount to about 15%.
However, a tryptic digest revealed substantial amounts of
serotransferrin, haptoglobin, hemopexin, and
α-1Bglycoprotein (MASCOT scores 892, 712, 382, and 64; HSA
itself was identified with a MASCOT score of 3503) as
contaminants, and it appears justifiable to regard these glycoproteins as
the source of the NaNa seen by NMR spectroscopy. These
(mostly smaller) glycoproteins bear two or more complex N-glycans
and thus may amount to 4-7% of the material. A glycosylated
HSA variant of about 15% would also be seen by intact protein
ESI-MS , but no such variant was found by this approach
(data not shown).
External calibration using a set of N-glycans of
known—preferably equal—concentrations appears to be the minimum
requirement for quantitative evaluation of glycomics data
obtained by ESI-MS of underivatized glycans. Without this
Fig. 4 Mass spectra of G2 and G2S2 in unspiked and spiked samples (A,
B). Note that the isotope-coded glycans emerge in unoccupied spaces in
the spectra. The spectra demonstrate the different isotope distributions of
natural glycans and 12 13C-containing glycans. Panels C confirm the
identical chromatographic behavior of the isotopologues. The
13C6galactose residues are labeled with red circles. EIC extracted ion
correction, the data are hardly more than incidental peak
height ratios that will in addition vary from day to day and
instrument to instrument. A recent attempt at external
calibration showed impressive linearity over several orders of
magnitude of permethylated glycans for a Thermo Fisher LTQ
Orbitrap instrument . Two nongalactosylated, neutral
glycans (GnGnbi and Gn[GnGn]bi in ProGlycAn nomenclature)
and one disialoglycan (Na6-4Na6-4) were the subjects of this
study. A larger panel of structures was used in a study that
introduced the application of stable-isotope-labeled glycans
for internal calibration . This work focused on IgG
glycans and thus on diantennary structures with zero to two
galactoses and fucose and up to two sialic acids. However, as
IgG glycans were analyzed by MALDI-TOF MS, no further
attention was paid to sialylated species.
In this work, we considered a broader range of N-glycans
from Man5 to tetrasialylated N-glycan (Fig. 1, Table 1). These
standards were prepared as reduced glycans suitable for
PGC-LC–ESI-MS, a method that is suitable for both neutral
and sialylated glycans and that has an unsurpassed ability to
separate isomeric forms (e.g., different Neu5Ac linkage 
or fucose linkage). The equimolar glyco tune mix can be used
to optimize the instrument tuning for glycan analysis and it
can be used to measure the different molar responses. By
application of the correction factors obtained to N-glycans of
identical or very similar composition, meaningful abundance
values for essentially all glycan species can be obtained.
Without correction factors, acceptable ratios were obtained
for complex glycans with zero to two sialic acids with the
Q-TOF MS system (Fig. 1, Table 2). However, this may only
hold true for this particular instrument and its tuning. In the
absence of any verification by a standard mixture, even these
common glycan species may give deviating ion abundance
Absolute quantitation could—in principle—be obtained by
external calibration. We noticed, however, that sample
preparation for LC–ESI-MS resulted in considerable loss (about
50%) of material. This problem can be overcome by the
addition of an internal standard at the earliest possible time during
sample processing (e.g., at the end of the enzymatic
deglycosylation). This standard must differ from the sample (i.e., it
must be stable isotope labeled). In the present work we
accomplished this task by a route that exclusively uses the tools of a
biochemistry laboratory. In fact, all enzymes required can be
obtained commercially. We assume, however, that such
isotope-labeled standards will find their most relevant
application not in absolute quantitation but rather in isomer assignment
by PGC-LC–ESI-MS as shown in Fig. 2. Much of the diversity
of the triantennary glycans shown in this example arises from
different sialic acid linkages, which could also be determined
via differential derivatization and MALDI-TOF MS . The
branching pattern of erythropoietin glycans and many other
isomer details can, however, be revealed only by LC–MS.
