Evaluating the Addition of Supercharging Reagents During CESI-MS for Identificationand Characterization of Biopharmaceuticals
Rajeswari Lakshmanan1; Chitra K. Ratnayake1; Rachel R. Ogorzalek Loo2; Joseph A. Loo2,1SCIEX Separations, AB SCIEX, Brea, CA; 2Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA
MATERIALS AND METHODS

Purpose: Previously, we reported on the integration of capillary electrophoresis (CE) withSample preparation: 100 µg of palivizumab was solubilized, reduced with DTT, alkylatedwith IAM, and digested overnight with trypsin. The sample was then dried down andresuspended in 100 μl of leading electrolyte (100 mM ammonium acetate at pH 4).

Results: Supercharging during CESI-MS showed a significant increase in the number ofCESI was connected on-line to a TripleTOF® 5600+ MS. Information dependent acquisition

higher charge states attained by the peptides. In addition, for glycopeptides, the intensity ofthe precursor ions as well as fragment ions increased with supercharging.

(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 collision energy wasused 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.

INTRODUCTIONThe integration of CE and ESI into CESI is achieved by making the end of theseparation of capillary porous to ion flow – this is achieved through an HF etchingprocess.

Data analysis: Data analysis was performed using AB SCIEX BioPharmaView™ andProteinPilot™ software. Glycopeptides were extracted manually and MS/MS spectra werechecked for diagnostic ions.

This tip is then inserted into a metal needle and filled with a static conductive liquid toestablish an electrical connection.

RESULTS

Analytes migrate accordingtotheirelectrophoretic
mobility inside the capillary
and when it reaches theporous sprayer tip, it iselectro-sprayed into MS.
The ultra-low flow of theprocess, yields very highionization efficiency andserves
to reduce ion
suppression.

ESI Needle
Static Conductive Liquid
Nanospray
Separation
capillaryPorous Capillary
Segment

Schematic of CESI porous tip sprayer
Supercharging reagents have been demonstrated to enhance the multiple charging ofproteins and peptides during ESI. This improves the mass measurement accuracy andfragmentation efficiency.
Recently, Bonvin et al., have added the supercharging reagents to the sheath liquidinterface during CE-MS to modulate charge state distribution of proteins.1 But, thesheath flow dilutes the analytes migrating from the capillary as well as increasing theflow. Alternatively, supercharging reagents can be directly added to the BGE during
CESI-MS, a ultra-low flow technique, to further maximize ionization.
Palivizumab, a humanized therapeutic antibody, targeted against respiratory syncytialvirus, is an ideal representation of IgG1 type monoclonal antibody. Tryptic peptides
generated from this antibody were analyzed by CESI-MS and supercharging reagentswere added to evaluate the efficiency of charge enhancement during CESI-MS.

Sequence
VTNMDPADTATYYCAR

Charge420

WQQGNVFSCSVMHEALHNHYTQK442

701.319423 Increased intensity of glycopeptide precursor ionsobtained from supercharging resulted in highly
intense fragment ions during tandem MS. The
intensity of [peptide+glycan] fragments increased byapproximately 5 to 10 fold for all three glycopeptides.

84

VTNMDPADTATYYCAR99

TTPPVLDSDGSFFLYSK

896.3872

216

TTPPVLDSDGSFF
937.4634
LYSK412217396GFYPSDIAVEWESNGQPENNYK374

ESGPALVKPTQTLTLTCTFSGF

GFYPSDIAVEWES
848.7163
NGQPENNYK395

3

ESGPALVKPTQTLTL
1178.1021
TCTFSGF27222Tandem MS spectra of G0F glycopeptide at 3+ charge
state compared with and without supercharging
reagents. The inset shows the relative abundance offragment ion at 1392 m/z.

622

Extracted ion electropherogram (EIE) of peptides from palivizumab HC ranging from 16 to 23 aminoacids. The table provides more information about each peptide in the EIE.

Average relative abundances ofglycopeptides at different charge statescompared with and without supercharging
4.00E+06
3.50E+06
3.00E+06

No Reagent
2.50E+06

1% m-NBA
2.00E+06

1% Sulfolane

1.50E+06
1.00E+06
5.00E+05
0.00E+00

G0F G1F G2F G0F G1F G2F
3+ 3+ 3+ 4+ 4+ 4+No Reagent

G0F

II. Charge state distribution of peptides during supercharging

EEQYNSTYR
EEQYNSTYR Small hydrophilic peptides, which
often lost in the void volume in RPLC can be identified well by CESIMS. Shown here are the peptides
with 2-5 residues identified fromboth heavy and light chains (HC &LC) of palivizumab.
Extracted ion electropherogram(EIE) of tryptic peptides frompalivizumab ranging from 2 to 5
amino acids. The table provides
more information about eachpeptide in the EIE.

No.

