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
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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