Improvedd CESI-MS sen
nsitivity
y and reepeatab
bility innGlycopep
ptide Analysis
As using
g a Dopant Enrriched Nitroge
en Gas1
Guinnevere S. M. Kammeijerr1, Isabelle Kohler
K, Bas C. Jansen1, Paul J. Hen
nsbergen1, O
Oleg A. Mayb
boroda1,1
2
1Daviid Falck , Stephen Lock , and Manfrred Wuhrer1
Leid
den Universiity Medical Center,
C
Centter for Proteomics and M
Metabolomiccs, P.O. Boxx 9600, 2300
0 RC Leiden,,
The Netherlandss, and 2SCIE
EX, United Kingdom
KIntrooduction

Materrials and MeethodsCESI-M
MS method: Trryptic digest sa
amplesElectroospray ionizationn (ESI)-mass spe
ectrometry (MS) is used routinely
y inproteoomics

researchh.

Often

is

it

combined

withnano-liquuidchrom
matography (LC)−−ESI-MS, whose
e flow rates are typically kept inn arange of 100−1000 nL
L/min, due to thee improved ioniz
zation efficiency at2(dissolvved in 250 mM
Mammon ium acetate at pH 4.0 (3:2, v//v)) were analyzzed using a barefused s ilica OptiMS CES
SI cartridge (30 μm ID x 91 cm, polymer coated).
Samplees were injected hydrodynamicallly (5 psi, 60 s eq
quivalent to 44 nL
nditions shown inor 6.9%% capillary volumme) 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 withw
Actionn

the prootein structure and function in mu
ultiple manners
Capillaary Electrosprayy Ionization (CEESI) is the integ
gration of capillaaryelectroophoresis (CE) and electrospra
ay ionization (EESI) into a singgle
1

processs in a single deevice (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

RinseeRinseeRinseeRinseeRinseeRinseeInjectioon
Separatiion
VoltageeTime(min)
2.5
2.5
44
360s
25s
355

Pressure(psi)
100
100
100
100
75
50.5
00Direction
Forw
ward
Forw
ward
Forw
ward
Forw
ward
Revverse
Forw
ward
Forw
ward
Forw
ward
Forw
ward

Voltage(kV)
0000000201Solution
0.1 M NaOH
0.1 M HClWater
10% Acetic acid
10% Acetic acid
Sample Vial
10% Acetic acid
10% Acetic acid
10% Acetic acidin ion suppression at the lower flow ratte. CESI-MS sep
parates analytes by
their ccharge and sizee 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 maass spectromete
erThis ddocument summa
arizes the work recently publishe
ed by the researrch
2group at Leiden Unive
ersity Medical Ce
enter . In this application
anote wew
will shhow how CESI-M
MS can be used to characterize glycopeptides
gfroommodell glycoproteins inncluding a polyclonal immunoglobulin G subclasss1(IgG1)). We will show how
ha dopant en
nriched nitrogen (DEN) gas supp
ply,
previoously shown to immprove peptide sensitivitysby a factor
fof ∼2.6-fold3

in commbination with an optimized innjection volume can be used toimprovve CESI-MS sennsitivity. In this work
wresults fromm CESI-MS will becompa
ared with a conve
entional nano-LC
C-ESI-MS approaach.

(Brukerr Daltonics) wass coupled to the CESI systemm using a source
adapterr available fromm Sciex. All na
ano- LC-ESI-MS
S and CESI-MSS
experimments were carrie
ed out in positivve mode using a capillary voltageof 1200
0 V, end plate offfset voltage 0 V, ion energy of 3.0
0 eV and collisioncell eneergy of 7.0 eV. M
MS source para
ameters were set at 1.2 L/min foorthe dryiing gas and 150
0°C for the sourcce temperature. MS spectra wereacquiredd between m/z 2
200 – 2000 at an
n acquisition rate
e of 1 Hz. For theDEN-ga
as experiments, aan in-house madde polymer cone was slid onto the2capillaryy housing whic h allowed a coa
axial sheath flow
w of the DEN-gas
around the CESI capillaary tip [the conce
entration of Acetoonitrile (MeCN) inEN-gas was e
experimentally ddetermined to be ∼4% (molethe DEpercenttage)]. Nano-LC separations werre carried out on an UltiMate 3000
System from Dionex ussing a core-shell Ascentis Expreess C18 nano-LCCcolumn preceded by a DDionex Acclaim P
PepMap100 C18
8 trap column with2a gradie
ent elution from 00.1% trifluoroaceetic acid to 96% a
acetonitrile .

