The Case for Proteomics and Phospho-Proteomics in Personalized Cancer Medicine

The concept of personalized medicine is predominantly been pursued through genomic and transcriptomic technologies, leading to the identification of multiple mutations in a large variety of cancers. However, it has proven challenging to distinguish driver and passenger mutations and to deal with tumor heterogeneity and resistant clonal populations. More generally, these heterogeneous mutation patterns do not in themselves predict the tumor phenotype. Analysis of the expressed proteins in a tumor and their modification states reveals if and how these mutations are translated to the functional level. It is already known that proteomic changes including posttranslational modifications are crucial drivers of oncogenesis, but proteomics technology has only recently become comparable in depth and accuracy to RNAseq. These advances also allow the rapid and highly sensitive analysis of formalin-fixed and paraffin-embedded biobank tissues, on both the proteome and phosphoproteome levels. In this perspective, pioneering mass spectrometry-based proteomic studies are highlighted that pave the way toward clinical implementation. It is argued that proteomics and phosphoproteomics could provide the missing link to make omics analysis actionable in the clinic

MAPU 2.0: high-accuracy proteomes mapped to genomes

The MAPU 2.0 database contains proteomes of organelles, tissues and cell types measured by mass spectrometry (MS)-based proteomics. In contrast to other databases it is meant to contain a limited number of experiments and only those with very high-resolution and -accuracy data. MAPU 2.0 displays the proteomes of organelles, tissues and body fluids or conversely displays the occurrence of proteins of interest in all these proteomes. The new release addresses MS-specific problems including ambiguous peptide-to-protein assignments and it provides insight into general functional features on the protein level ranging from gene ontology classification to comprehensive SwissProt annotation. Moreover, the derived proteomic data are used to annotate the genomes using Distributed Annotation Service (DAS) via EnsEMBL services. MAPU 2.0 is a model for a database specifically designed for high-accuracy proteomics and a member of the ProteomExchange Consortium. It is available on line at http://www.mapuproteome.com

Predicting post-translational lysine acetylation using support vector machines

Motivation: Lysine acetylation is a post-translational protein modification and a primary regulatory mechanism that controls many cell signaling processes. Lysine acetylation sites are recognized by acetyltransferases and deacetylases through sequence patterns (motifs). Recently, we used high-resolution mass spectrometry to identify 3600 lysine acetylation sites on 1750 human proteins covering most of the previously annotated sites and providing the most comprehensive acetylome so far. This dataset should provide an excellent source to train support vector machines (SVMs) allowing the high accuracy in silico prediction of acetylated lysine residues

PHOSIDA 2011: the posttranslational modification database

The primary purpose of PHOSIDA (http://www.phosida.com) is to manage posttranslational modification sites of various species ranging from bacteria to human. Since its last report, PHOSIDA has grown significantly in size and evolved in scope. It comprises more than 80 000 phosphorylated, N-glycosylated or acetylated sites from nine different species. All sites are obtained from high-resolution mass spectrometric data using the same stringent quality criteria. One of the main distinguishing features of PHOSIDA is the provision of a wide range of analysis tools. PHOSIDA is comprised of three main components: the database environment, the prediction platform and the toolkit section. The database environment integrates and combines high-resolution proteomic data with multiple annotations. High-accuracy species-specific phosphorylation and acetylation site predictors, trained on the modification sites contained in PHOSIDA, allow the in silico determination of modified sites on any protein on the basis of the primary sequence. The toolkit section contains methods that search for sequence motif matches or identify de novo consensus, sequences from large scale data sets

Mol. Syst. Biol.

We report a proteomic analysis of microdissected material from formalin-fixed and paraffin-embedded colorectal cancer, quantifying >7500 proteins between patient matched normal mucosa, primary carcinoma, and nodal metastases. Expression levels of 1808 proteins changed significantly between normal and cancer tissues, a much larger fraction than that reported in transcript-based studies. Tumor cells exhibit extensive alterations in the cell-surface and nuclear proteomes. Functionally similar changes in the proteome were observed comparing rapidly growing and differentiated CaCo-2 cells. In contrast, there was minimal proteomic remodeling between primary cancer and metastases, suggesting that no drastic proteome changes are necessary for the tumor to propagate in a different tissue context. Additionally, we introduce a new way to determine protein copy numbers per cell without protein standards. Copy numbers estimated in enterocytes and cancer cells are in good agreement with CaCo-2 and HeLa cells and with the literature data. Our proteomic data set furthermore allows mapping quantitative changes of functional protein classes, enabling novel insights into the biology of colon cancer

Primary Metabolic Chemistry.

