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3. HDX results in 3D structure and Heat map

1. Intact HDX

OVERVIEW This study is to investigate the conformational changes in calcium binding protein, calmodulin, utilizing a Waters nanoACQUITY UPLC® System with Hydrogen/Deuterium Exchange (HDX) Technology.

INTRODUCTION Hydrogen/deuterium exchange mass spectrometry (HDX MS) has proven to be a useful analytical method for the study of protein dynamics and changes to protein conformation. The applications in HDX MS require a system that can perform rapid chromatographic separations at 0 °C and accurate mass measurements of deuterium labeled proteins and peptides with small quantities of material. Recent improvements in LC-MS have made HDX MS an indispensible tool for discovery and development of protein drugs. In this study, a nanoACQUITY UPLC System with dedicated cooled module (HDX manager) was used as a robust HDX MS platform for protein conformational analysis. In this system, online pepsin digestion was coupled to highly reproducible separation performed at low temperature.1 High sequence coverage, up to 94% was obtained by the confident peptide identification. Precursor and fragment ions were acquired in a data-independent manner by alternating the collision energy between a low and elevated energy state on a Q-Tof Mass spectrometer (MSE). We report a recent HDX MS study of conformational changes in important intracellular protein, calmodulin with calcium bound (holo) and without calcium (apo).

METHODS Waters nanoACQUITY UPLC® with HDX Technology Binary Solvent Manager (BSM): Eluent A: 0.1 % formic acid in water,

Eluent B: 0.1 % formic acid in acetonitrile Auxiliary Solvent Manger (ASM): Eluent A: 0.05 % formic acid in water HDX manager : online digestion/desalting and separation at 0 °C Waters Xevo® QTof MS ESI positive mode was used. Capillary/Cone: 3.0 kV/37 V, Source/Desolvation temperature: 80 °C / 175 °C, Desolvation gas: 800 L/h Chromatography Peptides: The analytical column was an ACQUITY UPLC® BEH C18 1.7

µm 1.0 x 100 mm. The trap column was an ACQUITY UPLC 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.

On-line pepsin digestion : 2.1 x 30 mm pepsin column from Applied Biosystems was used. Protein preparation Calmodulin protein (CaM) from Sigma was prepared with calcium (Holo) and without calcium bound (apo).2 Labeling solution was prepared with 20 mM HEPES in D2O at pH 7.07 with CaCl2 for holo and without CaCl2 for Apo. The labeled protein was quenched after the timed incubation with 33 mM HCl to pH 2.5.

HYDROGEN/DEUTERIUM EXCHANGE MASS SPECTROMETRY SYSTEM FOR INVESTIGATING CONFORMATIONAL CHANGES IN CALMODULIN PROTEIN UPON CALCIUM BINDING Joomi Ahn 1, Michael Eggertson1, Martha Stapels1, Keith Fadgen1, Rebecca J. Rose2, 3, Ying Qing Yu1, Albert J. R. Heck2, 3, Jeffrey R. Mazzeo1 1Waters Corporation, 2Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Netherlands 3Netherlands Proteomics Centre, Utrecht, Netherlands

RESULTS & DISCUSSION

CONCLUSIONS

The nanoACQUITY UPLC System with HDX

Technology was very useful as a robust HDX MS platform for studying protein conformation.

Global and local conformation of CaM were

analyzed. The peptide-level comparison revealed the location of conformational changes. No visible change and minor change were observed upon various conditions for stability experiments.

This HDX information is useful to better

understand biophysical properties of the biomolecules.

References 1. Wales, et al. (2008) Anal. Chem., 80, 6815-6820 2. Zhu, et al. (2003) Biochem., 42, 15388-15397 3. www.hxms.com/mstools/ 4. www.hxms.com/HXExpress Acknowledgement Authors would like to thank Prof. John R. Engen and Dr. Tomas Wales for helpful input.

Figure 6. Heat map of the entire CaM sequence for comparing HDX results. Two sets of colored bands of heat map represent the % deuterium uptake of CaM time course for apo CaM (top) and holo (bottom). Darker color represents less exchange. The highlighted region of the black box indicates the region with conformational change; different color trend was observed between apo and holo. The red circle represents no change.

Figure 1. Workflow of HDX MS analysis for peptide level. Protein labeling and injection can be automated using Leap HDX automation manager (blue line). Digestion and separation can be done at 0 °C inside of a HDX manager.

COOLED

 TO 0˚C             

OP

TIO

NA

L A

UT

OM

ATIO

N

Online Pepsin digestion pH 2.5 at 0 °C

nanoACQUITY UPLC using 1.7 µm BEH column in fast separation

ESI Q‐Tof MS

Non‐labeled : PLGS IdentityE (MSE)

Protein in H2O, pH 7 at 20 °C

Protein labeling occurs

Labelling quenched chemically and back‐exchange minimised

Reduce pH to 2.5, temp to 0˚C

Add 20‐fold excess D2O

Local Analysis at Peptide Level

Global Analysis at Intact Level

Labeled : HDX Browser & HX‐Express

Peptide Map

HDX Result

Figure 5. Deuterium uptake information represented in CaM apo and holo 3D structures in time course at 10s, 1m, 10m, 60m, 240m labeling. At 10 s labeling, the same region of CaM were compared in black circles for apo and holo. Because of no calcium bound in apo, increased deuterium incorporation was found.

