Development of a trypsin-immobilized

monolithic polymer with pipette-tip

format for protein digestion

Proteomic research –primary structure of proteins, detection of post-translational modifications

MS/MS-based peptide sequencing for protein identification

Quality control (QC), quality assurance (QA) for the products in biotechnology, chemical synthesis and pharmaceutical industries1.

Wei Boon Hon1, Emily F. Hilder1, Kenneth C. Saunders2, and Paul R. Haddad1

1.Pfizer Analytical Research Centre (Parc) and ACROSS, School of Chemistry, University of Tasmania, Tasmania, Australia.2.Pfizer Global Research and Development, Sandwich, UK

wbhon@utas.edu.au

Acknowledgements: WBH gratefully acknowledges support from the State Government of Tasmania, Department of Economic Development and Tourism for scholarship support. We thank Dr Karsten Gömann (Central

Science Laboratory, UTAS) for assistance with SEM imaging. We also thank UTAS Conference Fund Scheme, Pfizer and OAI for providing travel grant for this poster presentation in HPLC 2010.

Enzyme-immobilized digestion

Fast

High efficiency

High-throughput

Eliminate autodigestion

Reproducible and reusable

The micrometer-sized pores and large surface area of monoliths could reduce the diffusion path length and provide low-pressure drop, leading to high digestion efficiency.

Low cost

Wide pH range

Biocompatible

Ease of preparation

High control of shape, porosity and selectivity

Synthesis of monoliths in situ in polypropylene pipette tips.

Surface modification of monolithic polymer via photografting to obtain reactive azlactone functionalities for trypsin immobilization.

To perform digestion of model proteins and samples spiked in serum using the immobilized enzymatic polypropylene pipette tip (IMEPP).

Enzyme immobilization on porous polymer monolith

Problems and challenges

The importance of protein digestion

Figure 1. SEM image of organic polymer monolith at a magnification of 13000X.

InstrumentationFigure 6. OAI deep UV illumination system (Model LS30/5) fitted with a 500W HgXe-lamp for the surface modification and in situ preparation of the organic polymer monolith in the polypropylene pipette tips, and photografting of reactive vinylazlactone group for trypsin immobilization.

In-solution digestion

Time-consuming

Low efficiency

Enzyme autodigestion Figure 7. SEM images of porous polymer monoliths inside a PP tube without surface modification (a) and the magnified part (b).

(a)

Figure 8. SEM images of single-step surface modification of PP tip with MMA/EDMA 1:1 with BP (3 wt. %) (a) and the magnified part (b).

Polymer gel (b)

(a)PP tipsVoid (b)

Surface modification of the PP tips

Effects of the VAL concentration and exposure time on IMEPP protein digestion of cytochrome c

Surface modification of the PP tips

Photografting of reactive azlactone functionalities for trypsin immobilization

References:1. J. Krenkova, N.A. Lacher, F. Svec, Anal. Chem. 81 (2009) 2004.2. T.B. Stachowiak, T. Rohr, E.F. Hilder, D.S. Peterson, M. Yi, F. Svec,

J.M.J. Frechet, Electrophoresis 24 (2003) 3689.3. Z. Altun, A. Hjelmstroem, M. Abdel-Rehim, L.G. Blomberg, J. Sep.

Sci. 30 (2007) 1964.

453.4 679.5

1546.0

1766.6

2061.0

2248.2

+MS, 0.3-0.7min #(24-53)

0

20

40

60

80

100

Intens.

[%]

250 500 750 1000 1250 1500 1750 2000 2250 m/z

129.1276.7350.7

453.4679.5

728.7817.8

971.2

1545.9

1766.5

2061.0

+MS, 0.2-0.5min #(12-34)

0

20

40

60

80

100

Intens.

[%]

250 500 750 1000 1250 1500 1750 2000 2250 m/z

261.2

454.3

536.3

584.8

648.9

779.5

907.6

957.2

1168.7

1296.8

1456.4

1546.0

1766.7

1894.0

2061.0

+MS, 0.8-1.9min #(56-144)

0

20

40

60

80

100

Intens.

