hx instrument

Capturing the essence of life

HX-IA Instrument™

Thorleif Lavold

Biomotif AB

The HX-IA Instrument™

1. H/D Exchange to study conformation,

binding sites, Epitope Mapping etc.

2. ElectroCapture technology

3. ”Findtheneedleinthehaystack” for on-line

pI separation of proteins

4. Ligand Screening

5. Compound Fishing-Epitope Imprinting

New HX-IA Instrument

HDX Amide Hydrogen Exchange

D2O

H’s & D’s

at backbone amide positions

H/D Exchange for Monitoring Protein Structural Changes

−D

FOLDED

UNFOLDED

m/z

Inte

nsity

m/z

Inte

nsity

mass shift

more D incorporated

IN HEAVY WATER

IN HEAVY WATER

After 10 min

HDX Amide Hydrogen Exchange

Folding Dynamics

vs

Ligand Binding

Conformational Changes

?

?

?

?

Aggregation

Relation to X-Ray, NMR

Epitope Mapping

To study dynamics of:

Protein folding &

Conformational changes

Localisation of:

— Epitope

— Mutation

— Aggregation

— Ligand binding site

Complementary information

to X-ray and NMR

Mutation

Pepsin column

Valve AValve B

Loop

Loading

Deuteration

Sample Elution

Mass Spectrometer

Waste lines

Solvent lines

Buffer and waste bottles

Gradient Pumps

10 mM NH4CH3COO

1 % Formic Ac.

Isocratic Pump

Autosampler Srynge Pumps

Power supply

0.05 % TFA

50 mM NH4CH3COO

25

mM

NH

4C

H3C

OO

Column

washing

Analytical Column

Isocratic Pump

Release/loop loading

TrypsincColumn wash.

LC-MS analysis

Electrolyte fluidic lines

Membrane –Assisted Sample Preparation in the HX-IA Instrument

Gradient 5-95% ACN in 45min

0.1%FA

D2O D2O D2O

100% D2O

Flow

Flo

wSample

Channel

Second

Solution

Semipermeable membrane separates sample channel from the deuterating

solution

Membrane-Assisted Sample Preparation

Semipermeable

Membrane

Semi-permeable Membranes

FlowSample

Channel

Second

Solution

Semipermeable membrane separates the sample channel from the deuterating

solution

Membrane-Assisted Sample Preparation

Membrane

0 % DeuterationPROTECTED

(Internal Pocket)

60 % DeuterationUnprotected

35 % DeuterationSemi-protected

15 % DeuterationPROTECTED

Different Deuteration Levels According to Protein Conformation

Different Deuteration Levels According to Protein Conformation

INTERLEIKIN 1β / ANTIBOBY H/D EXCHANGE EXPERIMENTS

Deuterated IL1β mass

IL1β alone IL1β +Fab

17451,57 17447,04

17451,85 17446,8

17451,3 17447,08

17452,1 17446,04

17451,33 17446,38

17451,71 17446,05

Average 17451,64 17446,56

Deuterons displaced by Fab binding = 5,08

Intact Mass MS

Literature example of irreversible oxidative modification of Myoglobin

Denature Digestion

Protein

Ligand

Surface Modification

Unmodified Epitope

M

Peptide Analysis

Where does it bind? Epitope mapping by MS using 1H2H exchange (HDX) or covalent modification

ConclusionsOnline Membrane-Assisted HDX

No D2O Dilution No Pippetting

Less sample consumption

Flow Injection Deuteration (no need for automated pipetting stations)

No need of liquid Nitrogen (online )

Acidification on-line with no dilution of sample

Tris or Phosphate buffer can be used

Can be coupled with any API-MS system

• Detection of non-covalently linked protein-ligand complexes

• Kd determination is possible to rank ligands

• Epitope mapping gives positional information

• Protein consumption is low• Gas Phase Ion mobility shows great potential for

investigating PPIs.– BUT…. How representative is the gas phase structure to

that in solution

INTERACTOMICS

Does it bind?How strongly does it bind?Where does it bind?What happens on binding?To what does it bind?What binds?

Electrocapture-based Separations

Charge particle


Electrocapture conditions will be fulfilled when

Ve ≥ Vf

Electrophoretic velocity is given by,

Ve = ue x E

Ve = Electrophoretic velocity

ue = Electrophoresis mobility

E = Electric field

Positive

Electrode

Negative

Electrode

Figure 1: This Figure is a schematic

of a dual membrane ES probe. Note

how there is no electrically connected

conductive surface in the sample

solution flow channel.

Dual Membrane Electrospray Probe

Electrocapture

Region

Membrane Section 2

Membrane

Section 1 Electrospray

Ion Source

Upstream Electrode

Down stream Electrode

Figure 1: This Figure is a schematic

of a dual membrane ES probe. Note

how there is no electrically connected

conductive surface in the sample

solution flow channel.

Dual Membrane Electrospray Probe

Electrocapture

Region

Membrane Section 2

Membrane

Section 1 Electrospray

Ion Source

Upstream Electrode

Down stream Electrode

New Flat membrane cell

New cell in PEEK

Protein band

Anodic chamber Cathodic chamber

Flow

Capillary electrophoresis

MALDI & ESI-MS

Micro reactions

Analytical Applications

Separations, μ-LC

Flow

Peek tubing

Conductivemembrane

Captured protein

125 m

Image downstream cathode junction during capture-device operation

Protein captured at 30 nL spot

ESI Total Ion Count of an Electrocapture-concentration Experiment

.