®

O- Ultra lo
ow flow ESI Interrface.
Figure 1: OptiMS
p1

Resuults

Limits oof Detection (LO Ds) were determ
mined for the G2F glycopeptide ofoThe b
best source connditions for the CESI-MS (deteermined based on
signal intensities, ba
ackground noisse, in-source frragmentation andrepeattability of the reelative abundances of tryptic glycopeptides
gfroompolyclo
onal IgG) indicatted that MeCN as dopant gave th
he best results andIgG moonoclonal antiboddy (mAb) 1 for bboth CESI-MS and nano-LC−ESIMS. Ta
able 2 highlights the lowest conce
entration that wa
as detected with aS/N rati o ≥3.

2similarr to what was reeported for nanoo-LC−ESI-MS . FigureF2, comparres

IgG conc (pg/µµL)

IgG injeccted amount (pg)the relative peak areas observed for glycopeptides
gwitth or without DEEN-Nanno LC-ESI-MS

250

250

gas annd demonstrate a ~2-fold enhancement for all glycopeptides when

CESI-MS75

3.3

CESI--MS + DEN gas

30.1

the DEN-gas was us
sed. Moreover, thet
addition of DEN-gas led too alower abundance of nooise and interfere
ences over the wholew
MS detection
range (i.e., m/z 200−2
2000), especiallyy in the region betweenbm/z 5000−800 w
which showed thee highest level of background intterferences (Figuure3).

Table 2
2. Limits of Deteection observed (lowest concenttration where thedetectedd S/N ratio wa s ≥3) for IgG G
G2F glycopeptid
de of IgGmAb1innano-LCC−ESI-MS and C
CESI-MS with annd without DEN-GGas.

PEP = pep
ptide sequence
EEQYNSTYR

depicted in Figure 4 which show
These rresults are also dws the differences
observeed in the S/N rattios between thee three methods at relatively highh,
mediumm, and low conce
entrations.

Figuree 2 (S-8). Differeences in peak arreas and signal-tto-noise ratio (S//N)

Figure 4. Peak areass of tryptic Fcc N-glycopeptide
e G1F from theIgGmAbb1 obtained witth nano-LC–ESI-MS, conventio
onal CE–ESI-MS
S,and CE
E–ESI-MS with DEN-gas at diffferent concentrrations. (A) Peakareas oobserved at rel atively high, medium, and low
w concentrationss.
Magnificcations of the mmiddle and low cconcentration is displayed in (BB)Peak arreas observed att 1.25 ng/μL andd (C) Peak areas observed at 0.13observved for the try
yptic fucosylated glycopeptidess from polyclon
nalantibo
ody IgG. CE–ES
SI-MS (light bluee) and CE–ESI-MS with DEN-gas
setup (dark blue) with (A) Absolute pea
ak areas and (B) S/N ratios.

Figuree 3 (S-9). Background spectra collectedcbetwee
en 4.5 and 5.5 minmon was below thng/μL. T
This concentratio
he LOD of nanoo-LC–ESI-MS andhence G1F was not d
detected (*). Errror bars represeent the standard
deviatioon (N = 3). Thee PEP illustratees the tryptic p
peptide sequence
EEQYN
NSTYR.

from tthe analysis of tryptict
glycopepttides. (A1) MS spectrumsobtained

At relatiively low concen
ntrations (Figure 4C), no signal w
was detected withwith coonventional CES
SI-MS setup and (B1) CESI-MS using
uDEN-gas.

the refe
erence method ((nano-LC−ESI-MMS) while CE−ES
SI-MS with DEN
Ngas led
d to higher S/N
N ratios (10-fold) compared to the conventiona
alp2

setup..

