Improved
d CESI-MS sen
nsitivity
y and re
epeatab
bility in
n
Glycopep
ptide Analysis
A
s using
g a Dopant Enrriched Nitroge
en Gas
1
Guin
nevere S. M. Kammeijerr1, Isabelle Kohler
K
, Bas C. Jansen1, Paul J. Hen
nsbergen1, O
Oleg A. Mayb
boroda1,
1
2
1
Daviid Falck , Stephen Lock , and Manfrred Wuhrer
1
Leid
den Universiity Medical Center,
C
Centter for Proteomics and M
Metabolomiccs, P.O. Boxx 9600, 2300
0 RC Leiden,,
The Netherlands
s, and 2SCIE
EX, United Kingdom
K
Intro
oduction
Materrials and Me
ethods
CESI-M
MS method: Trryptic digest sa
amples
Electro
ospray ionization
n (ESI)-mass spe
ectrometry (MS) is used routinely
y in
proteo
omics
research
h.
Often
is
it
combined
with
nano-liqu
uid
chrom
matography (LC)−
−ESI-MS, whose
e flow rates are typically kept in
n a
range of 100−1000 nL
L/min, due to the
e improved ioniz
zation efficiency at
2
(dissolvved in 250 mM
M
ammon ium acetate at pH 4.0 (3:2, v//v)) were analyzzed using a bare
fused s ilica OptiMS CES
SI cartridge (30 μm ID x 91 cm, polymer coated).
Sample
es were injected hydrodynamicallly (5 psi, 60 s eq
quivalent to 44 nL
nditions shown in
or 6.9%
% capillary volum
me) and then sep
parated using con
Table 1 .
these lower flow rattes. There is a growing intere
est in proteomiics
researrch for the characterization of gly
ycans, as glycans
s can interfere with
w
Action
n
the pro
otein structure and function in mu
ultiple manners
Capilla
ary Electrospray
y Ionization (CE
ESI) is the integ
gration of capilla
ary
electro
ophoresis (CE) and electrospra
ay ionization (E
ESI) into a sing
gle
1
processs in a single de
evice (Figure 1) . CESI-MS operates at lower flo
ow
rates than nano-LC--ESI-MS (10 – 25 nL/min) and offers several
advan
ntages which include increased io
onization efficiency and a reduction
Rinse
e
Rinse
e
Rinse
e
Rinse
e
Rinse
e
Rinse
e
Injectio
on
Separatiion
Voltage
e
Time
(min)
2.5
2.5
4
4
3
60s
25s
35
5
Pressure
(psi)
100
100
100
100
75
5
0.5
0
0
Direction
Forw
ward
Forw
ward
Forw
ward
Forw
ward
Revverse
Forw
ward
Forw
ward
Forw
ward
Forw
ward
Voltage
(kV)
0
0
0
0
0
0
0
20
1
Solution
0.1 M NaOH
0.1 M HCl
Water
10% Acetic acid
10% Acetic acid
Sample Vial
10% Acetic acid
10% Acetic acid
10% Acetic acid
in ion suppression at the lower flow ratte. CESI-MS sep
parates analytes by
their ccharge and size
e and is, thereffore, a complem
mentary separation
alysis of tryptic
Tablle 1: CESI separration conditions used for the ana
digestss.
mecha
anism to more trraditional techniq
ques, such as rev
verse phase nan
noLC-ES
SI-MS.
For MS
S analysis a UH R-QqTOF maXiss Impact HD ma
ass spectromete
er
This d
document summa
arizes the work recently publishe
ed by the researrch
2
group at Leiden Unive
ersity Medical Ce
enter . In this application
a
note we
w
will sh
how how CESI-M
MS can be used to characterize glycopeptides
g
fro
om
modell glycoproteins in
ncluding a polyclonal immunoglobulin G subclass
s1
(IgG1)). We will show how
h
a dopant en
nriched nitrogen (DEN) gas supp
ply,
previo
ously shown to im
mprove peptide sensitivity
s
by a factor
f
of ∼2.6-fold
3
in com
mbination with an optimized in
njection volume can be used to
improvve CESI-MS sen
nsitivity. In this work
w
results from
m CESI-MS will be
compa
ared with a conve
entional nano-LC
C-ESI-MS approa
ach.