The isotope labeling of N-glycans with three and more
antennae becomes increasingly difficult and economically
questionable. Hence, we suggest use of a Bbroad-range^
nonlabeled standard for instrument tuning and relative
quantitation. If desired, absolute quantitation can then easily be
achieved with just one or possibly two isotope-labeled glycans
(e.g., G2 and G2S2) as demonstrated with human plasma and
impurities of HSA.
This two-stage strategy can also be realized with the help of
isotope-labeled standards prepared by a different route as
offered by Asparia Glycomics (https://aspariaglycomics.com/).
A unique advantage of the 13C-galactose approach is the
possibility of generating asymmetrically labeled compounds,
where just one arm has received the heavy form, as such
compounds allow the inspection of the fragmentation
behavior under various conditions (data not shown). Another
unique option offered by the enzymatic approach is isotope
coding of glycopeptides or whole glycoproteins for isotope
dilution or pulse-chase experiments.
Taken together, our findings demonstrate the need to
consider the differing molar responses of sugars of different
composition, size, and sialic acid content. The suggested approach
is external calibration with an equimolar glycan mixture that
has already displayed its usefulness during instrument tuning.
Absolute quantitation by the addition of an isotope-labeled
internal standard can then be performed if required.
Acknowledgements Open access funding provided by University of
Natural Resources and Life Sciences Vienna (BOKU). This work, in
particular that of A. Thader, was supported by the Austrian Science
Fund (project number P22274).
Compliance with ethical standards Blood products were purchased
from the University Clinic for Blood Group Serology and Transfusion
Medicine, Graz, Austria). The fraction obtained accrues as waste in the
course of blood component manufacturing from expiring lots. Blood
donors gave their informed consent for the use of expiring lots by the
University Clinic or Blood Group Serology and Transfusion Medicine.
Open Access This article is distributed under the terms of the Creative
C o m m o n s A t t r i b u t i o n 4 . 0 I n t e r n a t i o n a l L i c e n s e ( h t t p : / /
creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a link
to the Creative Commons license, and indicate if changes were made.
Clemens Grünwald-Gruber is a
postdoctoral researcher in the
Laboratory for Glycobiology and
Analytical Biochemistry at the
University of Natural Resources
and Life Sciences, Vienna. He is
the chief operator of the mass
spectrometry equipment of the
proteomics core facility of the
u n i v e r s i t y ’s b i o t e c h n o l o g y
Andreas Thader was a doctoral
student and a postdoctoral
researcher in the working group of
F. Altmann at the University of
Natural Resources and Life
Sciences, Vienna. He specialized
in the cloning and expression of
glycosyltransferases and their
application to generate
stableisotope-labeled N-glycans of
Daniel Maresch works at the
Laboratory for Glycobiology and
Analytical Biochemistry at the
University of Natural Resources
and Life Sciences, Vienna, where
his responsibility is to provide
mass spectrometric know-how to
the participants of a doctoral
program in protein biotechnology.
Thomas Dalik is a long-time
member of the Laboratory for
Glycobiology and Analytical
Biochemistry at the University of
Natural Resources and Life
Sciences, Vienna. His specialty is
the preparation of glycopeptides,
glycans, and natural enzymes
required for the research work of the
group. He is responsible for the
production of the CCD-blocker
that can be used to prevent
glycoprotein-based false positives
in allergy diagnosis (http://www.
Friedrich Altmann is Head of
the Laboratory for Glycobiology
and Analytical Biochemistry at
t h e U n i v e r s i t y o f N a t u r a l
Resources and Life Sciences,
Vienna. His main interests are in
advancing the structural analysis
of glycoproteins and in the
biological impact of protein
glycosylation, for example, in the
context of allergy diagnosis, where
nonhuman glycosylation causes
false positive diagnostic results.