Sequencem/z observedCharge12345678910111213141516AKSRAPKEAK
EYK
VEPK
DTSK
VQWK
TISK
GQPR
VDKR
SCDK
ADYEK
QPPGK
LTISK
LTVDK

218.1503
262.152315.2031
347.1935
439.2193
236.6423
225.6136
280.6635
448.2772
457.2513
517.3084
255.1054
313.1451
263.6529
281.1845
288.1741

11111222
111222222

9515843611612

1011[M+3H]3+ = 878.686 Da

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

I. Identification of small hydrophilic and large hydrophobic peptides

1314 7

 Addition of either reagent showed a significant
increase in the relative abundance of all 3
glycopeptides at 3+ charge state.

m/zobserved

Antibodychain
HC
HC
LC
LC
HC
HC
HC
LC
HC
HC
HC
HC
LC
HC
HC
HC

EEQYNSTYR
EEQYNSTYR

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

EEQYNSTYR

EEQYNSTYR

EEQYNSTYR

EEQYNSTYREEQYNSTYR

EEQYNSTYR
EEQYNSTYRACBD

EEQYNSTYR

1% m-NBA

G0F[M+3H]3+ = 878.686 Da

 Small % of confident spectra at 5+ charges were present only when m-NBA or sulfolane wasadded.
Precursor Charge Distribution
Precursor Charge Distribution
Precursor Charge Distribution
with1%
Sulfolane
with 1% m-NBA
without
Reagent
60%
60%
60%
54.9%
 Addition of
50.4%
48.8%
50%
50%
50%
supercharging
41.4%
39.3%
40%
40%
40%
reagents results
27.7%
30%
30%
30%
in an increase in18.8%
20%
20%
20%
the average and
9.4%
10%
10%
10%
4.7%
maximum
3.0%
1.1%
0.3%
0.1%
0.0%
0.0%
0%
0%
0%
charges attained
123456123456123456Charge State
Charge State
Charge Stateby the peptides.
 Relative intensity of the highest observed charge
state also increases. For instance, in palivizumab HC,
a 63 amino acid tryptic peptide without any missedcleavage had 5 fold more intensity with 1% m-NBA.
 Consequently, this peptide was identified by MS/MS
with a total of 22 b & y fragment ions only when mNBA was present.

EEQYNSTYR

EEQYNSTYR

 With 1% sulfolane, the % of confident spectra at 3+ and 4+ charges increased compared towithout any reagent.
 With 1% m-NBA, 50.4% of confident spectra were obtained from 3+ charge state.

EEQYNSTYR

EEQYNSTYR

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

Percent of Confident Spectra

separated by CE and identified bytandem MS. Supercharging reagents were added to the CE background electrolyte (BGE).

No. ofaminoacids

WQQGNVFSCSVMHEALHNHYTQKPercent of Confident Spectra

Methods: Trypsin digested monoclonal antibody was

CESI-MS conditions: 50 nL of the sample was injected into the separation capillary andtransient-isotachophoresis focuses the sample during electrophoretic separation. 10%
acetic acid served as the BGE and a separation voltage of 20 kV at normal polarity wasapplied. For supercharging experiments, reagents such as m-NBA and sulfolane were addedto the BGE.

III. Glycopeptide identification and supercharging
 Relative intensities of glycopeptides (G0F, G1F, &G2F) from palivizumab with and without
supercharging were compared.

Percent of Confident Spectra

electrospray ionization (ESI) into one dynamic process which we have termed CESI. Our
purpose of this study is to evaluate the effect of supercharging reagents on peptide
ionization during CESI-MS.

 Electrophoretic separation is based on the charge-to-hydrodynamic volume ratio of the peptides
and not based on hydrophilic / hydrophobic interactions with the separation capillary. Largehydrophobic peptides can also be identified equally well, resulting in the high sequence
coverage obtainable by this technique.

Relative abundances calculated using peak areas

OVERVIEW

EEQYNSTYR

EEQYNSTYR

1% Sulfolane
G0F

EEQYNSTYR
EEQYNSTYR

EEQYNSTYR[M+3H]3+ = 878.686 Da

EEQYNSTYR
EEQYNSTYR

EEQYNSTYR
EEQYNSTYR

EEQYNSTYR

EEQYNSTYR

In addition, 4+ charge state for all 3 glycopeptides were present only with addition of m-NBA. This is beneficial
for glycopeptide analysis since it moves the high m/z glycopeptide precursor ions into the lower m/z region
where it can be easily selected for MS/MS, without any limitation from the mass analyzer.

CONCLUSIONS CESI-MS allowed efficient analysis of both short hydrophilic and long hydrophobic peptides, thereby providing
high sequence coverage for antibody identification.
 Charge state enhancement was observed for tryptic peptides during CESI-MS. Higher charge states fall in thelower m/z region and thus, can be fragmented without any limits for precursor selection during MS/MS.
 Addition of supercharging reagents during CESI-MS enhances glycopeptide identification. The relative
abundances of precursor and fragment ions significantly improved for different glycopeptides. Furthermore,
greater S/N ratio, improved peak shape, and mass resolution can potentially benefit automated glycopeptide
analysis.
REFERENCES
Bonvin, G., Rudaz, S., Schappler, J. Analytica Chimica Acta 813 (2014) 97-105.
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