unique selectivity of monolithic supports and their use ... · the selective of cim monolithic...

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Unique selectivity of monolithic supports and their use for separation of complex mixtures and isolation of

low abundance proteins

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Douglas HixsonCOBRE CCRD & Brown University, Providence, RI, USA

Marija Brgles, Institute of Immunology, Zagreb, Croatia

Feilei Huang, Marijana Rucevic and Lulu CaoCOBRE CCRD, Providence, RI, USA

James Clifton, Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI, USA

Egbert Mueller, Tosoh Bioscience, Stuttgart, Germany

Maja Pucic and Gordan Lauc, Faculty of Pharmacy & Biochemistry, University of Zagreb, Croatia

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Djuro JosicBrown Medical School

Proteomics CoreCOBRE Center for Cancer Research

DevelopmentProvidence, RI

USA

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CIM DEAE, 8mL column

CIM DEAE, 0.34mL disk

Capacity – 23-25 mg BSA/mL support

And – About 10-15 mg proteins from plasma/mL support (0.30 mL plasma/mL) support.

CIM, 0.100mL disk

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The selective of CIM monolithic supports for human plasma proteins

is a function of the sample load.

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SDS PAGE

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MW[kDa]CP 1 2 3 4 5 6 7 8 CP

Loading (cryopoor plasma)

1. CIM disk – 85µl

2. CIM tube – 2ml

3. CIM tube – 5ml

4. CIM tube – 10ml




8. CIM disk – 1.06ml

Elution with 0.155M NaCl (step 1)

15.0

15.0

37.5

75.0

112.5

150.0

187.5

187.5

mg protein/mlV

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SDS PAGE

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MW[kDa] CP 1 2 3 4 5 6 7 8 CP

1. CIM disk – 85µl

2. CIM tube – 2ml

3. CIM tube – 5ml





8. CIM disk – 1.06ml

Elution with 1.0M NaCl (step 2)

Loading (cryopoor plasma)

15.0

15.0

37.5

75.0

112.5

150.0

187.5

187.5

V mg protein/ml

8

0

5

10

15

20

25

30

35

0 2 4 6 8 10

Plasma load [ml plasma/ml support]

Cap

acity

[mg/

ml]

CIM-DEAE monolithCapacity as function of sample load

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Further experiments with CIM-DEAE 0.34 mL column

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1 2 3 4 5 6 7 8 9 10

196

104

60

42

28

21

15

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CIM DEAE – 0.34 ml

Elution with 0.15M NaCl

0.15 M chromatographic fractions of 30x diluted plasma on CIM DEAE

Samples, plasma load

1. std.

2. 1.1 mL

3. 2.2 mL

4. 3.3 ml

5. 6.7 mL

6. 13.3 mL

7. 16.7 mL

8. 25 mL

9. 35 mL

10. 62 mL

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196

104

60

42

28

2115

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1 2 3 4 5 6 7 8 9 10

CIM DEAE – 0.34 mL




1. std.

2. 1.1 mL

3. 2.2 mL

4. 3.3 ml

5. 6.7 mL

6. 13.3 mL

7. 16.7 mL

8. 25 mL

9. 35 mL

10. 62 mL

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1 2 3 4 5 6 7 8 9 10

196

104

60

42

28

21

15

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CIM DEAE – 0.34 mL




1. std.

2. 1.1 mL

3. 2.2 mL

4. 3.3 ml

5. 6.7 mL

6. 13.3 mL

7. 16.7 mL

8. 25 mL

9. 35 mL

10. 62 mL

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1.1 mL / 0.15 M

62 mL / 0.15 M

196

104

60

42

28

2115

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CIM DEAE – 0.34 mL2D EF

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1.1 mL / 0.2 M

62 mL / 0.2 M

196

104

60

42

28

21

15

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CIM DEAE – 0.34 mL2D EF

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Further investigations with different IE ligands

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CHROMATOGRAPHY OF CRYOPOOR PLASMA ON CIM QA

V (column) = 0.34 mLF = 3 mL/minp = 85 - 500 psi Binding buffer: 10 mM Tris, pH = 7.4Elution buffer: 1 M NaCl + 10 mM Tris, pH = 7.4

Capacity of the column was determined loading 50 mL of 30 x diluted cryopoor plasmam (protein in FT) = 74.5 mgm (protein in W) = 4.5 mgm (protein in E) = 9.65 mgm (protein loaded) = 86.8 mgrecovery = 102 %

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CIM QA0.34 mL monolithic column

After each chromatography column was regenerated with 0.5 M NaOH

Capacity of the column was determined also using BSA and was found to be:m (BSA) = 22.6 mg/mL

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Elution with 0.2M NaCl, fractions from CIM QA.