1. North SJ , Hitchen PG , Haslam SM , Dell A. Mass spectrometry in the analysis of N-linked and O-linked glycans . Curr Opin Struct Biol . 2009 ; 19 ( 5 ): 498 - 506 . doi:10.1016/j.sbi. 2009 .05.005.
2. Pabst M , Altmann F. Glycan analysis by modern instrumental methods . Proteomics . 2011 ; 11 ( 4 ): 631 - 43 . doi:10.1002/pmic.201000517.
3. Jensen PH , Karlsson NG , Kolarich D , Packer NH . Structural analysis of N- and O-glycans released from glycoproteins . Nat Protoc . 2012 ; 7 ( 7 ): 1299 - 310 . doi:10.1038/nprot.2012.063.
4. Bigge JC , Patel TP , Bruce JA , Goulding PN , Charles SM , Parekh RB . Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid . Anal Biochem . 1995 ; 230 ( 2 ): 229 - 38 . doi:10.1006/abio.1995.1468.
5. Tao S , Huang Y , Boyes BE , Orlando R. Liquid chromatographyselected reaction monitoring (LC-SRM) approach for the separation and quantitation of sialylated N-glycans linkage isomers . Anal Chem . 2014 ; 86 ( 21 ): 10584 - 90 . doi:10.1021/ac5020996.
6. Ahn J , Bones J , Yu YQ , Rudd PM , Gilar M. Separation of 2- aminobenzamide labeled glycans using hydrophilic interaction chromatography columns packed with 1.7 microm sorbent . J C h r o m a t o g r B . 2 0 1 0 ; 8 7 8 ( 3 - 4 ) : 4 0 3 - 8 . d o i : 1 0 . 1 0 1 6 / j . jchromb. 2009 .12.013.
7. Sethi MK , Thaysen-Andersen M , Smith JT , Baker MS , Packer NH , Hancock WS , et al. Comparative N-glycan profiling of colorectal cancer cell lines reveals unique bisecting GlcNAc and alpha-2,3- linked sialic acid determinants are associated with membrane proteins of the more metastatic/aggressive cell lines . J Proteome Res . 2014 ; 13 ( 1 ): 277 - 88 . doi:10.1021/pr400861m.
8. Bladergroen MR , Reiding KR , Hipgrave Ederveen AL , Vreeker GC , Clerc F , Holst S , et al. Automation of high-throughput mass spectrometry-based plasma N-glycome analysis with linkagespecific sialic acid esterification . J Proteome Res . 2015 ; 14 ( 9 ): 4080 - 6 . doi:10.1021/acs.jproteome.5b00538.
9. Biskup K , Braicu EI , Sehouli J , Tauber R , Blanchard V. The serum glycome to discriminate between early-stage epithelial ovarian cancer and benign ovarian diseases . Dis Markers . 2014 ; 2014 :238197. doi:10.1155/2014/238197.
10. Yuan J , Hashii N , Kawasaki N , Itoh S , Kawanishi T , Hayakawa T. Isotope tag method for quantitative analysis of carbohydrates by liquid chromatography-mass spectrometry . J Chromatogr A . 2005 ; 1067 ( 1-2 ): 145 - 52 .
11. Bowman MJ , Zaia J. Tags for the stable isotopic labeling of carbohydrates and quantitative analysis by mass spectrometry . Anal Chem . 2007 ; 79 ( 15 ): 5777 - 84 . doi:10.1021/ac070581b.
12. Bowman MJ , Zaia J. Comparative glycomics using a tetraplex stable-isotope coded tag . Anal Chem . 2010 ; 82 ( 7 ): 3023 - 31 . doi:10.1021/ac100108w.
13. Prien JM , Prater BD , Qin Q , Cockrill SL. Mass spectrometric-based stable isotopic 2-aminobenzoic acid glycan mapping for rapid glycan screening of biotherapeutics . Anal Chem . 2010 ; 82 ( 4 ): 1498 - 508 . doi:10.1021/ac902617t.