(Brukerr Daltonics) wass coupled to the CESI system
m using a source
adapterr available from
m Sciex. All na
ano- LC-ESI-MS
S and CESI-MS
S
experim
ments were carrie
ed out in positivve mode using a capillary voltage
of 1200
0 V, end plate offfset voltage 0 V, ion energy of 3.0
0 eV and collision
cell ene
ergy of 7.0 eV. M
MS source para
ameters were set at 1.2 L/min fo
or
the dryiing gas and 150
0°C for the sourcce temperature. MS spectra were
acquired
d between m/z 2
200 – 2000 at an
n acquisition rate
e of 1 Hz. For the
DEN-ga
as experiments, a
an in-house mad
de polymer cone was slid onto the
2
capillaryy housing whic h allowed a coa
axial sheath flow
w of the DEN-gas
around the CESI capilla
ary tip [the conce
entration of Aceto
onitrile (MeCN) in
EN-gas was e
experimentally d
determined to be ∼4% (mole
the DE
percenttage)]. Nano-LC separations werre carried out on an UltiMate 3000
System from Dionex ussing a core-shell Ascentis Expre
ess C18 nano-LC
C
column preceded by a D
Dionex Acclaim P
PepMap100 C18
8 trap column with
2
a gradie
ent elution from 0
0.1% trifluoroace
etic acid to 96% a
acetonitrile .
®
O
- Ultra lo
ow flow ESI Interrface.
Figure 1: OptiMS
p1
Resu
ults
Limits o
of Detection (LO Ds) were determ
mined for the G2F glycopeptide of
o
The b
best source con
nditions for the CESI-MS (dete
ermined based on
signal intensities, ba
ackground nois
se, in-source frragmentation and
repeattability of the re
elative abundances of tryptic glycopeptides
g
fro
om
polyclo
onal IgG) indicatted that MeCN as dopant gave th
he best results and
IgG mo
onoclonal antibod
dy (mAb) 1 for b
both CESI-MS and nano-LC−ESIMS. Ta
able 2 highlights the lowest conce
entration that wa
as detected with a
S/N rati o ≥3.
2
similarr to what was re
eported for nano
o-LC−ESI-MS . Figure
F
2, comparres
IgG conc (pg/µ
µL)
IgG injeccted amount (pg)
the relative peak areas observed for glycopeptides
g
witth or without DE
EN-
Nan
no LC-ESI-MS
250
250
gas an
nd demonstrate a ~2-fold enhancement for all glycopeptides when
CESI-MS
75
3.3
CESI--MS + DEN gas
3
0.1
the DEN-gas was us
sed. Moreover, the
t
addition of DEN-gas led to
o a
lower abundance of no
oise and interfere
ences over the whole
w
MS detection
range (i.e., m/z 200−2
2000), especially
y in the region between
b
m/z 50
00−
800 w
which showed the
e highest level of background intterferences (Figu
ure
3).
Table 2
2. Limits of Dete
ection observed (lowest concenttration where the
detected
d S/N ratio wa s ≥3) for IgG G
G2F glycopeptid
de of IgGmAb1in
nano-LC
C−ESI-MS and C
CESI-MS with an
nd without DEN-G
Gas.
PEP = pep
ptide sequence
EEQYNSTYR
depicted in Figure 4 which show
These rresults are also d
ws the differences
observe
ed in the S/N rattios between the
e three methods at relatively high
h,
medium
m, and low conce
entrations.
Figure
e 2 (S-8). Differe
ences in peak arreas and signal-tto-noise ratio (S//N)
Figure 4. Peak areass of tryptic Fcc N-glycopeptide
e G1F from the
IgGmAb
b1 obtained witth nano-LC–ESI-MS, conventio
onal CE–ESI-MS
S,
and CE
E–ESI-MS with DEN-gas at diffferent concentrrations. (A) Peak
areas o
observed at rel atively high, medium, and low
w concentrations
s.
Magnificcations of the m
middle and low cconcentration is displayed in (B
B)
Peak arreas observed att 1.25 ng/μL and
d (C) Peak areas observed at 0.13
observved for the try
yptic fucosylated glycopeptides
s from polyclon
nal
antibo
ody IgG. CE–ES
SI-MS (light blue
e) and CE–ESI-MS with DEN-gas
setup (dark blue) with (A) Absolute pea
ak areas and (B) S/N ratios.