1. STD2. 3 mL on CIM QA3. 6 mL on CIM QA4. 12 mL on CIM QA5. 24 mL on CIM QA6. 48 mL on CIM QA7. 60 mL on CIM QA8. STD

1 2 3 4 5 6 7 8

196

104

60

42

28

21

15

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CIM QA—0.34mL monolithic column

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1 2 3 4 5 6 7 8

196

104

60

42

28

21

15

7

CIM QA—0.34mL monolithic column

Elution with 0.3M NaCl, fractions from CIM QA.

1. STD2. 3 mL on CIM QA3. 6 mL on CIM QA4. 12 mL on CIM QA5. 24 mL on CIM QA6. 48 mL on CIM QA7. 60 mL on CIM QA8. STD

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Vitamin K-dependent proteins highly enriched in tightly bound fraction

Protein Plasma concentration (µg/ml)

Factor II 80-90

Factor IX 4.0

Factor X 6.4

Protein C 3.9-5.9

Protein S 25-35

Protein Z ~ 0.15

* Clotting factor VII (~0.5µg/ml) could not be detected. (See Josic et al., J. Chromatogr. B, 2003)

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0.2 M

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1. Std

2. 3 mL

3. 6 mL

4. 12 mL

5. 48 mL

6. 60 mL

7. std

CIM SO3 —0.34mL monolithic column

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0.3 M

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1. Std

2. 3 mL

3. 6 mL

4. 12 mL

5. 48 mL

6. 60 mL

7. std

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0.5 M

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1. Std

2. 3 mL

3. 6 mL

4. 12 mL

5. 48 mL

6. 60 mL

7. std

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1.0 M

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1. Std

2. 3 mL

3. 6 mL

4. 12 mL

5. 48 mL

6. 60 mL

7. std

1 2 3 4 5 6 7

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Did the tighter binding proteins displace the weaker

binding ones?

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1 mg of lysozyme on IgG saturated CIM SO3 analytical

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25015010075

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1 2 3 4 5 6 7 81. Std

2. FT

3. W

4. 0.1 M

5. 1 M-1

6. 1 M-2

7. Lysozyme

8. IgG (bovine)

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5 mg of lysozyme on IgG saturated CIM SO3 analytical

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1. Std

2. FT

3. W

4. 0.1 M

5. 1 M-1

6. 1 M-2

7. Lysozyme

8. IgG (bovine)

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1 2 3 4 5 6 7 8

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1 mg of IgG on lysozyme saturated CIM SO3 analytical.

5 mg of IgG on lysozyme saturated CIM SO3 analytical.

Part I

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SDS PAGE

1. std.

2. FT (1 mg)

3. W (1 mg)

4. 0.1 M (1 mg)

5. 1 M (1 mg)

6. FT (5 mg)

7. W (5 mg)

8. 0.1 M (5 mg)

9. 1.0 M (5 mg)

10. lysozyme+IgG (bovine)

1 2 3 4 5 6 7 8 9 10

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Experiments with 100 µL CIM SO3 monolithic columns

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1 2 3 4 5 6

1234

5

CIM SO3 -- 100µL


1. std.

2. 3 mL

3. 6 mL

4. 12 mL

5. 48 mL

6. 60 mL

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CIM SO3 -- 100µL

1 2 3 4 5

6 7891011

12


1. 0.9 mL

2. 1.8 mL

3. 3.6 mL

4. 14 mL

5. 18 mL

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1 2 3 4 5

131415

1617

CIM SO3 -- 100µL


1. 0.9 mL

2. 1.8 mL

3. 3.6 mL

4. 14 mL

5. 18 mL

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1 2 3 4 5

18

19

20

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CIM SO3 -- 100µL

Elution with 1.0 M NaCl

1. 0.9 mL

2. 1.8 mL

3. 3.6 mL

4. 14 mL

5. 18 mL

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Identified low abundant proteins (in eluate with 1.0 M NaCl)

1. Clotting Factor XII -- 30 pg/mL

2. Plasminogen -- 8 ng/mL

3. Gelsolin -- 250 µg/mL

Protein concentration

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How about bulk porous supports?