14. Varadi C , Mittermayr S , Millan-Martin S , Bones J. Quantitative twoplex glycan analysis using 12C6 and 13C6 stable isotope 2- aminobenzoic acid labelling and capillary electrophoresis mass spectrometry . Anal Bioanal Chem . 2016 . doi:10.1007/s00216- 016 - 9935 -8.
15. Ridlova G , Mortimer JC , Maslen SL , Dupree P , Stephens E. Oligosaccharide relative quantitation using isotope tagging and normal-phase liquid chromatography/mass spectrometry . Rapid Commun Mass Spectrom . 2008 ; 22 ( 17 ): 2723 - 30 . doi:10.1002 /rcm.3665.
16. Xia B , Feasley CL , Sachdev GP , Smith DF , Cummings RD . Glycan reductive isotope labeling for quantitative glycomics . Anal Biochem . 2009 ; 387 ( 2 ): 162 - 70 . doi:10.1016/j.ab. 2009 .01.028.
17. Gimenez E , Sanz-Nebot V , Rizzi A. Relative quantitation of glycosylation variants by stable isotope labeling of enzymatically released N-glycans using [12C]/[13C] aniline and ZIC-HILIC-ESITOF-MS . Anal Bioanal Chem . 2013 ; 405 ( 23 ): 7307 - 19 . doi:10.1007/s00216- 013 - 7178 -5.
18. Zhang P , Zhang Y , Xue X , Wang C , Wang Z , Huang L. Relative quantitation of glycans using stable isotopic labels 1-(d0/d5) phenyl-3-methyl-5-pyrazolone by mass spectrometry . Anal Biochem . 2011 ; 418 ( 1 ): 1 - 9 . doi:10.1016/j.ab. 2011 .07.006.
19. Sic S , Maier NM , Rizzi AM . Quantitative profiling of O-glycans by electrospray ionization- and matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry after in-gel derivatization with isotope-coded 1-phenyl-3-methyl-5-pyrazolone . Anal Chim Acta . 2016 ; 935 : 187 - 96 . doi:10.1016/j.aca. 2016 .06.032.
20. Hahne H , Neubert P , Kuhn K , Etienne C , Bomgarden R , Rogers JC , et al. Carbonyl-reactive tandem mass tags for the proteome-wide quantification of N-linked glycans . Anal Chem . 2012 ; 84 ( 8 ): 3716 - 24 . doi:10.1021/ac300197c.
21. Wang C , Wu Z , Yuan J , Wang B , Zhang P , Zhang Y , et al. Simplified quantitative glycomics using the stable isotope label Girard's reagent p by electrospray ionization mass spectrometry . J Proteome Res . 2014 ; 13 ( 2 ): 372 - 84 . doi:10.1021/pr4010647.
22. Gong B , Hoyt E , Lynaugh H , Burnina I , Moore R , Thompson A , et al. N-glycosylamine-mediated isotope labeling for mass spectrometry-based quantitative analysis of N-linked glycans . Anal Bioanal Chem . 2013 ; 405 ( 17 ): 5825 - 31 . doi:10.1007/s00216- 013 - 6988 -9.
23. Zhou S , Hu Y , Veillon L , Snovida SI , Rogers JC , Saba J , et al. Quantitative LC-MS/MS glycomic analysis of biological samples using aminoxyTMT . Anal Chem . 2016 ; 88 ( 15 ): 7515 - 22 . doi:10.1021/acs.analchem.6b00465.
24. Walker SH , Taylor AD , Muddiman DC . Individuality normalization when labeling with isotopic glycan hydrazide tags (INLIGHT): a novel glycan-relative quantification strategy . J Am Soc Mass Spectrom . 2013 ; 24 ( 9 ): 1376 - 84 . doi:10.1007/s13361- 013 - 0681 -2.
