1. No category

HYDROGEN/DEUTERIUM EXCHANGE MASS SPECTROMETRY (HX MS) SYSTEM FOR CAPTURING CONFORMATIONAL
CHANGES OF BIOTHERAPEUTICS
Joomi Ahn1,3, Keith Fadgen1, Damian Houde2, John R. Engen3
1
Waters Corporation, 2Biogen Idec, Inc., 3Northeastern University
RESULTS
INTRODUCTION
Hydrogen/deuterium exchange mass spectrometry
(HX MS) has proven to be a useful analytical method
for the study of protein dynamics and changes to
protein conformation. The applications in HX MS
require a system that can perform rapid
chromatographic separations at 0 °C and accurate
mass measurements of labeled proteins and peptides
with small quantities of material.1 Recent
improvements in LC-MS have made HX MS an
indispensable tool for biotherapeutic proteins. In this
study, we describe comprehensive HX MS workflow
recently adopted by a biopharmaceutical laboratory.
This workflow utilizes a nanoACQUITY / Synapt HDMS
interfaced with a newly developed chiller module. In
this system, online pepsin digestion was coupled to
highly reproducible UPLC performed at low
temperature. MSE analyses show high confidence
peptide identification, up to 100% linear sequence
coverage, and reproducible peptic peptide peak area
(less than 10 % RSD) in large scale replicate analyses
of phosphorylase b. To illustrate how these data are
useful for HX MS, we describe a recent study of
conformational changes in recombinant monoclonal
IgG1 antibody upon post-translational modifications.2
Label & Quench
Syringe MS Data Collection and Processing:
Undeuterated peptides as control: MSE data were collected for
all analyses. Data were processed using ProteinLynx Global
Server(PLGS) 2.4 with IdentityE
Deuterated peptides : HX-Express was used to measure the
mass shift and peak width upon the deuterium uptake of
peptides.
2
1
3
6
Pepsin column
2.1 x 50 mm
5
HTM 2
Waste (B)
CM
1
3
6
4
Analytical column
1.0 x 100 mm
MS 5
BSM Intact
BSM
Peptides
NanoACQUITY UPLC using 1.7 µm
BEH column Fast separation
ASM
Non-labeled : PLGS Identity E (MSE)
Labeled : HX Express
Chromatographic separation at
0 °C in Chiller Module (CM)
ESI Q-Tof MS
Figure 1. HX MS Workflow from protein labeling to data analysis.5
Peak Area of Phosphorylase b peptides found > 26 times, N=31
Figure 3. MS schematic. The peptide precursor ions
were time-aligned with all of their MSE fragments.
Figure 2. (A) Fluidic schematic for online pepsin digestion
in nanoACQUITY. (B) NanoACQUITY UPLC with Chiller
Module (left). Inside view of Chiller Module (right)
Deuterated peptides
(B)
400000
On‐line Pepsin digestion of phosphorylase b % Coverage
400
Figure 8 Deuterium incorporation information modeled to the
structure of IgG1. Panel A shows the model structure of IgG1
illustrating the location of CH1, CH2, and CH3 domains. The
relative percent deuterium incorporation is shown at 10 sec, 1,
10, 60, 240 min. (B-F, respectively) 2
240 min
Labeling
250000
350
200000
150000
300
100000
e
ga 250
r
e
v 200
o
C
%150
50000
15‐22
37‐52
67‐84
84‐89
87‐99
89‐99
112‐119
124‐139
127‐139
146‐152
181‐196
181‐198
182‐202
202‐215
219‐235
222‐240
224‐240
243‐252
256‐264
273‐285
326‐337
337‐350
366‐373
384‐393
396‐405
405‐418
405‐425
444‐454
471‐491
510‐515
523‐529
523‐530
533‐545
548‐562
566‐580
586‐597
586‐604
604‐618
618‐624
624‐640
625‐640
652‐661
661‐681
687‐696
699‐708
712‐721
717‐730
730‐738
730‐740
779‐785
800‐819
0
Sequence in amino acid #
16
14
12
10
8
6
4
2
0
60 min
400
Deuterium Level of FSTKDSSAAW peptide
354
10 min
100
50
% RSD of peak area
17 % RSD of in‐solution pepsin digestion
0 sec
control
94
0
% Linear % Overlapping, % Redundancy, coverage, N=5
N=5
N=5
5.2 % RSD of on‐line pepsin digestion
(C)...
1 min
10 sec
...