80-fold magnification!

m/z1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900

%

0

100

alfa-detergent-a 46 (1.037) Sm (Mn, 2x8.00); Cm (2:46) TOF MS ES+ 1.64e31235

11511084

1236

1388

1387

1237

1237

1238

1239

1239

1241

1257

15401388

1540

1539

1389

1390

1391

1391

1409

1844

1844

1541

1692

1692

1542

1543

1691

1543

1544

1544

16411550

1693

1843

1694

1743

1695

1845

1845

1846

1945

1938

1936

1859

2148

2047

1996

1996

19972127

2061

2452

2249

2225

23762300

2350

2755

2528

2499

2604

2553

2680

26542694

2832

2816

2805

29372907 29982982

n-octyl β-D glucopyranoside

ESI-MS Analysis of α-Lactalbumin in a solution containing 0.5% Non-Ionic Detergent

Detergent clusters

m/z1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900

%

0

100

alfa-peak-a 204 (4.871) Sm (Mn, 2x8.00); Cm (200:208) TOF MS ES+ 1152127

2117

2114

2111

2108

1850

1847

1845

12171208

1203

1190

1227

15041379

1861

1864

2130

2480

2136

2139

2470

24592166

2175

2206 23242285

2484

2491

25262974

ESI-MS Analysis of α-Lactalbumin in 0.5% Non-Ionic Detergent after Electrocapture

After Electrocapture

Protein peaks

200

Vf = Ve = ue x E

E (V/cm)

Time

Vf > Ve = ue x E

Flow


myo-alfa-rybo

m/z600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

proteinmix-a 3253 (61.724) Cm (3192:3270) TOF MS ES+ 2822041

20401786

17861781

20371791

2046

2049

2052


17021548

15441700

204317961957 2052

22042057


17041552

15491549

15561702

1709 1892

1711

18961897

1969


170415521550

1549

15541709 18961710 1967

1969


1709

17091522

1519 1524

1714 1953

1717 1953 1959

Electrocapture-based Separations of Proteins

120 V

90 V

60 V

Ribonuclease

Beta-casein

Beta-lactoglobulin

Findtheneedleinthehaystack Instrument™

2D-EC “NeedleInTheHaystack”

Cell #1 is held at 170V/cm Cell #2 at 171V/cm

Green, grey and purple fractions

above 171 V/cm goes to waste


The yellow molecules are the only ones captured between

170V/cm-171V/cm in Cell #2 and further concentrated

And or separated before MS-detection.

Any Voltage fractions can be selected for targeted “Compound

Fishing” experiments with 2D-EC NeedleInTheHaystack.

+ +

--

+ -

Electric Field

Hydrodynamic flow Mass Spec

Inject Ligand Cocktail

Using the protein as an “immobilised stationary phase”

Compounds elute in order of increasing affinity

Ligand screening using Solution Phase Ion Mobility

Flow

Peek tubing

Conductivemembrane

Captured protein

125 m

Image downstream cathode junction during capture-device operation

Protein captured at 30 nL spot

rt 7.5 min

BSAafterrelease of voltage

BSA

SB00396

• Compound Fishing-Epitope Imprinting

The principle on the formation of MIP

phase

1. Mixing a template corresponding to the analyte/handle of interest with a compound (functional monomer) having the optimal bonding sites for the formation of hydrogen donor – acceptor interaction.

2. The functional monomer easily form at polymer in the presence of the template/handle, affording the correct cavity and bonding properties to the handle. Afterwards the template is removed.

This is analogous to a key in a lock.

Template = our handle

Principle of “our” MIP cavity

bonding• The cavity match the functional

group

• The group has strong interaction

due to hydrogen bonding.

• X = reactive group that depending

on its nature can “selectively”

form a covalent bond to a

functional group of the analyte of

interest. Compound fishing .

Compound fishing

If the analyte of interest has a functional group such as following examples:

• -COOH

• -NH2

• -CO-

• and many more

The X can be selected to form a covalent bond with the particular group of

the analyte, as shown in following example.

What is special with our approach?

So far every analyte of interest most often require the formation of a

dedicated MIP phase.

Our approach is generic, only one MIP phase is needed to capture almost

any targeted organic compound .

We can selectively pick a compound or a group of compounds through

derivatisation.

The MIP phase can be employed as a packed column for LC-MS, or

on a surface using MALDI-MS.

MIP as biosensor (QCM, etc.).

Beneficial….

• Open up completely new diagnostic tool to analyze “stuff” not being possible before.

• In addition analyze biomarkers that could not be monitored before at the required low levels.

• New dimension for medical diagnostic purposes.

• For a patients, both urine- and blood- samples can be analysed.

• Urine samples by direct derivatisation.

• Blood samples, by a simple purification step such as ultra

filtration removing the proteins followed by derivatisation.

Fee For Service

• H/D Exchange

• Epitope Mapping

• Patent filing cases

• ElectroCapture pre-concentration

Target is all Pharma and Biotech companies

working with Ab, Proteins, Peptides etc.

THANK YOU!

• Thorleif Lavold CEO, Biomotif AB

• [email protected]

• www.biomotif.com

mailto:[email protected]

hx instrument

Documents

hxia instrumenthd exchange

dilution of sample tris

1h2h exchange hdx

hxia instrumentgradient

study conformation

deuterating solutionmembrane

ligandsepitope mapping

binding sites