25. Hecht ES , Scholl EH , Walker SH , Taylor AD , Cliby WA , Motsinger-Reif AA , et al. Relative quantification and higherorder modeling of the plasma glycan cancer burden ratio in ovarian cancer case-control samples . J Proteome Res . 2015 ; 14 ( 10 ): 4394 - 401 . doi:10.1021/acs.jproteome.5b00703.
26. Zhang X , Wang Y , Qian Y , Wu X , Zhang Z , Liu X , et al. Discovery of specific metastasis-related N-glycan alterations in epithelial ovarian cancer based on quantitative glycomics . PLoS One . 2014 ; 9 ( 2 ) : e87978 . doi:10.1371/journal.pone.0087978.
27. Alvarez-Manilla G , Warren NL , Abney T , Atwood 3rd J , Azadi P , York WS , et al. Tools for glycomics: relative quantitation of glycans by isotopic permethylation using 13CH3I . Glycobiology. 2007 ; 17 ( 7 ): 677 - 87 . doi:10.1093/glycob/cwm033.
28. Atwood 3rd JA , Cheng L , Alvarez-Manilla G , Warren NL , York WS , Orlando R. Quantitation by isobaric labeling: applications to glycomics . J Proteome Res . 2008 ; 7 ( 1 ): 367 - 74 . doi:10.1021 /pr070476i.
29. Hu Y , Desantos-Garcia JL , Mechref Y. Comparative glycomic profiling of isotopically permethylated N-glycans by liquid chromatography/electrospray ionization mass spectrometry . Rapid Commun Mass Spectrom . 2013 ; 27 ( 8 ): 865 - 77 . doi:10.1002 /rcm.6512.
30. Echeverria B , Etxebarria J , Ruiz N , Hernandez A , Calvo J , Haberger M , et al. Chemo-enzymatic synthesis of 13C labeled complex N-glycans as internal standards for the absolute glycan quantification by mass spectrometry . Anal Chem . 2015 ; 87 ( 22 ): 11460 - 7 . doi:10.1021/acs.analchem.5b03135.
31. Dong X , Zhou S , Mechref Y. LC-MS/MS analysis of permethylated free oligosaccharides and N-glycans derived from human, bovine, and goat milk samples . Electrophoresis . 2016 ; 37 ( 11 ): 1532 - 48 . doi:10.1002/elps.201500561.
32. Pabst M , Altmann F. Influence of electrosorption, solvent, temperature, and ion polarity on the performance of LC-ESI-MS using graphitic carbon for acidic oligosaccharides . Anal Chem . 2008 ; 80 ( 19 ): 7534 - 42 . doi:10.1021/ac801024r.
33. Pabst M , Bondili JS , Stadlmann J , Mach L , Altmann F. Mass + retention time = structure: a strategy for the analysis of N-glycans by carbon LC-ESI-MS and its application to fibrin N-glycans . Anal Chem . 2007 ; 79 ( 13 ): 5051 - 7 . doi:10.1021/ac070363i.
34. Abrahams JL , Packer NH , Campbell MP . Relative quantitation of multi-antennary N-glycan classes: combining PGC-LC-ESI-MS with exoglycosidase digestion . Analyst . 2015 ; 140 ( 16 ): 5444 - 9 . doi:10.1039/c5an00691k.
35. Mehta N , Porterfield M , Struwe WB , Heiss C , Azadi P , Rudd PM , et al. Mass spectrometric quantification of N-linked glycans by reference to exogenous standards . J Proteome Res . 2016 . doi:10.1021/acs.jproteome.6b00132.
36. Shubhakar A , Kozak RP , Reiding KR , Royle L , Spencer DI , Fernandes DL , et al. Automated high-throughput permethylation for glycosylation analysis of biologics using MALDI-TOF-MS . Anal Chem . 2016 ; 88 ( 17 ): 8562 - 9 . doi:10.1021/acs.analchem. 6 b01639.
