Evaluating the Addition of Supercharging Reagents During
CESI-MS for Identification & Characterization ofBiopharmaceuticals
Rajeswari Lakshmanan1; Chitra K. Ratnayake1; Rachel R. Ogorzalek Loo2; Joseph A. Loo2; Edna Betgovargez1
1SCIEX Separations, AB SCIEX, Brea, CA; 2Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA
OVERVIEW
Purpose: Our purpose of this study is to evaluate the effect ofsupercharging reagents on peptide ionization during CESI-MS.
CESI refers to the integration of capillary electrophoresis (CE)with electrospray ionization (ESI) into one dynamic process
within the same devise.
Methods: Trypsin digested monoclonal antibody (mAb) wasanalyzed by tandem MS. Supercharging reagents were added tothe CE background electrolyte (BGE).

EIE of 63-mer tryptic peptide without any reagent (A) and with
1% m-NBA (C). (B) and (D) show the mass spectrum without
and with 1% m-NBA respectively.

Results: Supercharging during CESI-MS showed a significant
increase in the number of higher charge states attained by thepeptides. In addition, for glycopeptides, the intensity of theprecursor ions as well as fragment ions increased withsupercharging.
INTRODUCTION


Electrophoretic separation is based on the charge-tohydrodynamic volume ratio of the peptides and not based onhydrophilic / hydrophobic interactions with the separation
capillary. Large hydrophobic peptides can also be identified
equally well, resulting in the high sequence coverageobtainable by this technique.

Supercharging reagents have been demonstrated toenhance the multiple charging of proteins and peptides
during ESI. This improves the mass measurement accuracy
and fragmentation efficiency.
Recently, Bonvin et al., have added the supercharging
reagents to the sheath liquid interface during CE-MS tomodulate charge state distribution of proteins.1 But, thesheath flow dilutes the analytes migrating from the capillary
as well as increasing the flow. Alternatively, supercharging
reagents can be directly added to the BGE during CESIMS, a ultra-low flow technique, to further maximizeionization.
Palivizumab, a humanized therapeutic antibody, targetedagainst respiratory syncytial virus, is an ideal representationof IgG1 type monoclonal antibody. Tryptic peptidesgenerated from this antibody were analyzed by CESI-MSand supercharging reagents were added to evaluate theefficiency of charge enhancement during CESI-MS.

GFYPSDIAVEWESNGQPE
NNYK

Small hydrophilic peptides, which are often lost in the void
volume in RP-LC can be identified well by CESI-MS. Shown
here are the peptides with 2-5 residues identified from bothheavy and light chains (HC & LC) of palivizumab.

Relative intensities of glycopeptides (G0F, G1F, & G2F)
from palivizumab
with/without
supercharging
werecompared. Addition of either reagent showed a significant
increase in the relative abundance of all 3 glycopeptides at3+ charge state.

Increased intensity of glycopeptide precursor ions obtained
from supercharging resulted in highly intense fragment ions
during tandem MS. The intensity of [peptide+glycan]
fragments increased by approximately 5 to 10 fold for all
three glycopeptides.

ESGPALVKPTQTLTLTCTFSGF

Extracted ion electropherogram (EIE) of peptides frompalivizumab HC ranging from 16 to 23 amino acids. The tableprovides more information about each peptide in the EIE.
m/z
# ofCharge
observed
aaSequence420

WQQGNVFSCSVMHEALHNHYTQK442
8499396412

VTNMDPADTATYYCARTTPPVLDSDGSFFLYSK

423896.3872

216937.4634

217848.7163

322271178.1021

222

GFYPSDIAVEWESNGQPENNYK

6701.319

395374ESGPALVKPTQTLTLTCTFSGF

Sulfolane and m-NBA were added to the BGE (10% aceticacid) at 1% concentration (v/v).

Number of spectra obtained at 95% confidence was plotted
as a function of charge states for each instance.

With 1% sulfolane, the % of confident spectra at 3+ and 4+charges increased compared to without any reagent.

With 1% m-NBA, 50.4% of confident spectra were obtained
from 3+ charge state.

Small % of confident spectra at 5+ charges were present
only when m-NBA or sulfolane was added.

Addition of supercharging reagents results in an increase in
the average and maximum charges attained by thepeptides. Relative intensity of the highest observed charge
state also increases. For instance, in palivizumab HC, a 63
amino acid tryptic peptide without any missed cleavage had
5 fold more intensity with 1% m-NBA.

Consequently, this peptide was identified by MS/MS with atotal of 22 b & y fragment ions only when m-NBA waspresent.

RESULTSI. Identification of small hydrophilic and large hydrophobicpeptides

D

TTPPVLDSDGSFFLYSKII. Charge state distribution of peptides during supercharging
Data analysis: Data analysis was performed using SCIEX
BioPharmaView™ and ProteinPilot™ software. Glycopeptides
were extracted manually and MS/MS spectra were checked fordiagnostic ions.

BIII. Glycopeptide identification and supercharging

VTNMDPADTATYYCARSample preparation: 100 µg of palivizumab was solubilized,
reduced with DTT, alkylated with IAM, and digested overnightwith trypsin. The sample was then dried down and resuspended
in 100 μl of leading electrolyte (100 mM ammonium acetate atpH 4).

Beckman Coulter CESI 8000 was connected on-line to aTripleTOF® 5600+ MS (SCIEX, Framingham, MA). Information
dependent acquisition (IDA) mode consisting of 250 msec TOF
MS survey scan and 50 msec IDA on the top 30 ions which
exceed 150 cps was utilized for data acquisition. Rolling collisionenergy was used to induce fragmentation and the dynamic
exclusion time was set to 5 sec. The total run time was set to 60
min. Auto-calibration was enabled once every 3 runs.

CWQQGNVFSCSVMHEALHNHYTQKMATERIALS AND METHODSCESI-MS conditions: 50 nL of the sample was injected into theseparation capillary and transient-isotachophoresis focuses thesample during electrophoretic separation. 10% acetic acidserved as the BGE and a separation voltage of 20 kV at normal
polarity was applied. For supercharging experiments, reagents
such as m-NBA and sulfolane were added to the BGE.

AExtracted ion electropherogram (EIE) of tryptic peptides
from palivizumab ranging from 2 to 5 amino acids. The tableprovides more information about each peptide in the EIE.

TRADEMARKS/LICENSINGREFERENCES
Bonvin, G., Rudaz, S., Schappler, J. Analytica Chimica Acta 813 (2014) 97-105.

For Research Use Only. Not for use in diagnostic procedures.
© 2015 SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd.
or their respective owners. SCIEX is being used under license.

Tandem MS spectra of G0F glycopeptide at 3+ charge statecompared with and without supercharging reagents. The inset
shows the relative abundance of fragment ion at 1392 m/z.

CONCLUSIONS CESI-MS allowed efficient analysis of both short hydrophilic
and long hydrophobic peptides, thereby providing highsequence coverage for antibody identification.
 Charge state enhancement was observed for tryptic peptides
during CESI-MS. Higher charge states fall in the lower m/z
region and thus, can be fragmented without any limits forprecursor selection during MS/MS.
 Addition of supercharging reagents during CESI-MS
enhances
glycopeptide
identification.
The
relative
abundances of precursor & fragment ions significantly
improved for different glycopeptides. Furthermore, greater
S/N ratio, improved peak shape, and mass resolution canpotentially benefit automated glycopeptide analysis.