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1 2 3 4 5 6 7 8 9 10

1.STD2.–--3.FT14.FT25.FT36.0.15 M7.0.2 M8.0.3 M9.0.5 M10.1 M

150100

250

50

25

75

20

15

10

60 mL of 10 x diluted cryopoor plasma (07/09) on Toyopearl DEAE 650 M 1 mL column [load-432 mg proteins]

SDS-PAGE (4-12 %) of fractions from chromatography.

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Conclusions:

If monolithic supports were used, the composition of bound and eluted proteins is dependent on column loading.Under overloading conditions, the weakly bound proteins seem to be displaced by stronger binding proteins. This phenomenon was not dependent on column size.Small monolithic columns are ideal supports for development of new methods for separation of complex mixtures and sample preparation for MS analyses.

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High throughput sample preparation and rapid method

development

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MediaScout® 96 array technology

Setting of up to 96 individual pre-packed, ready-to-use columns on one platform according to the selected application

Packed with any desired chromatography media

Professionally packed to manufacturers‘ recommendation

MediaScout® MiniColumns are available in different volumesI.D. 5 mmBed height 2.5 – 30 mm (50 – 600 µl CV)

50 µl200 µl 100 µl

Column operational mode: liquid is forced through the column by1. elevated gravity / CentriColumnor positive liquid displacement (PLD) / continuous liquid feed2. Pipet / PipetColumn; 3. Robot / RoboColumn

1. 2. 3.

1. Small Scale Parallel Chromatography1.2 Concept / Experimental Setup

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MediaScout® StarterKit

for the first steps in parallel chromatography

Electrical PLD-Pipett e.g. Xstream, Eppendorf

MediaScout®

PipetColumn

Setting of up to 8 individual prepacked columns

Continuous liquid flow

Flow-rate controlled by dispensing speed

Simple operational mode

manual method development

PipetColumn RoboColumnAutomation

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Robotic System Configuration

E MTP-Reader

B Robotic Manipulator (RoMa)C Te-Chrom™ Plate Holder

A Liquid Handling Arm (LiHa)

D Te-Slide™Transport Mechanism

A B

CD

E

Aspirate / DispenseTransport x-y-z axisIntegration of 96 MediaScout®RoboColumn array plate Transport of elution plateEvaluation of elution plate

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Can monolithic discs be used instead of small columns packed with bulk support?

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Yes

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ELISA plate containing 96 monolithic discs (0.100µL column volume)

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High-throughput screening

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IgG glycosylation analysis

Proteomics 2009, 9, 882-913

Carolin Huhn, Maurice H. J. Selman, L. Renee Ruhaak, André M. Deelder and Manfred Wuhrer

Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands

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Fc glycosylation monitoring for human IgG at the level of tryptic glycopeptides.

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Comparison of IgG1 glycosylation profiles.

C1, control 1; C2, control 2; RA1 rheumatoid arthritis patient 1; RA2, rheumatoid arthritis patient 2; pep, peptide moiety.

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CIM Protein G 96 monolithic plate

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CP PL FT E FT E FT E CP

100 kDa

20 kDa

250 kDa150 kDa

75

kDa

37 kDa

50 kDa

25 kDa

15 kDa

10 kDa

CP – Precision Plus Protein StandardPL – Plasma sampleFT – Flow Through fractionE – Eluate fraction

SDS-PAGE

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Conclusions:

Monolithic columns can be used in development of new methods for separation of complex mixtures and sample preparation for high-throughput analyses.

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This work was supported by National Institute of Health, Centers for Biochemical Research Development (COBRE), Grant No. P20RR017695.

unique selectivity of monolithic supports and their use ... · the selective of cim monolithic...

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