37. Green ED , Adelt G , Baenziger JU , Wilson S , Van Halbeek H. The asparagine-linked oligosaccharides on bovine fetuin. structural analysis of N-glycanase-released oligosaccharides by 500-megahertz 1H NMR spectroscopy . J Biol Chem . 1988 ; 263 ( 34 ): 18253 - 68 .
38. Liebminger E , Huttner S , Vavra U , Fischl R , Schoberer J , Grass J , et al. Class I -mannosidases are required for N-glycan processing and root development in Arabidopsis thaliana . Plant Cell . 2009 ; 21 ( 12 ): 3850 - 67 . doi:10.1105/tpc.109.072363.
39. Altmann F. Determination of amino sugars and amino acids in glycoconjugates using precolumn derivatization with ophthalaldehyde . Anal Biochem . 1992 ; 204 ( 1 ): 215 - 9 . doi:10.1016 / 0003 -2697(92)90164- 3 .
40. Pabst M , Grass J , Toegel S , Liebminger E , Strasser R , Altmann F. Isomeric analysis of oligomannosidic N-glycans and their dolichollinked precursors . Glycobiology . 2012 ; 22 ( 3 ): 389 - 99 . doi:10.1093 /glycob/cwr138.
41. Pabst M , Grass J , Fischl R , Leonard R , Jin C , Hinterkorner G , et al. N u c l e o t i d e a n d n u c l e o t i d e s u g a r a n a l y s i s b y l i q u i d chromatography-electrospray ionization-mass spectrometry on surface-conditioned porous graphitic carbon . Anal Chem . 2010 ; 82 ( 23 ): 9782 - 8 . doi:10.1021/ac101975k.
42. Stavenhagen K , Hinneburg H , Thaysen-Andersen M , Hartmann L , Varon Silva D , Fuchser J , et al. Quantitative mapping of glycoprotein micro-heterogeneity and macro-heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides . J Mass Spectrom . 2013 ; 48 ( 6 ): 627 - 39 . doi:10.1002/jms.3210.
43. Bones J , Mittermayr S , O'Donoghue N , Guttman A , Rudd PM . Ultra performance liquid chromatographic profiling of serum Nglycans for fast and efficient identification of cancer associated alterations in glycosylation . Anal Chem . 2010 ; 82 ( 24 ): 10208 - 15 . doi:10.1021/ac102860w.
44. Anderson EP , Maxwell ES , Burton RM. Enzymatic synthesis of C14-labeled uridine diphosphoglucose, galactose 1-phosphate and uridine diphosphogalactose . J Am Chem Soc . 1959 ; 81 : 6514 - 7 .
45. Bulter T , Elling L. Enzymatic synthesis of nucleotide sugars . Glycoconj J . 1999 ; 16 ( 2 ): 147 - 59 .
46. Satomi Y , Shimonishi Y , Hase T , Takao T. Site-specific carbohydrate profiling of human transferrin by nano-flow liquid chromatography/electrospray ionization mass spectrometry . Rapid Commun Mass Spectrom . 2004 ; 18 ( 24 ): 2983 - 8 . doi:10.1002/rcm.1718.
47. Schubert M , Walczak MJ , Aebi M , Wider G . Posttranslational modifications of intact proteins detected by NMR spectroscopy: application to glycosylation . Angew Chem Int Ed. 2015 ; 54 ( 24 ): 7096 - 100 . doi:10.1002/anie.201502093.
48. Peach RJ , Brennan SO . Biochim Biophys Acta . 1991 ; 1097 ( 1 ): 49 - 54
49. Carlson J. et al. Proc Natl Acad Sci U S A . 1992 ; 89 ( 17 ): 8225 - 8229
50. Muneeruddin K , Thomas JJ , Salinas PA , Kaltashov IA . Characterization of small protein aggregates and oligomers using size exclusion chromatography with online detection by native electrospray ionization mass spectrometry . Anal Chem . 2014 ; 86 ( 21 ): 10692 - 9 . doi:10.1021/ac502590h.