Protein Function Microarrays: Design, Use and Bioinformatic Analysis in Cancer Biomarker Discovery and Quantitation

Abstract

Protein microarrays have many potential applications in the systematic, quantitative analysis of protein function, including in biomarker discovery applications. In this chapter, we review available methodologies relevant to this field and describe a simple approach to the design and fabrication of cancer-antigen arrays suitable for cancer biomarker discovery through serological analysis of cancer patients. We consider general issues that arise in antigen content generation, microarray fabrication and microarray-based assays and provide practical examples of experimental approaches that address these. We then focus on general issues that arise in raw data extraction, raw data preprocessing and analysis of the resultant preprocessed data to determine its biological significance, and we describe computational approaches to address these that enable quantitative assessment of serological protein microarray data. We exemplify this overall approach by reference to the creation of a multiplexed cancer-antigen microarray that contains 100 unique, purified, immobilised antigens in a spatially defined array, and we describe specific methods for serological assay and data analysis on such microarrays, including test cases with data originated from a malignant melanoma cohort.

Supplementary Material

3.1.1 Methodology

3.1.1.1 Cloning of Cancer/Testis Antigen Genes

In total, 100 proteins were cloned and expressed for printing on the CT100 array. Seventy-two of these were CT antigen proteins, while the remaining twenty-eight were other cancer-associated proteins/proteins of interest. All antigens were cloned into baculoviral expression vectors and expressed in insect cells.

The following procedure was carried out for insect cell-expressed proteins. The gene encoding the E. coli biotin carboxyl carrier protein (BCCP) domain – amino acids 74–156 of the E. coli accB gene (Athappilly and Hendrickson 1995; Chapman-Smith and Cronan 1999) – was amplified by PCR from an E. coli genomic DNA preparation and cloned downstream of a viral polyhedrin promoter in an E. coli vector to create the transfer vector pJB1. This E. coli transfer vector system is derived from pTriEx-1.1 (Novagen). Flanking this polh-BCCP expression cassette were the baculoviral 603 and 1,629 genes (Zhao et al. 2003), which enabled the subsequent homologous recombination of the construct into a replication-deficient baculoviral genome.

Synthetic genes for each of the antigens of interest were obtained from Origene, Open Biosystems or GeneService. PCR primers were designed for each CT antigen cDNA such that the stop codon would be removed, enabling it to be cloned into the pJB1 transfer vector upstream of and in-frame with the 3′-BCCP tag via ligation-independent cloning methods, replacing the ORF region between the Spe I and Nco I sites of pJB1 in the process (all primers synthesised by IDT, UK) (Yang et al. 1993). Each resulting transfer vector thus encoded an individual antigen fused to a C-terminal BCCP tag.

The PCR amplification of each synthetic gene; ligation-independent cloning of these products into a BCCP tag-containing transfer vector, pJB30; and transformation of this vector into E. coli DH5α were all carried out according to standard recombinant DNA protocols (Sambrook et al. 2001) and are accordingly not described here in detail. Successful PCR amplification of each antigen was confirmed by gel electrophoresis, while successful cloning was determined by sequencing the relevant regions of each transfer vector (i.e. the region containing the ligated PCR products as well as the junctions between these and the BCCP tags) according to standard protocols and verified against the RefSeq database.

3.1.1.2 Maintenance and Co-transfection of Sf21 Cells

A replication-incompetent baculovirus vector, bacmid pBAC10:KO₁₆₂₉ (Zhao et al. 2003), was propagated in E. coli HS996 cells, and bacmid DNA was prepared according to standard procedures. pBAC10/KO₁₆₂₉ was then linearised by restriction with Bsu361 (New England Biolabs) for 5 h at 37°C, after which Bsu361 was heat killed at 80°C for 15 min. Five hundred nanograms of undigested pJB1 transfer vector was then combined with 500 ng linearised bacmid, and the total volume made up to 12 μl with water. Twelve microlitres Lipofectin (diluted 2:1 in H₂O) was then added to this DNA mix, and the tube was incubated at room temperature for 30 min. One millilitre serum-free media (InsectXpress, Lonza) was added to the Lipofectin/DNA mixture. A 6-well plate containing 1  ×  10⁶ Sf21 cells/well (Invitrogen) was prepared and incubated at 27°C for 1 h to allow the cells to adhere. Excess media were aspirated from the Sf21 cells and replaced with the Lipofectin/DNA/serum-free mix. The transfected cells were incubated at 27°C overnight. The media was then replaced with 2 ml InsectXpress media supplemented with 2% FBS and incubated at 27°C without agitation for a further 72 h. Cells were resuspended by physical agitation and then pelleted by centrifugation at 1,000 × g for 10 min. The supernatant containing recombinant baculovirus was transferred to a fresh tube and stored at 4°C; this was the P₀ stock. The general baculoviral system used here was adapted from the work of Prof Ian Jones (Reading University, UK) (Zhao et al. 2003).

