Sam Hanash

Protein based microarrays: A tool for probing the proteome of cancer cells and tissues

A novel proteomic approach for probing cell and tissue proteome, which combines liquid phase protein separations with microarray technology has been developed. Proteins in cell and tissue lysates or in cellular subfractions are separated using any one of a number of separation modes which may consist of ion exchange liquid chromatography (LC), reverse phase LC, carrier ampholyte based separations, e.g. the use of Rotofor, affinity based separations, or gel based separations. Each first‐dimension fraction obtained using one separation mode can be further resolved using one or more of the other separation modes to yield either purified protein in solution or liquid fractions with substantially reduced protein complexity. The advantage of a liquid based separation system is that proteins in hundreds of individual fractions can be arrayed directly and used as targets for a variety of probes. Constituent proteins in reactive fractions are identified by mass spectrometry and may be further resolved to determine the nature of the reactive protein(s). We present in this report initial data based on microarray analysis of individual Rotofor fractions obtained from lung adenocarcinoma cell line A549 lysates which have been probed with antibodies against specific proteins.

Amplification and overexpression of the IGF2 regulator PLAG1 in hepatoblastoma

There is evidence that 8q amplification is associated with poor prognosis in hepatoblastoma. A previous comparative genomic hybridization analysis identified a critical region in chromosomal bands 8q11.2–q13. Using restriction landmark genomic scanning in combination with a virtual genome scan, we showed that this region is delineated by sequences within contig NT_008183 of chromosomal subbands 8q11.22–q11.23. A real‐time PCR–based genomic copy number assay of 20 hepatoblastomas revealed gain or amplification in this critical chromosomal region in eight tumors. The expression of four genes and expressed sequence tags (ESTs) within this newly defined region was assayed by real‐time reverse transcriptase polymerase chain reaction (RT‐PCR) in four tumors with and six tumors without gain or amplification. The PLAG1 oncogene was found to be highly expressed in all but one tumor compared to normal liver tissue. Furthermore, quantitative RT‐PCR revealed that the expression level of the developmentally regulated transcription factor PLAG1 was 3–12 times greater in hepatoblastoma tumors and cell lines compared to age‐matched normal liver and comparable to the expression in fetal liver tissue. PLAG1 has been shown be a transcriptional activator of IGF2 in other tumor types. Using luciferase reporter assays, we demonstrated that PLAG1 transactivates transcription from the embryonic IGF2 promoter P3, also in hepatoblastoma cell lines. Thus, our results provide evidence that PLAG1 overexpression may be responsible for the frequently observed up‐regulation of IGF2 in hepatoblastoma and therefore may be implicated in the molecular pathogenesis of this childhood neoplasia.

HUPO Initiatives Relevant to Clinical Proteomics

The past few years have seen a tremendous interest in the potential of proteomics to address unmet needs in biomedicine. Such unmet needs include more effective strategies for early disease detection and monitoring and more effective therapies, in addition to developing a better understanding of disease pathogenesis. Proteomics is particularly suited for investigating biological fluids to identify disease-related alterations and to develop molecular signatures for disease processes. However, much of the effort undertaken in clinical proteomics to date represents either demonstrations of principles or relatively small-scale studies when compared with genomics effort and accomplishments or more pertinently when contrasted with the tremendous untapped potential of clinical proteomics. Clearly, we are in the early stages. What seems to be urgently needed is an organized effort to build a solid foundation for proteomics that includes developing a much needed infrastructure with adequate resources. The Human Proteome Organization (HUPO) is fostering an organized international effort in proteomics that includes initiatives around organ systems and biological fluids that have disease relevance as well as development of proteomics resources.