[%]

250 500 750 1000 1250 1500 1750 2000 2250 m/z

Figure 9. ESI-TOF mass spectrum obtained from cytochrome c digestion using pipette tips (a) without VAL photografting; (b) photografting under UV light at an exposure time of 2 min with 15% VAL in photografting mixture; (c) exposure time of 30 min with 25% VAL in photografting mixture.

(b)(a)

(c)

Protein digestion steps

Protein digestion and mass spectrometry

Figure 14. ESI-TOF MS spectra of peptides obtained by digestion of six proteins using IMEPP. Other conditions as in Fig. 9.

Table 1. Results of sequence coverage for the digestion of proteins and samples spiked in rat serum using IMEPP.

Protein MW (Da)

Sequence Coverage (%)

Soluble trypsinIMEPP

(0.5mg/mL)

IMEPP (Spiked

sample 0.5 mg/mL)

Igg -1 36106 51 31 5

Igg -2 35901 45 37 22

Igg -3 41287 39 36 32

Igg -4 35941 34 42 27

Igg -kappa 11609 80 68 61

Igg -lambda 11237 66 75 75

Table 2. Comparison of sequence coverage identification of hIgG using soluble trypsin , IMEPP and IMEPP for sample spiked in rat serum.

Figure 15. ESI-TOF MS spectra of peptides obtained by digestion of four proteins spiked in rat serum (~70g proteins in 100 L) using IMEPP. Other conditions as in Fig. 9.

Monoliths can be synthesized in situ in polypropylene pipette tips.

Surface modification of monolithic polymer can be used to create reactive azlactone functionalities for trypsin immobilization.

These tips can be used to perform digestion of proteins and peptides in standard buffer and in rat serum.

LC-MS/MS separation to evaluate the efficiency and selectivity of the tips.

Characterization of the tips (e.g. amount of grafted VAL and enzyme immobilized)

Digestion of bioanalytical samples.

Integration of an automated 96-tip robotic device to allow enzymatic digestion of samples within a few minutes.

Protein digestion and LC-ESI-TOF MS

Figure 11. Separation of peptides resulting from the cytochrome c digestion using the IMEPP and soluble enzyme. Digestion and separation conditions as in Fig. 10.

LC-MS separation was performed with an Agilent 1200 Series LC (Agilent Technologies, Palo Alto, CA) coupled to the micrOTOF-Q-MS from Bruker Daltonics (Bremen, Germany), operating at a resolution of 10000. Separation conditions: 2.1 x 100mm i.d. Dionex C16 column; injection volume, 5 µ L. Mobile phases: A, 0.1% formic acid in water; B, 0.1% formic acid in acetonitrile.

Cytochrome C (0.01mg/mL)

BSA (0.5mg/mL)

hIgG (0.5mg/mL)

Figure 12. Separation of peptides resulting from the BSA digestion using the IMEPP and soluble enzyme. Digestion and separation conditions as in Fig. 10. Gradient: 0 min, 10% B and then ramp from 10% B to 50% B in 35 min.

Figure 13. Separation of peptides resulting from the BSA digestion using the IMEPP and soluble enzyme. Digestion and separation conditions as in Fig. 12.

+MS, 0.3-0.4min #(15-24)

0

20

40

60

Intens.

[%]

250 500 750 1000 1250 1500 1750 2000 2250 m/z

Calcitonin Gene Related Peptide rat (0.005 mg/mL)

+MS, 0.3-0.5min #(18-30)

0

20

40

60

80

Intens.

[%]

250 500 750 1000 1250 1500 1750 2000 2250 m/z

Cytochrome c (0.01 mg/mL)

+MS, 0.4-0.6min #(25-37)

0

20

40

60

80

100

Intens.

[%]

250 500 750 1000 1250 1500 1750 2000 2250 m/z

+MS, 0.2-0.3min #(14-18)

0

5

10

15

Intens.

