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Solid‐Phase Profiling of Proteins

Hon‐chiu Eastwood Leung¹, Sau‐mei Leung¹, Alex Karavanov¹

¹Ciphergen Biosystems Inc, Fremont, California

Publication Name:

Current Protocols in Protein Science

Unit Number:

Unit 3.9

DOI:

10.1002/0471140864.ps0309s26

Online Posting Date:

February, 2002

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Abstract

Protein array technologies refer to any fabrication and use of any arrays containing multiple proteins captured on solid surfaces. This valuable tool is used for high-throughput protein-protein interaction studies, protein marker discovery and other applications. This unit provides basic protocols for biomarker discovery and protein-protein interactions. Different kinds of recently emerged protein array technologies and related detection methods are reviewed.

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Unit Introduction
Other Protein Array Technologies
Strategic Planning
Basic Protocol 1: Serum Protein Profiling Using ProteinChip Array
Basic Protocol 2: Protein-Protein Interaction Analysis Using ProteinChip Arrays
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Serum Protein Profiling Using ProteinChip Array

Materials

8 M electrophoresis grade urea (Sigma) in 1% CHAPS/1× phosphate buffered saline (PBS), pH 7.2 to 7.4 (appendix 2E): prepare fresh or use aliquot stored at –70°C (do not freeze and thaw the buffer more than once)
Serum sample, human
Cibacron blue 3GA immobilized on 4% beaded agarose Type 3000 (Sigma)
1× PBS, pH 7.2 (appendix 2E)
1 M urea/0.125% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)/1× PBS (appendix 2E): prepare fresh or use aliquot stored at –70°C (do not freeze and thaw the buffer more than once)
Buffers:
pH 4: 50 mM sodium acetate: adjust pH with acetic acid; store up to 3 months at room temperature
pH 6: 50 mM sodium phosphate: adjust pH with 1/7.3 (v/v) 50 mM Na₂HPO₄/50 mM NaH₂PO₄; store up to 3 months at room temperature
pH 8: 50 mM Tris×HCl: adjust pH with HCl; store up to 3 months at room temperature
pH 10: 100 mM 3-cyclohexamino-1-propanesulfonic acid (CAPS): adjust pH according to manufacturers' recommendations; store up to 3 months at room temperature
Saturated sinapinic acid (SPA) in 0.5% trifluoroacetic acid (TFA)/50% acetonitrile; prepare fresh daily
Spin column with 0.45-µm cellulose acetate micro-spin filter (E & K Scientific)
SAX2, WCX2 ProteinChip Arrays (Ciphergen Biosystems)
8-well bioprocessor (Ciphergen Biosystems)
Adhesive tape
Pap pen (i.e., a hydrophobic pen; Zymed)
PBSII chip reader (Ciphergen Biosystems)

NOTE: HPLC-quality reagents are recommended for preparation of all buffers.

Basic Protocol 2: Protein-Protein Interaction Analysis Using ProteinChip Arrays

Materials

1× phosphate buffered saline (PBS), pH 7.2 (appendix 2E)
1 M ethanolamine, pH 8.2
1× PBS, pH 7.2/0.5% and 0.1% Triton X-100
5 M NaCl (appendix 2E)
25% (v/v) ethylene glycol (PS1 array)
Cell extract, lysate, or conditioned media (³1 mg/ml total protein) with or without a molar excess of either the purified protein of interest or a control protein in 1× PBS, pH 7.2
Saturated -cyano-4-hydroxycinnamic acid (CHCA) or sinapinic acid (SPA) in 0.5% TFA/50% acetonitrile

PS1 or PS2 ProteinChip array (Ciphergen Biosystems)
PAP pen (PS1 array)
Humidified chamber: empty pipet-tip box with damp filter paper fixed on top of the stand
15-ml plastic screw-cap centrifuge tube (Corning)
Platform rocker
PBSII chip reader (Ciphergen Biosystems)

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Figures

Figure 3.9.1

Types of ProteinChip array surfaces. From top to bottom, chromatographic surfaces include hydrophobic surfaces (C¹⁶ aliphatic hydrocarbon chain), hydrophilic surfaces (silicon dioxide), metal binding surfaces, anionic (carboxylate) surfaces, and cationic surfaces (quaternary amine).