Integral protein microarrays for the identification of lung cancer antigens in Sera that induce a humoral immune response

The identification of biomarkers (both molecules and profiles) in patient sera offers enormous interest for the diagnosis of cancers. In this context, the detection of antibodies to tumor cell autologous antigens possesses great potential. The humoral immune response represents a form of biological amplification of signals that are otherwise weak because of very low concentrations of antigen, especially in the early stages of cancers. Herein we present the use of integral microarrays spotted with tumor-derived proteins to investigate the antibody repertoire in the sera of lung cancer patients and controls. The use of two-dimensional liquid chromatography allowed us to separate proteins from the lung adenocarcinoma cell line A549 into 1760 fractions, which were printed in duplicate, along with various controls, onto nitrocellulose coated slides. The sensitivity and specificity of the microarrays to detect singular antibodies in fluids were first validated through the recognition of fractions containing a lung marker antigen by antibody probing. Twenty fractions were initially selected as highly reactive against the anti-PGP9.5 antibody, and subsequent mass spectrometry analyses confirmed the identity of PGP9.5 protein in four of them. As a result, the importance of neighboring fractions in microarray detection was revealed due to the spreading of proteins during the separation process. Next, the microarrays were individually incubated with 14 serum samples from patients with lung cancer patients, 14 sera from colon cancer patients, and 14 control sera from normal subjects. The reactivity of the selected fractions was analyzed, and the level of immunoglobulin bound to each fraction by each serum sample was quantified. Eight of the 20 fractions offered p values < 0.01 and were recognized by an average of four reacting patients, whereas no serum from normal individuals was positive for those fractions. Protein microarrays from tumor-derived fractions hold the diagnostic potential of uncovering antigens that induce an immune response in patients with certain types of cancers.

Mining the plasma proteome for disease applications across seven logs of protein abundance

The current state of proteomics technologies has sufficiently advanced to allow in-depth quantitative analysis of the plasma proteome and development of a related knowledge base. Here we review approaches that have been applied to increase depth of analysis by mass spectrometry given the substantial complexity of plasma and the vast dynamic range of protein abundance. Fractionation strategies resulting in reduced complexity of individual fractions followed by mass spectrometry analysis of digests from individual fractions has allowed well in excess of 1000 proteins to be identified and quantified with high confidence that span more than seven logs of protein abundance. Such depth of analysis has contributed to elucidation of plasma proteome variation in health and of protein changes associated with disease states.

Brain-specific Proteins Decline in the Cerebrospinal Fluid of Humans with Huntington Disease

We integrated five sets of proteomics data profiling the constituents of cerebrospinal fluid (CSF) derived from Huntington disease (HD)-affected and -unaffected individuals with genomics data profiling various human and mouse tissues, including the human HD brain. Based on an integrated analysis, we found that brain-specific proteins are 1.8 times more likely to be observed in CSF than in plasma, that brain-specific proteins tend to decrease in HD CSF compared with unaffected CSF, and that 81% of brain-specific proteins have quantitative changes concordant with transcriptional changes identified in different regions of HD brain. The proteins found to increase in HD CSF tend to be liver-associated. These protein changes are consistent with neurodegeneration, microgliosis, and astrocytosis known to occur in HD. We also discuss concordance between laboratories and find that ratios of individual proteins can vary greatly, but the overall trends with respect to brain or liver specificity were consistent. Concordance is highest between the two laboratories observing the largest numbers of proteins.

Distinct sequences on 11q13.5 and 11q23–24 are frequently coamplified with MLL in complexly organized 11q amplicons in AML/MDS patients

Amplification within chromosome arm 11q involving the mixed‐lineage leukemia gene (MLL) locus is a rare but recurrent aberration in acute myeloid leukemia and myelodysplastic syndrome (AML/MDS). We and others have observed that 11q amplifications in most AML/MDS cases have not been restricted to the chromosomal region surrounding the MLL gene. Therefore, we implemented a strategy to characterize comprehensively 11q amplicons in a series of 13 AML/MDS patients with MLL amplification. Analysis of 4 of the 13 cases by restriction landmark genomic scanning in combination with virtual genome scan and by matrix‐based comparative genomic hybridization demonstrated that the 11q amplicon in these four cases consisted of at least three discontinuous sequences derived from different regions of the long arm of chromosome 11. We defined a maximally 700‐kb sequence around the MLL gene that was amplified in all cases. Apart from the core MLL amplicon, we detected two additional 11q regions that were coamplified. Using fluorescence in situ hybridization (FISH) analysis, we demonstrated that sequences in 11q13.5 and 11q23–24 were amplified in 8 of 13 and 10 of 12 AML/MDS cases, respectively. Both regions harbor a number of potentially oncogenic genes. In all 13 cases, either one or both of these regions were coamplified with the MLL amplicon. Thus, we demonstrated that 11q amplicons in AML/MDS patients display a complex organization and have provided evidence for coamplification of two additional regions on the long arm of chromosome 11 that may harbor candidate target genes.

