Gene expression analysis by signature pyrosequencing.

We describe a novel method for transcript profiling based on high-throughput parallel sequencing of signature tags using a non-gel-based microtiter plate format. The method relies on the identification of cDNA clones by pyrosequencing of the region corresponding to the 3'-end of the mRNA preceding the poly(A) tail. Simultaneously, the method can be used for gene discovery, since tags corresponding to unknown genes can be further characterized by extended sequencing. The protocol was validated using a model system for human atherosclerosis. Two 3'-tagged cDNA libraries, representing macrophages and foam cells, which are key components in the development of atherosclerotic plaques, were constructed using a solid phase approach. The libraries were analyzed by pyrosequencing, giving on average 25 bases. As a control, conventional expressed sequence tag (EST) sequencing using slab gel electrophoresis was performed. Homology searches were used to identify the genes corresponding to each tag. Comparisons with EST sequencing showed identical, unique matches in the majority of cases when the pyrosignature was at least 18 bases. A visualization tool was developed to facilitate differential analysis using a virtual chip format. The analysis resulted in identification of genes with possible relevance for development of atherosclerosis. The use of the method for automated massive parallel signature sequencing is discussed.

Amplification of mRNA populations by a cDNA tag strategy.

Here we describe an amplification method for global transcript analysis. The strategy relies on amplification of cDNA tags (signature tags) achieved by random fragmentation of the cDNAs to short tags of similar length, isolation of the 3' ends and then PCR amplification of the 3'-end signature tag population. This method minimizes biased amplification that may occur during parallel amplification of long and short templates. The amplified tags can be either cloned and sequenced or labeled and hybridized to DNA arrays to identify the expressed transcripts. To verify that the relative levels between transcripts in different mRNA/cDNA populations are maintained during the amplification protocol, we have used the Affymetrix oligonucleotide platform and real-time PCR.

Selective enrichment of monospecific polyclonal antibodies for antibody-based proteomics efforts.

A high stringency protocol, suitable for systematic purification of polyclonal antibodies, is described. The procedure is designed to allow the generation of target protein-specific antibodies suitable for functional annotation of proteins. Antibodies were generated by immunization with recombinantly produced affinity-tagged target proteins. To obtain stringent recovery of the antibodies, a two-step affinity chromatography principle was devised to first deplete the affinity tag-specific antibodies followed by a second step for affinity capture of the target protein-specific antibodies. An analytical dot-blot array system was developed to analyze the cross-reactivity of the affinity-purified antibodies. The results suggest that the protocol can be used in a highly parallel and automated manner to generate mono-specific polyclonal antibodies for large-scale, antibody-based proteomics efforts, i.e. affinity proteomics.

Antibody performance in western blot applications is context-dependent.

An important concern for the use of antibodies in various applications, such as western blot (WB) or immunohistochemistry (IHC), is specificity. This calls for systematic validations using well-designed conditions. Here, we have analyzed 13 000 antibodies using western blot with lysates from human cell lines, tissues, and plasma. Standardized stratification showed that 45% of the antibodies yielded supportive staining, and the rest either no staining (12%) or protein bands of wrong size (43%). A comparative study of WB and IHC showed that the performance of antibodies is application-specific, although a correlation between no WB staining and weak IHC staining could be seen. To investigate the influence of protein abundance on the apparent specificity of the antibody, new WB analyses were performed for 1369 genes that gave unsupportive WBs in the initial screening using cell lysates with overexpressed full-length proteins. Then, more than 82% of the antibodies yielded a specific band corresponding to the full-length protein. Hence, the vast majority of the antibodies (90%) used in this study specifically recognize the target protein when present at sufficiently high levels. This demonstrates the context- and application-dependence of antibody validation and emphasizes that caution is needed when annotating binding reagents as specific or cross-reactive. WB is one of the most commonly used methods for validation of antibodies. Our data implicate that solely using one platform for antibody validation might give misleading information and therefore at least one additional method should be used to verify the achieved data.

Microfluidic analysis of antibody specificity in a compact disk format.

A new and flexible technology for high throughput analysis of antibody specificity and affinity is presented. The method is based on microfluidics and takes advantage of compact disks (CDs) in which the centrifugal force moves fluids through microstructures containing immobilized metal affinity chromatography columns. Analyses are performed as a sandwich assay, where antigen is captured to the column via a genetically attached His6-tag. The antibodies to be analyzed are applied onto the columns. Thereafter, fluorescently labeled secondary antibodies recognize the bound primary antibodies, and detection is carried out by laser-induced fluorescence. The CDs contain 104 microstructures enabling analysis of antibodies against more than 100 different proteins using a single CD. Importantly, through the three-dimensional visualization of the binding patterns in a column it is possible to separate high affinity from low affinity binding. The method presented here is shown to be very sensitive, flexible and reproducible.

A human protein atlas for normal and cancer tissues based on antibody proteomics.

Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, approximately 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.

Genome-based proteomics.

Protein-protein interactions play crucial roles in various biological pathways and functions. Therefore, the characterization of protein levels and also the network of interactions within an organism would contribute considerably to the understanding of life. The availability of the human genome sequence has created a range of new possibilities for biomedical research. A crucial challenge is to utilize the genetic information for better understanding of protein distribution and function in normal as well as in pathological biological processes. In this review, we have focused on different platforms used for systematic genome-based proteome analyses. These technologies are in many ways complementary and should be seen as various ways to elucidate different functions of the proteome.

An improved dual-expression concept, generating high-quality antibodies for proteomics research.

A novel, improved dual bacterial-expression system, designed for large-scale generation of high-quality polyclonal antibody preparations intended for proteomics research, is presented. The concept involves parallel expression of cDNA-encoded proteins, as a fusion with two different tags in two separate vector systems. Both systems enable convenient blotting procedures for expression screening on crude bacterial cell cultures and single-step affinity purification under denaturing conditions. One of the fusion proteins is used to elicit antibodies, and the second fusion protein is used in an immobilized form as an affinity ligand to enrich antibodies with selective reactivity to the cDNA-encoded part, common for the two fusion proteins. To evaluate the system, four cDNA clones from putative nuclear proteins from the non-biting midge Chironomus tentans were expressed. Antibodies to these cDNA-encoded proteins were generated, enriched and used in blotting and immunofluorescence procedures to determine expression patterns for the native proteins corresponding to the cDNAs. The four antibody preparations showed specific reactivity to the corresponding recombinant cDNA-encoded proteins, and three of the four antibodies gave specific staining in Western-blot analysis of nuclear cell extracts. Furthermore, two of the antibody preparations gave specific staining in immunofluorescence analysis of C. tentans cells. We conclude that the dual-vector concept presented offers a highly stringent strategy for the generation of monospecific polyclonal antibodies, which are useful in proteomics research.

Affinity proteomics for systematic protein profiling of chromosome 21 gene products in human tissues.

Here we show that an affinity proteomics strategy using affinity-purified antibodies raised against recombinant human protein fragments can be used for chromosome-wide protein profiling. The approach is based on affinity reagents raised toward bioinformatics-designed protein epitope signature tags corresponding to unique regions of individual gene loci. The genes of human chromosome 21 identified by the genome efforts were investigated, and the success rates for de novo cloning, protein production, and antibody generation were 85, 76, and 56%, respectively. Using human tissue arrays, a systematic profiling of protein expression and subcellular localization was undertaken for the putative gene products. The results suggest that this affinity proteomics strategy can be used to produce a proteome atlas, describing distribution and expression of proteins in normal tissues as well as in common cancers and other forms of diseased tissues.