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Figure 3.9.2

Protein profiles of serum using WCX ProteinChip array and four different pH buffer conditions. Spectrum (A), (C), (E), and (G) are serum spiked with a known protein and spectrum (B), (D), (F), and (H) are serum without the spiked protein. The difference (indicated by the arrow) of the two samples can be clearly seen in pH 6 (spectrum C versus D) and pH 8 (spectrum E versus F) buffer conditions.

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Figure 3.9.3

Protein profiles of crude rat liver lysate in three different ProteinChip arrays and wash conditions. The upper panel depicts hydrophobic surface H4 chip using a 10% acetonitrile wash. The middle panel is a strong anionic exchanger surface (SAX) chip with a pH 8.5 buffer wash. The lower panel is a weak cationic exchange (WCX) chip with a pH 4.5 buffer wash. Arrows indicate the most prominent proteins retained on one surface but not the other.

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Figure 3.9.4

(A) Profile of PTN fraction purified on heparin-Sepharose using a normal phase chip (NP-2). (B to D) ALK ECD covalently bound to active spots on preactivated surface (PS1) chip and overlaid with: (B) PTN, (C) PTN+ALK ECD ~1/1.66 molar ratio, and (D) PTN+TGF receptor ~1/1.7 molar ratio.

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Literature Cited

Literature Cited
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	Haab, B.B., Dunham, M.J., and Brown, P.O. 2000. Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions. Genome Biology. 2: research0004.1-0004.13 (available online at http://genomebiology.com/2001/2/2/research/0004).
	Human Genome Project (HGP) 2001. The human genome. Science genome map. Science 291:1219-1224.
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	Moch, H., Schraml, P., Bubendorf, L., Mirlacher, M., Kononen, J., Gasser, T., Mihatsch, M.J., Kallioniemi, O.P., and Sauter, G. 1999. High-throughput tissue microarray analysis to evaluate genes uncovered by cDNA microarray screening in renal cell carcinoma. Am. J. Pathol. 154:981-986.
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	Roberts, R.W. 1999. Totally in vitro protein selection using mRNA-protein fusions and ribosome display. Curr. Opin. Chem. Biol. 3:268-273.
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	Schweitzer, B., Wiltshire, S., Lambert, J., O'Malley, S., Kukanskis, K., Zhu, Z., Kingsmore, S.F., Lizardi, P.M., and Ward, D.C. 2000. Immunoassays with rolling circle DNA amplification: A versatile platform for ultrasensitive antigen detection. Proc. Natl. Acad. Sci. U.S.A. 97:10113-10119.
	Stoica, G.E., Kuo, A., Aigner, A., Sunitha, I., Soutton, B., Malerczyk, C., Caughey, D.J., Wen, D., Karavanov, A., Riegel, A.T., and Wellstein, A. 2001. Identification of ALK (anaplastic lymphoma kinase) as a receptor for the growth factor pleiotrophin. J. Biol. Chem. In press.
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	Vasiliskov, A.V., Timofeev, E.N., Surzhikov, S.A., Drobyshev, A.L., Shick, V.V., and Mirzabekov, A.D. 1999. Fabrication of microarray of gel-immobilized compounds on a chip by copolymerization. Biotechniques 27:592-594, 596-598, 600 passim.
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	Wiltshire, S., O'Malley, S., Lambert, J., Kukanskis, K., Edgar, D., Kingsmore, S.F., and Schweitzer, B. 2000. Detection of multiple allergen-specific IgEs on microarrays by immunoassay with rolling circle amplification. Clin. Chem. 46:1990-1993.
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The authors are grateful for helpful comments provided by Christine Yip and to Dr. Huw Davies for providing Figure 3.9.2.