[%]

250 500 750 1000 1250 1500 1750 2000 2250 m/z

hIgG (0.5 mg/mL)

BSA(0.5 mg/mL)

Digestion of proteins spiked in rat serum

0 5 10 15 20 25 30Time [min]

(a) Soluble trypsin (b) IMEPP (c) Proteins spiked in rat serum + IMEPP

0 5 10 15 20 25 30 35

Time [min]

(a)

(b)

(c)

(a)

(b)

(c)

Protein MW (Da)Concentration

(mg/mL)

Sequence Coverage (%)Soluble trypsin

IMEPPIMEPP (Spiked

sample)Melittin 2848 0.005 100 100 100

Rat CGRP 3806 0.005 100 100 100Cytochrome c 11702 0.01 97 95 87

myoglobin 16951 0.1 95 89 66BSA 69294 0.5 92 88 84

0 2 4 6 8 10 12 14 16Time [min]

(a)

(b)

(c)

(a)

(b)

(c)

Preparation of polymer monolith in situ in PP tips

Photografting of 2-vinyl-4,4-dimethylazlactone

Trypsin immobilization

Surface modification & monolith preparation

H2N

Svec, F., 2006. Electrophoresis, 27 (5-6), 947.

Photografted reactive monomer: vinylazlactone

Immobilized protein

Covalent attachment of protein to surface

(a)

(b)

Polymerization mixture

BuMA

Monomers: 16 wt%

Crosslinker: 24 wt%

1-Propanol

Porogenic solvents: 60 wt%

Initiator: 1 wt% w.r.t. monomer

DMAP

40:60 mixture

EDMAOH

OH

HO

1,4-Butanediol

40 min 25 min

UV UV

Protein prospector database

Protein sample

Immobilized trypsin on porous polymer

monolith

Peptides

Proteins are denatured, reduced, alkylated and then transferred to

the IMEPP

100µ L of protein solution pipetted for 15 times (~6-8 min) and

digestion occurs

Digested peptides are collected and analyzed

LC-MS and Direct infusion ESI-MS analysis

Figure 5. Schematic diagram of protein digestion.

Figure 4. Schematic diagram of the photopatterning process1.

Figure 3. Schematic diagram showing in situ preparation of polymer monoliths in polypropylene pipette tips3.

Figure 2. Schematic diagram of the photoinduced surface modification and preparation of a monolith in an empty pipette tip2.

Rat CGRP

0 5 10 15 20 25 30

Time [min]

(b)

(c)

Rat CGRP (0.005mg/mL)

Figure 10. Separation of peptides resulting from the Rat CGRP digestion using the IMEPP. Digestion condition as in Fig 9. Separation conditions: Gradient: 0 min, 20% B and then ramp from 20% B to 55% B in 35 min; flow rate, 0.2 mL/min. BPC, base peak chromatogram.

Polypropylene tip Surface modification mixture:(1:1 methyl methacrylate: ethylene glycol dimethacrylate + 3% benzophenone)

Free double bonds

UV

(a) (b)

35

35

+MS, 1.0-1.9min #(57-114)

0

20

40

60

80

100

Intens.

[%]

250 500 750 1000 1250 1500 1750 2000 2250 m/z

+MS, 0.3-1.4min #(16-84)

0

20

40

60

80

100

Intens.

[%]

250 500 750 1000 1250 1500 1750 2000 2250 m/z

Melittin (0.005 mg/mL)2.8kDa

Calcitonin Gene Related Peptide (rat) (0.005 mg/mL)3.8kDa

+MS, 0.5-1.0min #(41-78)

0

20

40

60

80

100

Intens.

[%]

250 500 750 1000 1250 1500 1750 2000 2250 m/z

Cytochrome c (0.01 mg/mL)13kDa

+MS, 0.3-0.4min #(15-23)

0

20

40

60

80

100

Intens.

[%]

250 500 750 1000 1250 1500 1750 2000 2250 m/z

BSA(0.5 mg/mL)66kDa

+MS, 0.2-0.3min #(9-19)

0

10

20

30

Intens.

[%]

250 500 750 1000 1250 1500 1750 2000 2250 m/z

hIgG (0.5 mg/mL)150kDa

+MS, 0.3-0.7min #(18-39)

0

20

40

60

80

100

Intens.

[%]

250 500 750 1000 1250 1500 1750 2000 2250 m/z

Myoglobin (0.1 mg/mL)17kDa

Altun et al. J Chromatogr A 31 (2008) 743

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