0   10     20    30     40    50    60    70    80 

Relative % deuterium uptake

Figure 2. (A) HDX analyses presented in this study utilized Waters nanoACQUITY UPLC system with HDX technology. The system consists of HDX manager (top unit), nano binary solvent manager (middle), and nano auxiliary solvent manager (bottom unit). (B) Xevo QTof MS ESI-MS (C) ProteinLynx Global Server™ (PLGS) ver.2.4 with IdentityE

for accurate peptide identification.

(A)

2.4 (C)

(B)

Figure 3(A) Intact protein analysis is shown for global HDX MS. Intact spectra of apo and holo CaM showed the mass shift from the control to each time point at charge state 10+. Apo CaM showed greater mass shift than the holo (red arrows) due to the conformational change.

(A)

m/z1680 1700 1720

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100 1692.3646

1716.0874

1692.4429

1716.2711

1691.4786

1715.3264

1690.4626

1714.1721

1688.0149

1711.8644

1685.9591

1679.9030

1703.7478m/z

1680 1700 1720

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100 1691.5048

1715.4052

1689.8114

1713.6997

1687.5203

1711.4712

1686.6616

1710.5801

1684.5024

1682.9686

1706.9659

1679.9291

240 m

60 m

10 m

5 m

1 m

10 s

Control0 s

Apo CaM HoloCaM Deuterium uptake of intact level

0

20

40

60

80

100

120

140

0 1 10 100 1000

Rela

tive D

eu

teri

um

(D

a)

Time (min)

APO

HOLO

36 Da increase at 1 min labeling

2. Peptide HDX

Figure 4(A) Peptide analysis is shown for local HDX MS. The chromatograms of CaM peptides digested by online pepsin column was shown in left panel and The spectra for one of the peptides was shown in the right panel. The increased mass shift was seen from control (0s) to each time point (10s, 1m, 10m, 60m, and 240m) due to longer time exposure in deuterium labeling. (B) 94 % sequence coverage was achieved with many overlapping peptides. These peptides were identified by PLGS and the map was constructed 3 with common peptides exist both in apo and holo.

(A) Spectra

(B)

(C) HDX Browser

Peptide Map : 94 % coverage

(D)

0

2

4

6

8

10

12

0.10 1.00 10.00 100.00 1000.00Rel

ativ

e D

eute

rium

Lev

el (

Da)

Time (min)

128-140

APO HOLO

0123456789

10

0.10 1.00 10.00 100.00 1000.00Rel

ativ

e D

eute

rium

Lev

el (

Da)

Time (min)

128-138

APO HOLO

0123456789

0.10 1.00 10.00 100.00 1000.00Rel

ativ

e D

eute

rium

Lev

el (

Da)

Time (min)

37-47

APO HOLO

0123456789

0.10 1.00 10.00 100.00 1000.00Rel

ativ

e D

eute

rium

Lev

el (

Da)

Time (min)

35-45

APO HOLO

YES 

No 

Has conformation changed?

at [M+10H]10+

Deuterium uptake of peptide level

Time3.00 4.00 5.00 6.00 7.00 8.00

%

0

100

3.00 4.00 5.00 6.00 7.00 8.00%

0

100

3.00 4.00 5.00 6.00 7.00 8.00

%

0

100

3.00 4.00 5.00 6.00 7.00 8.00

%

0

100

3.00 4.00 5.00 6.00 7.00 8.00

%

0

100

3.00 4.00 5.00 6.00 7.00 8.00

%

0

100

Labeling time

240 m

60 m

10 m

1 m

10 s

0 s

m/z600 605 610

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

Chromatogram

Figure 4(C) Waters HDX Browser software was used to link the labeled raw data with peptide information and to display them in m/z and time. This software significantly reduced the data processing time. (D) The deuterium uptake curves for apo and holo comparison in peptide level. Top curves (peptide 128-140 and 128-138) illustrate that there are distinct difference in deuterium uptake between apo and holo. This means that there are obvious conformational changes in these regions. The bottom curves for peptides 37-47 and 35-45 showed no change in deuterium uptake. HX-Express was used to measure the deuterium uptake.4

10 sec

1 min

10 min

60 min

control

control

ICFD.pdb 1PRW.pdb

36 % exchanged

APO HOLO

6 % exchanged

Apo

Holo

Apo

Holo

Apo

Holo

Apo

Holo

Change No Change

Figure 3(B) The deuterium uptake curves in intact protein comparison is shown. The mass shift in y-axis was calculated from the deconvoluted intact mass. The global level conformational change was revealed by running fast intact HDX screening.

(B)