CE-MS is usuallyuconsiderred to have lowwer concentration

Concclusions

sensitivity than chrom
matographic app
proaches (due tot the significan
ntly
lower loading levels (nanoliter versu
us microliter rannge). However, byA CESII-MS method inttegrating the use
e of a DEN-gass supply has bee
encombining CESI-MS with a DEN-g
gas supply andd an on-line prre-investig
gated for the use
e in the detectionn of glycopeptide
es. CESI-MS withconcentration

DEN-gaas offered severa
al advantages inccluding:-

technique,CESI-MSS

showedbetter

concentration

sensitivity than the state-of-the-artt nano-LC−ESI-MS approachees,making it a very com
mpetitive and attractive technique for glycopeptide
analyssis.

to CESI-MS w
without DEN-gas.
Finallyy repeatability annd intermediate precision was innvestigated at loow,
mediu
um, and high cooncentrations forr the three mosst abundant trypptic

were lower than 4% for all studied concentrations,cwhichw
is similar to4

resultss reported for nano-LC−ESI-MS .
RelativeRabundanceeLower LODs than observed w
with state-of-the--art nano-LC−ESSIMS methods.

glycoppeptides from IgG
GmAb1. Tables 3 displays the areas
aobserved forfrepeattability (intraday variability). Relative standards deviations (RSDDs)

Improved sen
nsitivities for glyccopeptide analysis when compareed

Excellent reppeatability for gllycopeptide deteection with RSD
Dslower than 4%%.

For furtther information on this topic wee would like to reefer readers to thhe2

full scie
entific publication on which this ap
pplication note iss based .

Referrences

Concc, (ng/µL)

GOF

G1F

G2F

0.03

373 % (3.2%)

51 % (2.6%)

12 % (1.5%)

0.30

373 % (1.1%)

51 % (0.9%)

12 % (2.7%)

Elecctrospray Ioniza
ation Mass Specctrometry:

3.00

373 % (0.6%)

51 % (0.4%)

12 % (1.0%)

Cheem., (2010), 82, 9476-9483.

1. Bussnel, J-M., et. al., “High Capa
acity Capillary ElectrophoresisSheeath-lessInterfface

withupling a Porous
Cou

Tran
nsient-Isotachopphoresis”,

Anal.

2. Kammmeijer, G. S. M
M., Kohler, I., Jaansen, B. C., Hensbergen, P. J.,
Table 2. Repeatability (N = 3) of Tryptic Fc N-Glycopep
ptides fromIgGmA
Ab1 with CE−ESI-MS Using the DEN-Gas.
DMayyboroda, O. A.,, Falck, D. and Wuhrer, M. “DDopant enrichednitroogen gas comb
bined with sheatthless capillary eelectrophoresis−−elecctrospray ionizattion-mass Specttrometry for improved sensitivity
yand
d repeatability inn glycopeptide AAnalysis”, Anal C
Chem. 2016 Jun
7;88
8(11):5849-56.
3. Meyyer, J. and Kommives, E. A. “Chharge State Coa
alescence during
elecctrospray ionizattion improves ppeptide identifica
ation by tandem
masss spectrometry””. J. Am. Soc. M
Mass Spectrom. 22012, 23, 1390−−13999.
4. Selmman, M. H. J.; Derks, R. J. E.; Bondt, A.; Palmblad, M.;Schhoenmaker, B.; K
Koeleman, C. A. M.; van de Geijnn, F. E.; Dolhain,R. JJ. E. M.; Deeldeer, A. M.; Wuhrrer, M. J. Proteoomics 2012, 75,
131 8−1329.

Doccument number: RU
UO-MKT-02-4920-A

p3