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TOP-DOWN SEQUENCE ANALYSIS OF PROTEINS VIA
ION-MOBILITY TIME-OF-FLIGHT MASS SPECTROMETRY
Asish B. Chakraborty, Weibin Chen, and Jeff Mazzeo
Biopharmaceutical Sciences, Waters Corporation, Milford, MA 01757
OVERVIEW
TOP-DOWN FRAGMENTATION OF G3PDH
WATERS SYNAPT G2 HDMS
Top-down Sequencing of LC after SEC Separation
 Top-down or middle-down MS methodology directly
UV280
HC
fragments intact proteins or protein subunit to
obtain
sequence
information
for
protein
characterization or identification.
LC
 The many different types of fragments from direct
MW: 36 kDa
Top-down Fragmentation of rhGH to Probe the
Sequence Variation
rhGH,CAA23779.1
2+ to 5+
Salts
rhGH,P01241
HC
fragmentation of large proteins mean that spectral
interpretation
and
sequence
deduction
is
challenging.
LC
Salts
TIC
 Waters SYNAPT® G2 HDMS™ System combines
TOP-DOWN PROTEIN SEQUENCING
BY SYNAPT G2 HDMS
Top-down Fragmentation of LC followed by IMS Separation
TOF Analyzer
Antibody
fragments
from UPLC
SEC Separation

m/z
 
Data
Processing
Drift Time
Select a few charge states of protein or the light
 The LC/MS system was configured with a Waters
ACQUITY UPLC® chromatography system and a Waters
SYNAPT G2 HDMS quadrupole ion-mobility time-offlight mass spectrometer.
Top-down spectra of glyceraldehyde-3-phosphate
dehydrogenase (precursor m/z 943.5, +38). Spectra contain
multiple charge states that complicates the sequence
interpretation. The 3D viewer (below) helps to disentangle some
of the complexity by clarifying the view for the user.
TOF mode (resolution mode) for all analyses.
MassLynx™ 4.1 software was used for instrument
control and data processing.
 The LC and HC of mAb were generated from a limited
DTT reduction.
Chromatographic separations were
performed on a 4.6 x150 mm, ACQUITY UPLC® BEH200
SEC, 1.7 µm column.
ACQUITY UPLC/TUV
Column: SEC BEH 4.6 x 150 mm, 1.7 µm
Flow Rate: 0.3 mL/min
Solvent: 30% ACN in 0.1%TFA and 0.1%FA
Column Temperature: 30 °C
TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS
b32
b34
b35
b36
3858.99 3988.03
3729.96
4059.07
0
GH FRACTION 6POSITION H6_TOF_MSMS_06AC 785 (13.428) M3 [Ev-763401,It50,En2] (0.050,200.00,0.200,1400.00,6,Cmp); Cm (1:1166)
m/z
TOF MSMS 1307.59ES+
1.07e6
3391.74
100
+1 charge state
3262.71
Dr
ift
2971.58
Tim
e
3667.84
3134.65
3538.80
2870.54
y96
y967+
A
y957+
y977+
y966+
a2
b4
b7
y976+
b6
y8 y9
2642.39 2755.50
y11
y13
2500
2600
2700
2800
3961.95
2900
3000
3100
3200
3300
3400
3500
3600
3700
3800
3900
4000
4100
4200
4300
4400
4500
mass
4600
M -NH2
CONCLUSIONS
 We have demonstrated the performance of Synapt G2
C-term sequence:
HQGLSSPVTKSFNRGEC
y7
S
3867.94
Figure 5. MaxEnt 3 deconvoluted spectrum showing a portion of
the spectrum, using the 2+ to 5+ charged fragment ions of rhGh.
Comparison of the fragment masses reveals the sequence
difference of the two GH variants.
N-term sequence:
DIQMTQSPSSLSASVGDR
b3
3796.90
7+
0
y978+
b5
-
b31
3325.78
2799.56 2914.59
states, sizes and masses by ion mobility
 UPLC SEC Conditions for MS Analysis
b33
3453.84
b28
3061.66
1: TOF MSMS 1302.40ES+
2.88e5
3582.89
2686.44
Separate CID fragment ions based on the charge
 The SYNAPT G2 HDMS was operated in the mobility-
b29
b24
the TRAP cell
fragmentation data
b27
b26
y968+
Process data with software tools to simplify the
3144.70
100
higher charge
states
chain or using the quadrupole
Fragment the ions using CID in
b30
091115-UCA012-WBC-08-GH 150 (4.965) M3 [Ev-633598,It50,En1] (0.050,200.00,0.200,1400.00,5,Cmp); Cm (148:154)
100513_UCA064_WCAC_LYSC_DIG_PEAK 1_LC_IMS_MSMS_02.raw : 1
Figure 1.

Figure 4. Top-down sequencing of rhGH variants.
%
 In this work, we demonstrate the top-down analysis
of
recombinant
human
growth
hormone,
Glyceraldehyde-3-phosphate
dehydrogenase
(G3PDH) and an antibody subunit (LC) for the
structural characterization of the proteins on a
SYNAPT G2 HDMS.
Figure 2. Separation of LC from HC using Waters Acquity BEH200
SEC 1.7 µm column (p/n:186005225).
%
QuanTof™ technology and enhanced ion-mobility
separation (IMS) to provide a unique instrument
platform for successful top-down protein analysis.
y15
y17
B
Figure 3 (A). Spectrum of higher charge state fragment ions
selected from the IMS display (DriftScope™); (B). Spectrum of 1+
charged fragment ions (precursor m/z: 1079.4, 22+) following ion
mobility separation.
HDMS for the top-down sequencing of G3P
dehydrogenase, rhGH and the light chain of a
monoclonal antibody.
 The post-fragmentation IMS separation enabled the
separation of different charge states of fragment ions,
produced easily interpretable spectra from selected ion
fragments, and increased sequence coverage.
 The QuanTof technology offers improved MS resolution
and accurate mass measurement for fragment ions
with higher charge states. When coupled with
enhanced IMS resolution, it offers a practical top-down
methodology
for
comprehensive
protein
characterization.