Thioesters and acyl phosphates are important metabolites throughout the biosphere. This may imply that they were involved in prebiotic chemistry. The study of the reactivity of thioesters and acyl phosphates may therefore provide some insights into the possible roles for these molecules in prebiotic chemistry. The work described in this thesis demonstrates that acetyl phosphate and inorganic phosphate react to form pyrophosphate in the presence of salts of some divalent metals. Divalent metal ions and nitrogen containing compounds act in synergy to promote pyrophosphate formation in some cases. Ferrous salts were much more efficient at promoting the formation of pyrophosphate than similar reactions containing magnesium ions, Addition of pyridine, or a variety of other nitrogen containing compounds, did not enhance the pyrophosphate yield. The promotion of pyrophosphate formation at near neutral pH by ubiquitous metal salts is considered to be a feasible route for prebiotic production of pyrophosphate. One chemoautotrophic origin of life theory concentrates on the oxidative formation of pyrite (FeS2) from ferrous sulfide and hydrogen as a possible source of prebiotic reductive power. N-Phenyl acetamide can be prepared from mercaptoacetic acid and aniline in water using FeS/H2S as a reagent system. We have established that one possible intermediate, N-phenyl mercaptoacetamide does react to give the product, and that this reaction is fast. Ferrous ions were observed to promote the formation of N-acetyl alanine from alanine and thioacetic acid. Zinc and cadmium ions on the other hand, promote the hydrolysis of thioacetic acid to acetic acid in preference to the N-acylation reaction. Both ferrous ions and ferro cyanide ions were observed to promote the peptide bond formation between protected amino acid derivatives. The results described in this thesis are consistent with the proposal that iron chemistry may have been important in prebiotic chemistry

Acute WNT signalling activation perturbs differentiation within the adult stomach and rapidly leads to tumour formation

A role for WNT signalling in gastric carcinogenesis has been suggested due to two major observations. First, patients with germline mutations in adenomatous polyposis coli (APC) are susceptible to stomach polyps and second, in gastric cancer, WNT activation confers a poor prognosis. However, the functional significance of deregulated WNT signalling in gastric homoeostasis and cancer is still unclear. In this study we have addressed this by investigating the immediate effects of WNT signalling activation within the stomach epithelium. We have specifically activated the WNT signalling pathway within the mouse adult gastric epithelium via deletion of either glycogen synthase kinase 3 (GSK3) or APC or via expression of a constitutively active β-catenin protein. WNT pathway deregulation dramatically affects stomach homoeostasis at very short latencies. In the corpus, there is rapid loss of parietal cells with fundic gland polyp (FGP) formation and adenomatous change, which are similar to those observed in familial adenomatous polyposis. In the antrum, adenomas occur from 4 days post-WNT activation. Taken together, these data show a pivotal role for WNT signalling in gastric homoeostasis, FGP formation and adenomagenesis. Loss of the parietal cell population and corresponding FGP formation, an early event in gastric carcinogenesis, as well as antral adenoma formation are immediate effects of nuclear β-catenin translocation and WNT target gene expression. Furthermore, our inducible murine model will permit a better understanding of the molecular changes required to drive tumourigenesis in the stomach

Phospho.ELM: a database of phosphorylation sites—update 2011

The Phospho.ELM resource (http://phospho.elm.eu.org) is a relational database designed to store in vivo and in vitro phosphorylation data extracted from the scientific literature and phosphoproteomic analyses. The resource has been actively developed for more than 7 years and currently comprises 42 574 serine, threonine and tyrosine non-redundant phosphorylation sites. Several new features have been implemented, such as structural disorder/order and accessibility information and a conservation score. Additionally, the conservation of the phosphosites can now be visualized directly on the multiple sequence alignment used for the score calculation. Finally, special emphasis has been put on linking to external resources such as interaction networks and other databases

ProteomeScout: A repository and analysis resource for post-translational modifications and proteins

ProteomeScout (https://proteomescout.wustl.edu) is a resource for the study of proteins and their post-translational modifications (PTMs) consisting of a database of PTMs, a repository for experimental data, an analysis suite for PTM experiments, and a tool for visualizing the relationships between complex protein annotations. The PTM database is a compendium of public PTM data, coupled with user-uploaded experimental data. ProteomeScout provides analysis tools for experimental datasets, including summary views and subset selection, which can identify relationships within subsets of data by testing for statistically significant enrichment of protein annotations. Protein annotations are incorporated in the ProteomeScout database from external resources and include terms such as Gene Ontology annotations, domains, secondary structure and non-synonymous polymorphisms. These annotations are available in the database download, in the analysis tools and in the protein viewer. The protein viewer allows for the simultaneous visualization of annotations in an interactive web graphic, which can be exported in Scalable Vector Graphics (SVG) format. Finally, quantitative data measurements associated with public experiments are also easily viewable within protein records, allowing researchers to see how PTMs change across different contexts. ProteomeScout should prove useful for protein researchers and should benefit the proteomics community by providing a stable repository for PTM experiments