Combined restriction landmark genomic scanning and virtual genome scans identify a novel human homeobox gene, ALX3, that is hypermethylated in neuroblastoma

Restriction landmark genome scanning (RLGS) allows comparative analysis of several thousand DNA fragments in the genome and provides a means to identify CpG islands that are altered in tumor cells as a result of amplification, deletion, or methylation changes. We have developed a novel informatics tool, designated virtual genome scan (VGS), that makes it possible to predict automatically the sequence of fragments in RLGS patterns by matching to the human genome sequence. A combination of RLGS and VGS was utilized to identify changes of chromosome 1–derived fragments in neuroblastoma. A NotI‐EcoRV fragment was found to be absent frequently in neuroblastoma cell line RLGS patterns. Sequence prediction by VGS as well as cloning of the fragment showed that it contained a CpG island that is part of the human orthologue of the hamster homeobox gene Alx3. Expression analysis in a panel of human and mouse tissues showed predominant expression of ALX3 in brain tissue. Methylation‐sensitive sequence analysis of the promoter region in neuroblastoma cell lines indicated that methylation of specific sequences correlated with repression of the ALX3 gene. Expression was re‐induced after treatment with the methylation inhibitor 5‐aza‐2′‐deoxycytidine. Promoter methylation analysis of ALX3 in primary neuroblastoma tumors, using methylation‐sensitive polymerase chain reaction, found preferential ALX3 methylation in advanced‐stage tumors. The VGS approach we have implemented in combination with RLGS is useful for the identification of genomic CpG island–related methylation changes or deletions in cancer.

Identification of proteins from two-dimensional gel electrophoresis of human erythroleukemia cells using capillary high performance liquid chromatography/electrospray-ion trap-reflectron time-of-flight mass spectrometry with two-dimensional topographic map analysis of in-gel tryptic digest products.

Protein spots from two-dimensional (2-D) gel electrophoresis of a human erythroleukemia cell line have been identified by analysis of the in-gel tryptic digests using capillary high performance liquid chromatography (HPLC) separation with on-line detection using electrospray ionization mass spectrometry (ESI-MS). This is performed using an electrospray/ion trap storage/reflectron time-of-flight mass spectrometer system (ESI-IT-reTOFMS). A 2-D topographic mapping display developed to process the on-line data acquired with this TOF system has been used to obtain mass identification of each peptide, even though the capillary HPLC only provides limited separation capability of the tryptic peptide mixtures studied herein. Using this method, a substantial fraction of the protein sequence can be covered and identified using the tryptic map. It is demonstrated that by entering the cell species, the approximate MW and pI range as determined by 2-D gel electrophoresis, and the tryptic peptide map into the database a unique match for identification of the protein generally results. It is also demonstrated that a much improved coverage of the protein sequence is obtained by this method relative to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).

Autoantibody Profiling for Cancer Detection

Despite substantial progress in the understanding of the pathogenesis of cancer, the development and implementation of strategies for early cancer detection have lagged behind. Harnessing the immune response to tumor antigens is particularly useful for early detection because the immune response occurs early during tumor development and affords signal amplification with the end product, namely reactive immunoglobulins, being released into the circulation allowing easy access through the blood. This article presents recent developments in autoantibody profiling with a focus on proteomic approaches and applications to lung cancer.