Surfing transcriptomic landscapes. A step beyond the annotation of chromosome 16 proteome.

The Spanish team of the Human Proteome Project (SpHPP) marked the annotation of Chr16 and data analysis as one of its priorities. Precise annotation of Chromosome 16 proteins according to C-HPP criteria is presented. Moreover, Human Body Map 2.0 RNA-Seq and Encyclopedia of DNA Elements (ENCODE) data sets were used to obtain further information relative to cell/tissue specific chromosome 16 coding gene expression patterns and to infer the presence of missing proteins. Twenty-four shotgun 2D-LC-MS/MS and gel/LC-MS/MS MIAPE compliant experiments, representing 41% coverage of chromosome 16 proteins, were performed. Furthermore, mapping of large-scale multicenter mass spectrometry data sets from CCD18, MCF7, Jurkat, and Ramos cell lines into RNA-Seq data allowed further insights relative to correlation of chromosome 16 transcripts and proteins. Detection and quantification of chromosome 16 proteins in biological matrices by SRM procedures are also primary goals of the SpHPP. Two strategies were undertaken: one focused on known proteins, taking advantage of MS data already available, and the second, aimed at the detection of the missing proteins, is based on the expression of recombinant proteins to gather MS information and optimize SRM methods that will be used in real biological samples. SRM methods for 49 known proteins and for recombinant forms of 24 missing proteins are reported in this study.

Evaluation of homo- and hetero-functionally activated glass surfaces for optimized antibody arrays.

Antibody arrays hold great promise for biomedical applications, but they are typically manufactured using chemically functionalized surfaces that still require optimization. Here, we describe novel hetero-functionally activated glass surfaces favoring oriented antibody binding for improved performance in protein microarray applications. Antibody arrays manufactured in our facility using the functionalization chemistries described here proved to be reproducible and stable and also showed good signal intensities. As a proof-of-principle of the glass surface functionalization protocols described in this article, we built antibody-based arrays functionalized with different chemistries that enabled the simultaneous detection of 71 human leukocyte membrane differentiation antigens commonly found in peripheral blood mononuclear cells. Such detection is specific and semi-quantitative and can be performed in a single assay under native conditions. In summary, the protocol described here, based on the use of antibody array technology, enabled the concurrent detection of a set of membrane proteins under native conditions in a specific, selective, and semi-quantitative manner and in a single assay.

Chromosome 19 annotations with disease speciation: a first report from the Global Research Consortium.

A first research development progress report of the Chromosome 19 Consortium with members from Sweden, Norway, Spain, United States, China and India, a part of the Chromosome-centric Human Proteome Project (C-HPP) global initiative, is presented ( http://www.c-hpp.org ). From the chromosome 19 peptide-targeted library constituting 6159 peptides, a pilot study was conducted using a subset with 125 isotope-labeled peptides. We applied an annotation strategy with triple quadrupole, ESI-Qtrap, and MALDI mass spectrometry platforms, comparing the quality of data within and in between these instrumental set-ups. LC-MS conditions were outlined by multiplex assay developments, followed by MRM assay developments. SRM was applied to biobank samples, quantifying kallikrein 3 (prostate specific antigen) in plasma from prostate cancer patients. The antibody production has been initiated for more than 1200 genes from the entire chromosome 19, and the progress developments are presented. We developed a dedicated transcript microarray to serve as the mRNA identifier by screening cancer cell lines. NAPPA protein arrays were built to align with the transcript data with the Chromosome 19 NAPPA chip, dedicated to 90 proteins, as the first development delivery. We have introduced an IT-infrastructure utilizing a LIMS system that serves as the key interface for the research teams to share and explore data generated within the project. The cross-site data repository will form the basis for sample processing, including biological samples as well as patient samples from national Biobanks.

Biomarker discovery by novel sensors based on nanoproteomics approaches.

During the last years, proteomics has facilitated biomarker discovery by coupling high-throughput techniques with novel nanosensors. In the present review, we focus on the study of label-based and label-free detection systems, as well as nanotechnology approaches, indicating their advantages and applications in biomarker discovery. In addition, several disease biomarkers are shown in order to display the clinical importance of the improvement of sensitivity and selectivity by using nanoproteomics approaches as novel sensors.

High-throughgput phage-display screening in array format.

Emerging technologies for the design and generation of human antibodies require improved approaches enabling their screening, characterization and validation. Currently, strategies based on ELISA or western blot are used to that aim. However, the ever increasing number of novel antibodies generated would benefit from the development of new high-throughput (HT) platforms facilitating rapid antibody identification and characterization. Herein, we describe a protein chip bearing recombinant phage particles and based on a large phage antibody library. In this paper we have set forth a novel implementation which provides a powerful and simple methodology enabling the identification of single-chain variable fragments (scFv). As a proof-of-principle of this method, we tested it with recombinant antigen (human recombinant interleukin 8). Additionally, we developed a novel bioinformatics tool that serves to compare this novel strategy with traditional methods. The method described here, together with associated informatics tools, is robust, relatively fast and represents a step-forward in protocols including phage library screenings.

Protein microarrays: technological aspects, applications and intellectual property.

Over the last decade, proteomics has undergone remarkable progress thanks to the technical advances made in the field. Improvements in the design of the protein microarrays, including more types of chemical groups for surface functionalization, new capture agents and novel detection strategies, among others, have allowed the detection of proteins in a robust, specific, sensitive, real time and high throughput manner. However, there are still problems that hinder the analysis of low abundance proteins or those present in complex samples. For this reason, the development of patents related to the features mentioned above has an important relevance. In this review, we focus on the study of recently approved patents that try to solve the existing problems. Thanks to them, it is expected that the identification of disease biomarkers can be made in a suitable and reliable way, and above all, biocompatible and environmentally friendly.

Protein arrays as tool for studies at the host-pathogen interface.

Pathogens and parasites encode a wide spectrum of multifunctional proteins interacting to and modifying proteins in host cells. However, the current lack of a reliable method to unveil the protein-protein interactions (PPI) at the host-pathogen interface is retarding our understanding of many important pathogenic processes. Thus, the identification of proteins involved in host-pathogen interactions is important for the elucidation of virulence determinants, mechanisms of infection, host susceptibility and/or disease resistance. In this sense, proteomic technologies have experienced major improvements in recent years and protein arrays are a powerful and modern method for studying PPI in a high-throughput format. This review focuses on these techniques analyzing the state-of-the-art of proteomic technologies and their possibilities to diagnose and explore host-pathogen interactions. Major technical advancements, applications and protocol concerns are presented, so readers can appreciate the immense progress achieved and the current technical options available for studying the host-pathogen interface. Finally, future uses of this kind of array-based proteomic tools in the fight against infectious and parasitic diseases are discussed.

Data Analysis Strategies for Protein Microarrays.

Microarrays constitute a new platform which allows the discovery and characterization of proteins. According to different features, such as content, surface or detection system, there are many types of protein microarrays which can be applied for the identification of disease biomarkers and the characterization of protein expression patterns. However, the analysis and interpretation of the amount of information generated by microarrays remain a challenge. Further data analysis strategies are essential to obtain representative and reproducible results. Therefore, the experimental design is key, since the number of samples and dyes, among others aspects, would define the appropriate analysis method to be used. In this sense, several algorithms have been proposed so far to overcome analytical difficulties derived from fluorescence overlapping and/or background noise. Each kind of microarray is developed to fulfill a specific purpose. Therefore, the selection of appropriate analytical and data analysis strategies is crucial to achieve successful biological conclusions. In the present review, we focus on current algorithms and main strategies for data interpretation.