(B) The % Coverage of on-line pepsin digestion of phosphorylase b. The linear coverage was 94 % and the overlapping coverage
was 400 %, which was calculated by the normalized number of amino acid residues that repeatedly found from the overlapped
peptides. The redundancy score was calculated by the normalized sum of amino acid count that repeatedly found in the sequence.
(C) A part of phosphorylase b sequence map was shown to illustrate the overlapping coverage and redundancy score.
TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS
CONCLUSIONS
4.0
3.5
3.0
 NanoACQUITY UPLC with newly improved
Chiller Module was used in HD exchange MS.
2.5
2.0
1.5
1.0
0.5
0.0
0.1
1.0
10.0
100.0
 HD exchange MS analysis is a useful analytical
method for protein conformational studies.
Time (min)
Sequence in amino acid #
Figure 5. (A) Reproducibility of peak area for peptic peptides in replicates of
phosphorylase b in nanoACQUITY system. This indicated that the majority of peptides
produced from online digestion were well below 17 % RSD peak area in in-solution offline digestion.
Relative Deuterium Level (Da)
Peak Area
FSTKDSSAAW peptide at 5.2min
240 min
Labeling
350000
300000
Figure 4. Accurate peptide
identification based on MSE.
First, the undeuterated
(control) peptides were identified in this manner, then
deuterated peptides of same
species were confirmed with
retention time.
E
Online Pepsin Digestion
533‐545
730‐740
181‐198
182‐202
717‐730
699‐708
779‐785
366‐373
618‐624
337‐350
687‐696
523‐529
586‐597
566‐580
219‐235
523‐530
67‐84
112‐119
444‐454
84‐89
224‐240
146‐152
124‐139
256‐264
652‐661
326‐337
273‐285
202‐215
405‐425
661‐681
89‐99
730‐738
222‐240
127‐139
625‐640
243‐252
15‐22
384‐393
624‐640
548‐562
800‐819
396‐405
87‐99
405‐418
471‐491
181‐196
37‐52
604‐618
712‐721
510‐515
586‐604
On-line pepsin digestion : 2.1 x 50 mm immobilized pepsin
columns packed in-house3 and 2.1 x 30 mm pepsin column
purchased from Applied Biosystems
Undeuterated peptides
ASM (A)
Data Analysis
At Eluting Mode (Default) 4
% RSD of peak area
Chromatography
Peptides: The analytical column was an ACQUITY UPLC® BEH
C18 1.7 µm 1.0 x 150 mm. The trap column was an ACQUITY
VanGuard® Pre-column, BEH C18, 1.7 µm 2.1 x 5 mm.
Intact Protein : The analytical column was an ACQUITY
UPLC® BEH C4 1.7 µm 2.1 x 50 mm. The desalting column
was used a MassPREP Desalting cartridge column 2.1 x 5 mm.
Waters ESI Q‐TOF MS
Injection Needle METHODS
LC / MS system:
Waters nanoACQUITY UPLC®
Chiller Module (CM) and Electric Module (EM)
Binary Solvent Manager (BSM)
Auxillary Solvent Manger (ASM)
Waters SynaptTM HDMSTM
ESI positive mode
Capillary / Cone : 3.0 kV / 37 V
Source / Desolvation : 80 °C / 175 °C
Desolvation gas : 800 L/h
DISCUSSION
NanoACQUITY UPLC with Chiller Module
(A)
On-line pepsin digestion pH 2.5
in Chiller Module at 0 °C
&
0 sec
control
Figure 6. BPI chromatograms and spectra of interferon
alpha-2b. The reproducible chromatograms with
undeuterated (0 sec) and deuterated (10 sec, 1, 10, 60, and
240 min. D2O labeling, respectively) peptides were shown.
The higher mass shift was observed upon deuterium uptake
at longer time of continuous labeling.
References
Figure 7. Deuterium incorporation calculation in HXExpress.4 The relative deuterium level (in Da) was
plotted in right panel. The data shows the relative mass
difference between control and each time point, which
was determined by centroid masses in green bar.
1.
2.
3.
4.
5.
Wales et.al. (2008) Anal. Chem., 80, (17) 6815-6820
Houde et al. (2009) Anal. Chem., 81 (7) 2644-2651
Wang, L et al. (2002). Mol Cell Proteomics 1(2):132-8
Weis et al. (2006). J. Amer.Soc. Mass Spectrom. 17 (12) 1700-1703
Mitchell, J. L. & Engen, J. R. (2009). Protein Analysis with HydrogenDeuterium Exchange Mass Spectrometry. In “Protein Mass Spectrometry”,
Elsevier. J. Whitelegge, Editor. Compr. Anal. Chem. 52, 83-102
720003334en