Recombinant baculoviral particles were amplified according to standard procedures. Briefly, a 6-well plate was set up with 1  ×  10⁶ Sf21 cells/well and incubated at 27°C for 1 h. Excess media were removed and replaced with 500 μl of P₀ virus plus 500 μl InsectXpress media supplemented with 2% FBS and incubated at 27°C without agitation for a further 72 h. P₁ virus was harvested as described above. A 150-ml tissue culture flask was seeded with 20 ml of 1  ×  10⁶ Sf21 cells/ml and incubated at 27°C for 1 h. Excess media were removed and replaced with 500 μl of P₁ virus plus 3 ml InsectXpress media supplemented with 2% FBS and incubated at 27°C for 1 h, after which a further 25 ml InsectXpress media supplemented with 2% FBS were added and cells incubated without agitation for 72 h. P₂ virus was harvested as described above. The titre of the P₂ viral stock was determined by a SybrGreen-based quantitative PCR assay versus a stock of known titre determined by plaque assay. Stocks that were found to have low titre were re-amplified.

3.1.1.3 Expression of BCCP-Tagged CT Antigens

A 24-well deep well plate containing 6  ×  10⁶ Sf21 cells/well suspended in 3 ml InsectXpress media supplemented with 2% FBS and 50 μM biotin was used. 200 μl of P₂ virus was added, and the plate was incubated at 27°C for 72 h with agitation. Cells were harvested by centrifugation of the plate prior to lysis. Cells were gently resuspended and washed in 3 ml of PBS buffer for 5 min, the plate was recentrifuged and the supernatant was discarded; this was repeated three times in total. Pellets were gently resuspended in 350 μl of freezing buffer (25 mM HEPES, 50 mM KCl, pH 7.5) ensuring thorough mixing of the cells. Cells were aliquoted in 50 μl volumes and stored at −80°C until required for cell lysis. For cell lysis, aliquots were thawed and 50 μl lysis buffer (25 mM HEPES pH 7.5, 20% glycerol, 50 mM KCl, 0.1% Triton X-100, 0.1% BSA, 250 U/ml protease inhibitor cocktail and 1 mM DTT plus 10 U Benzonase (Novagen)) was added to each; this was then incubated on ice with agitation for 30 min. Cell debris was removed by centrifugation at 13,000 × g for 30 min at 4°C, the supernatant collected and then stored on ice for up to 24 h prior to printing.

The protein concentration of the soluble, crude protein extract was determined by Bradford assay (Bradford 1976) to confirm that effective cell lysis had occurred. Antigen expression and biotinylation were analysed by Western blot according to standard protocols (Sambrook et al. 2001). Antigen expression was confirmed using a mouse anti-c-myc antibody (Sigma-Aldrich) at 1:5,000 followed by a 1:25,000 dilution of goat anti-mouse IgG HRP conjugate (KPL). For more rapid processing, dot blots were sometimes used (same conditions as for Western blots) to assess expression prior to array fabrication. Biotinylation of the antigens was confirmed using a streptavidin–HRP conjugate probe (GE Healthcare) at 1:10,000.

3.1.1.4 Fabrication of Protein Microarrays

3.1.1.4.1 Preparation of Streptavidin-Coated Slides for Printing

A Nexterion Slide H microarray slide (Schott, Germany) was equilibrated to room temperature and removed from the foil package. A 1 mg/ml streptavidin solution was made up in 150 mM of Na₂HPO₄ buffer (pH 8.5). The microarray surface was immersed in approximately 5 ml of the streptavidin solution for 1 h at room temperature. The slide was removed from the streptavidin solution (which can be reusable successively up to 10 times) and then washed for 1 h at room temperature in 10 ml of 150 mM Na₂HPO₄ buffer (pH 8.5) containing 50 mM of ethanolamine to deactivate any remaining amine-reactive groups. The slide was washed for 3  ×  5 min in 10 ml wash buffer and then for 5 min in 10 ml water. The slide was then placed in a 50-ml Falcon tube and centrifuged at 1,000 × g for 5 min at 20°C until dry. Streptavidin-coated slides were placed into slide boxes, sealed in Ziploc bags and stored at −20°C.

