The human protease CLIP-CHIP: Genomic analysis of all 715 human protease and inhibitor gene transcripts in human breast carcinoma Reinhild Kappelhoff ,Tom Ewart+, Xose S. Puente*, Arun Seth+, Carlos López-Otín* and Christopher M. Overall  † Departments of Biochemistry and Molecular Biology †, Oral Biological and Medical Sciences , University of British Columbia, Vancouver, BC, Canada +Department of Molecular Pathology, Sunnybrook & Women's College Health Sciences Centre, Toronto, ON, Canada, *Departamento de Bioquimica y Biologia Molecular, Universidad de Oviedo, Oviedo, Spain Introduction The availability of the human genome sequence has enabled the identification the degradome—the complete repertoire of proteases produced in man. By using primary information retrieved from public and private sequencing projects, together with data from the MEROPS, InterPro and Ensembl databases we have annotated a total of 558 genes encoding proteases or protease-homologs in the human genome and 157 inhibitors (Xose S. Puente, Luis M. Sanchez, Christopher M. Overall, and Carlos Lopez-Otin, Nature Rev Genetics 4, , 2003). To date there are no complete protease microarray chips reported. Hybridization, scanning and analyzing the CLIP-CHIP Hybridization of labeled cDNA to the microarray was at 25  C for 16 h in 5x SSC, 0.3 % SDS. Washing steps were carried out at 42  C. The CLIP-CHIP was scanned in an Affymetrix 428 (MWG) microarray laser scanner using 532 nm for the cyanine 3 channel and 635 nm for the cyanine 5 channel. Photo multiplier gains were set individually for each channel. Conclusions and outlook The development of the CLIP-CHIP represents the first complete dedicated human protease and inhibitor chip reported to date. The CLIP-CHIP will prove to be an extremely valuable and versatile tool for the genomic analysis of proteolytic function in vivo. The advantage of using a custom glass chip is that new protease or inhibitor oligonucleotides can be added in an agile manner at each chip printing for minimal cost. Moreover, having a dedicated subarray containing oligonucleotides relevant to the experimental question being addressed further focuses the experiment without distraction from analyses of many thousands of genes that is possible in larger arrays or the multiple 20mer oligonucleotides per gene necessary for the Affymetrix chips. Work is in progress to analyze RNA from an additional 50 breast cancer tissues of different disease stages to analyze their protease and inhibitor expression profiles. The CLIP-CHIP A dedicated human protease chip CLIP CHIP Serine (177) Threonine (28) Inhibitors (157) Aspartic (21) Cysteine (148) Metallo (184) (+) Controls (46)(-) Controls (7) (A) (B) Inhibitors Serine Cysteine Metallo Aspartate Threonine (C) low expression level high Figure 1. The human protease CLIP-CHIP. ( A) Overview of the CLIP-CHIP microarray layout. The human microarray is spotted in duplicate (1, 2). In addition 192 control oligonuclotides and a dedicated subarray with 217 breast cancer related and prognosis oligonucleotides are included (left side). (B) The five classes of proteases are logically arranged together on the microarray. Thus, the microarray contains oligonucleotides for 21 aspartic proteases, 148 cysteine proteases, 184 metallo proteases, 177 serine proteases, 28 threonine proteases and 157 inhibitors as well as a further 46 positive and 7 negative controls (light areas of the array) spotted in triplicate. (C) Representative expression pattern of proteases and inhibitors in human mammary epithelial cells (cyanine 3 channel). The color bar shows the expression level: black no expression to white high expression. (1) (2) CLIP-CHIP Oligonucelotides (571) for each protease, inactive homologue and inhibitor were obtained from the Human Genome Oligo Set v2.0 (Qiagen). Oligonucleotides were synthesized as 70-mers with a 5’-C6 amino linker for attachment to an epoxy silane glass matrix. Oligonucleotides for the 144 genes not represented in the Human Oligo Set were synthesized after extensive BLAST analyses to identify unique sequences. 715 protease and inhibitor oligonucleotides plus 46 positive and 7 negative control oligonucleotides were arranged in logical groups and spotted in triplicate (Figure 1). A dedicated subarray is customized according to the experiment. Here we used 217 oligonucleotides for breast cancer related and prognosis genes as well as 192 control oligonucleotides (96 positive, 80 negative, 16 Array Controls/Ambion). Figure 3. Signal Intensities of proteases and inhibitors highly expressed in normal human mammary epithelial cells (Normal 1, 2) and invasive ductual breast carcinoma cells (Cancer 1,2) are listed. Normal1/Cancer1 and Normal2/Cancer2 were hybridized to the same slide. Normal mammary epithelial cells is from one source (BioCain); Cancer 1 and 2 are from two different patients with invasive ductual carcinoma Figure 2. Expression levels of the 23 MMPs in normal human mammary epithelia and breast cancer cells. Matrix metalloproteinases (MMPs) are an important family of proteases that correlate with tumor grade. MMPs traditionally were thought to degrade the ECM in metastasis, but the large number of bioactive mediators that are precisely processed by MMPs reveals that MMPs are important signaling proteases and so may explain the side effects of MMPI trials.