Figure
e 3 (S-9). Background spectra collected
c
betwee
en 4.5 and 5.5 min
m
on was below th
ng/μL. T
This concentratio
he LOD of nano
o-LC–ESI-MS and
hence G1F was not d
detected (*). Errror bars represe
ent the standard
deviatio
on (N = 3). The
e PEP illustrate
es the tryptic p
peptide sequence
EEQYN
NSTYR.
from tthe analysis of tryptic
t
glycopepttides. (A1) MS spectrum
s
obtained
At relatiively low concen
ntrations (Figure 4C), no signal w
was detected with
with co
onventional CES
SI-MS setup and (B1) CESI-MS using
u
DEN-gas.
the refe
erence method ((nano-LC−ESI-M
MS) while CE−ES
SI-MS with DEN
Ngas led
d to higher S/N
N ratios (10-fold) compared to the conventiona
al
p2
setup..
CE-MS is usually
u
considerred to have low
wer concentration
Conc
clusions
sensitivity than chrom
matographic app
proaches (due to
t the significan
ntly
lower loading levels (nanoliter versu
us microliter ran
nge). However, by
A CESII-MS method inttegrating the use
e of a DEN-gass supply has bee
en
combining CESI-MS with a DEN-g
gas supply and
d an on-line prre-
investig
gated for the use
e in the detection
n of glycopeptide
es. CESI-MS with
concentration
DEN-ga
as offered severa
al advantages inccluding:-
technique,
CESI-MS
S
showed
better
concentration
sensitivity than the state-of-the-artt nano-LC−ESI-MS approache
es,
making it a very com
mpetitive and attractive technique for glycopeptide
analyssis.

to CESI-MS w
without DEN-gas.

Finallyy repeatability an
nd intermediate precision was in
nvestigated at lo
ow,
mediu
um, and high co
oncentrations forr the three mos
st abundant tryp
ptic
were lower than 4% for all studied concentrations,
c
which
w
is similar to
4
resultss reported for nano-LC−ESI-MS .
Relative
R
abundance
e
Lower LODs than observed w
with state-of-the--art nano-LC−ES
SIMS methods.

glycop
peptides from IgG
GmAb1. Tables 3 displays the areas
a
observed for
f
repeattability (intraday variability). Relative standards deviations (RSD
Ds)
Improved sen
nsitivities for glyccopeptide analysis when compare
ed
Excellent rep
peatability for gllycopeptide dete
ection with RSD
Ds
lower than 4%
%.
For furtther information on this topic we
e would like to re
efer readers to th
he
2
full scie
entific publication on which this ap
pplication note iss based .
Referrences
Concc, (ng/µL)
GOF
G1F
G2F
0.03
37
3 % (3.2%)
51 % (2.6%)
12 % (1.5%)
0.30
37
3 % (1.1%)
51 % (0.9%)
12 % (2.7%)
Elecctrospray Ioniza
ation Mass Specctrometry:
3.00
37
3 % (0.6%)
51 % (0.4%)
12 % (1.0%)
Che
em., (2010), 82, 9476-9483.
1. Bussnel, J-M., et. al., “High Capa
acity Capillary ElectrophoresisShe
eath-less
Interfface
with
upling a Porous
Cou
Tran
nsient-Isotachop
phoresis”,
Anal.
2. Kam
mmeijer, G. S. M
M., Kohler, I., Ja
ansen, B. C., Hensbergen, P. J.,
Table 2. Repeatability (N = 3) of Tryptic Fc N-Glycopep
ptides from
IgGmA
Ab1 with CE−ESI-MS Using the DEN-Gas.
D
Mayyboroda, O. A.,, Falck, D. and Wuhrer, M. “D
Dopant enriched
nitro
ogen gas comb
bined with sheatthless capillary e
electrophoresis−
−
elecctrospray ionizattion-mass Specttrometry for improved sensitivity
y
and
d repeatability in
n glycopeptide A
Analysis”, Anal C
Chem. 2016 Jun
7;88
8(11):5849-56.
3. Meyyer, J. and Kom
mives, E. A. “Ch
harge State Coa
alescence during
elecctrospray ionizattion improves p
peptide identifica
ation by tandem
masss spectrometry””. J. Am. Soc. M
Mass Spectrom. 2
2012, 23, 1390−
−
139
99.
4. Selm
man, M. H. J.; Derks, R. J. E.; Bondt, A.; Palmblad, M.;
Sch
hoenmaker, B.; K
Koeleman, C. A. M.; van de Geijn
n, F. E.; Dolhain,
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Doccument number: RU
UO-MKT-02-4920-A
p3