EVALUATING MULTIPLEX FRAGMENTATION AND ION MOBILITY SEPARATIONS TO IMPROVE THE
QUALITY OF RAPID LCMS PEPTIDE MAPPING ANALYSES FOR BIOTHERAPEUTIC PROTEINS.
Scott J. Berger, Henry Shion, Asish Chakraborty, St John Skilton, and Weibin Chen, Waters Corporation, Milford, MA
 LCMS peptide mapping remains the fundamental
technique for defining primary structure for
biotherapeutic proteins.
 Improvements in separations, mass detection, and
informatics have reduced typical map acquisition
times to below 90 min., and reduced data processing
from days to hours.
 While
ftraditional
maps
are
invaluable
for
biotherapeutic characterization, they lack the
throughput required of effective screening tools for
early (clone screening, QbD) and late (Formulations,
Stability) development activities.
 Modern TOF analyzers have enabled the rapid
collection of accurate mass peptide mapping data,
but accurate mass only peptide identifications can be
ambiguous, and constitute insufficient evidence for
confident verification of new variants.
 Here, we investigate how MSE multiplexed
fragmentation and ion mobility LCMS could provide
more confident fast peptide map assignments.
UNDERSTANDING LC/HDMSE ANALYSIS
m/z
Accurate mass precursor and multiplexed fragmentation data
were acquired using LC/MSE acquisition methodology.

For conventional 90 min acquisitions, the precursor/
fragmentation (MS/MSE) duty cycle was 1 sec, equally
divided, but reduced to 0.4 sec cycle time during fast map
analyses. This enabled sufficient data points across ion peaks
to facilitate precursor identification, quantification, and
chromatographic linkage of precursors to their fragments.


In selected experiments, SYNAPT ion mobility functionality
was enabled (HDMSE), permitting additional ion separation
prior to CID fragmentation, other parameters unchanged.
TRASTUZUMAB 5 MIN LC/HDMSE PEP MAP
LC/MSE (Low Energy Data)
LC/MSE (Low Energy Data)
m/z
m/z
Waters SYNAPT G2 HDMS
QTof System Schematic
Retention Time (min)
Retention Time (min)
Retention Time (min)
LC/MSE (Elevated Energy Data)
(Fragment Ions)
m/z
E
E
LC/MS data for the 10 min LC/MS map processed by
BiopharmaLynx demonstrated coverage for both mAb
chains comparable to the 90 min run.
Retention Time (min)
Analytical-scale UPLC-QTof MS methods were developed for
rapid (5, 10 min gradient), and typical (90 min) peptide maps
of a biotherapeutic IgG1 monoclonal antibody.

TRASTUZUMAB 10 MIN LC/HDMSE PEP MAP
LC/MSE (Low Energy Data)
(Peptide Ions)
METHODS

TRASTUZUMAB 90 MIN LC/MSE PEP MAP
LC/MSE (Elevated Energy Data)
Summed (6.1-6.3 min) LC/MSE Fragment Data
LC/MSE data for the 5 min LC/MSE map processed by
BiopharmaLynx evidenced slightly reduced map coverage
for both mAb chains.
Heavy Chain T22 (CAM-Cys)
LC/MSE
Heavy Chain T22 (CAM-Cys)
LC/HDMSE
m/z
LC/MSE data acquisition: The Triwave device acts as a
simple collision cell and alternates between low (MS) and
elevated (MSE) energies to collect precursors and multiplexed
fragment ion data in a single experiment.
LC/HDMSE data acquisition: The central Ion Mobility region
is activated to enable gas phase peptide precursor ion
separations (based on mass, charge, shape of peptides). Ion
mobility drift resolved peptides are fragmented in the Transfer
region prior to TOF MS analysis.
Automated data processing was accomplished using the
BiopharmaLynx 1.3.2 software package. Coverage maps
shown include peptides with >2 confirmatory b/y fragments
(>10 ppm error precursor and fragments, 1 missed cleavage,
Mods: CAM-Cys, MetOx, N-deamidation, G0F N-Glycosylation)
In Source CID Fragments
LC/MSE data processed by BiopharmaLynx
demonstrated high coverage for both mAb chains.
Heavy Chain T6 (Deamidated)
LC/MSE
RT 22.18 min
6.1 to 6.3 min
Heavy Chain T6 (Deamidated)
LC/MSE
Heavy Chain T6 (Deamidated)
LC/HDMSE
T6D
T11
OVERVIEW
Ion Mobility Drift (bins)
Heavy Chain T11 (CAM-Cys)
LC/MSE
Heavy Chain T11 (CAM-Cys)
LC/HDMSE
Heavy Chain T11 (CAM-Cys)
LC/MSE
RT 22.15 min
LC/MSE data: software data processing associates peptide
precursor and product (fragment) ions that share common
chromatographic elution profiles.
LC/HDMSE data: Precursors and fragment ion assignments
also must share common ion mobility drift time profiles.
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MSE Fragmentation data for two peptides that
nearly coelute (RT diff 0.03 min) are sufficiently
resolved to yield distinct fragmentation spectra.
Red= y-ion, blue= b-ion, green= neutral loss
LC/MSE fragmentation data remain sufficient for confirming
most
peptide
identifications.
HDMSE
spectra
prove
increasingly useful for simplifying spectra for visual
inspection, given a higher background signal and increased
incidence of peptide coelution. Some additional fragments
appear, as interfering ions were resolved by ion mobility.
CONCLUSIONS
 Sub-10 min LC/MSE mAb peptide maps can yield high
sequence coverage peptide maps, containing accurate
mass fragmentation data sufficient to validate accurate
mass peptide assignments.
 Higher spectral background and greater incidence of
chimeric fragmentation are seen as map times compress.
 This chimeracy does not preclude automated data
Peptides HC T6D and T11 now effectively coelute (6.20 and 6.19 min)
and share some common MSE fragments (Top Left, boxed region) that
are still assigned to the proper precursor by accurate mass. These are
highlighted (PINK ions + arrows) in the corresponding MSE spectra.
Ion mobility enables the gas phase separation of these two peptides
(Drift of 52.4 and 62.0, respectively), enabling the software to assign
fragments directly to the correct precursor.
analysis using physical quality metrics (e.g. min number
or % of b/y ion intensity) to confirm peptide assignment.
 Ion Mobility capabilities of the SYNAPT HDMS QTof (LC/
HDMSE) reduced or eliminated fragment ion chimeracy,
and improved visual data clarity, but were not essential
for MSE fragment ion confirmation in these studies.
©2012 Waters Corporation