As a QC test, one streptavidin-coated slide per batch was incubated for 1 h with a solution of Cy5-biotinylated BSA (10 μg/ml in PBS), washed and scanned; this demonstrated that with this procedure, we can readily achieve CVs of 2–3% across the print area of the slide surface, judged by analysis of a virtual grid of 576 evenly distributed spots.

3.1.1.4.2 CT Antigen Microarray Fabrication

The expression and biotinylation of the various antigens were confirmed using SDS-PAGE- or dot blot based Western blot analysis prior to printing and crude lysates were then diluted with PBS containing 40% sucrose (sucrose was included to increase the surface tension and to reduce spreading of printed droplets). Forty microlitres of the crude protein extract for each BCCP-tagged protein to be arrayed was transferred into individual wells of a 384-well V-bottom plate. The plate was centrifuged at 4,000 rpm for 2 min at 4°C to pellet any cell debris that may have carried over from cell lysis. The plate was then stored on ice prior to the microarray print run, and during printing it was kept at 4°C.

Replica CT100 arrays were printed in a 4-plex format (i.e. 4 replica arrays per slide), using crude cell lysates. Each of the 72 CT antigens and the 28 TA antigens were printed in triplicate within each array. Several different controls were also included in each array. The positive controls included 50 ng/μl biotinylated human IgG (Rocklands Immunochemicals Inc.). The negative controls included biotinylated 200 ng/μl sheep IgG (Rocklands Immunochemicals Inc.) and an ‘empty vector’ lysate control consisting of a crude insect cell lysate containing the BCCP-tag alone with no recombinant fusion partner. In addition, three different concentrations (5, 10 and 15 ng/μl) of biotinylated Cy5-BSA were included in each sub-array for slide orientation and signal normalization purposes.

Each CT100 array was printed on home-made streptavidin-coated microarray slides (prepared as above) using a Genetix QArray2 robotic arrayer (Genetix Ltd., UK) equipped with 8  ×  300 μm flat-tipped solid pins. Each array was printed as a set of eight 7  ×  7 blocks, with each block printed by a different pin. The printing procedures were carried out at room temperature, while the source plate was kept at 4°C, and the atmosphere in the print chamber was humidified to ∼50%. The arrays were printed using the following key QArray2 settings: inking time  =  500 ms, microarraying pattern  =  7  ×  7, 500 μm spacing, maximum stamps per ink  =  1, number of stamps per spot  =  2, printing depth  =  150 μm, water washes  =  60 s wash and 0 s dry, ethanol wash  =  10 s wash and 1 s dry.

After printing, each slide was washed for 30 min with 50 ml prechilled blocking solution (25 mM HEPES pH 7.5, 20% glycerol, 50 mM KCl, 0.1% Triton X-100, 0.1% BSA, 1 mM DTT and 50 μM biotin) and then stored at −20°C submerged in storage buffer (25 mM HEPES pH 7.5, 50% glycerol, 50 mM KCl, 0.1% Triton X-100, 0.1% BSA and 1 mM DTT).

3.1.1.4.3 Verification of Immobilisation of BCCP-Tagged Proteins to Array Surface

Following standard protocols for Western blots, it is possible to verify the successful immobilisation of biotinylated proteins to the array surfaces, as follows. Mouse anti-c-myc antibody was diluted 1:1,000 in 1 ml PBST containing 5% fat-free milk powder. The protein array was removed from wash buffer and equilibrated in PBST at room temperature for 5 min. The PBST was drained away and 5 ml antibody solution was added to the array, which was then incubated with gentle agitation at room temperature for 30 min. The array was washed for 3  ×  5 min with 1 ml of PBST. Goat anti-mouse antibody-HRP conjugate was diluted 1:1,000 in 1 ml milk/PBST. The antibody solution was added to the array, and the array was incubated with gentle agitation at room temperature for 30 min. The array was washed for 3  ×  5 min with 1 ml of PBST and then submerged in 5 ml of chemiluminescent detection reagents (Pierce). After 1 min, the slide was placed in a 50-ml Falcon tube and centrifuged for 30 s to dry. In a dark room, the array was placed against autoradiography film for varying lengths of time before developing the film.