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Technical phosphoproteomic and bioinformatic tools useful in cancer research.

ABSTRACT: Reversible protein phosphorylation is one of the most important forms of cellular regulation. Thus, phosphoproteomic analysis of protein phosphorylation in cells is a powerful tool to evaluate cell functional status. The importance of protein kinase-regulated signal transduction pathways in human cancer has led to the development of drugs that inhibit protein kinases at the apex or intermediary levels of these pathways. Phosphoproteomic analysis of these signalling pathways will provide important insights for operation and connectivity of these pathways to facilitate identification of the best targets for cancer therapies. Enrichment of phosphorylated proteins or peptides from tissue or bodily fluid samples is required. The application of technologies such as phosphoenrichments, mass spectrometry (MS) coupled to bioinformatics tools is crucial for the identification and quantification of protein phosphorylation sites for advancing in such relevant clinical research. A combination of different phosphopeptide enrichments, quantitative techniques and bioinformatic tools is necessary to achieve good phospho-regulation data and good structural analysis of protein studies. The current and most useful proteomics and bioinformatics techniques will be explained with research examples. Our aim in this article is to be helpful for cancer research via detailing proteomics and bioinformatic tools.

SUBMITTER: Lopez E

PROVIDER: S-EPMC3195713 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Technical phosphoproteomic and bioinformatic tools useful in cancer research.

López Elena E Wesselink Jan-Jaap JJ López Isabel I Mendieta Jesús J Mendieta Jesús J Gómez-Puertas Paulino P Muñoz Sarbelio Rodríguez SR

Journal of clinical bioinformatics 20111003

Reversible protein phosphorylation is one of the most important forms of cellular regulation. Thus, phosphoproteomic analysis of protein phosphorylation in cells is a powerful tool to evaluate cell functional status. The importance of protein kinase-regulated signal transduction pathways in human cancer has led to the development of drugs that inhibit protein kinases at the apex or intermediary levels of these pathways. Phosphoproteomic analysis of these signalling pathways will provide importan ...[more]

PMID: 21967744

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Project description:An encouraging approach for the diagnosis and effective therapy of immunological pathologies, which would include cancer, is the identification of proteins and phosphorylated proteins. Disease proteomics, in particular, is a potentially useful method for this purpose. A key role is played by protein phosphorylation in the regulation of normal immunology disorders and targets for several new cancer drugs and drug candidates are cancer cells and protein kinases. Protein phosphorylation is a highly dynamic process. The functioning of new drugs is of major importance as is the selection of those patients who would respond best to a specific treatment regime. In all major aspects of cellular life signalling networks are key elements which play a major role in inter- and intracellular communications. They are involved in diverse processes such as cell-cycle progression, cellular metabolism, cell-cell communication and appropriate response to the cellular environment. A whole range of networks that are involved in the regulation of cell development, differentiation, proliferation, apoptosis, and immunologic responses is contained in the latter. It is so necessary to understand and monitor kinase signalling pathways in order to understand many immunology pathologies. Enrichment of phosphorylated proteins or peptides from tissue or bodily fluid samples is required. The application of technologies such as immunoproteomic techniques, phosphoenrichments and mass spectrometry (MS) is crucial for the identification and quantification of protein phosphorylation sites in order to advance in clinical research. Pharmacodynamic readouts of disease states and cellular drug responses in tumour samples will be provided as the field develops. We aim to detail the current and most useful techniques with research examples to isolate and carry out clinical phosphoproteomic studies which may be helpful for immunology and cancer research. Different phosphopeptide enrichment and quantitative techniques need to be combined to achieve good phosphopeptide recovery and good up- and-down phospho-regulation protein studies.

Project description:BackgroundThe mitogen activated protein kinase (MAPK) pathways are known to be deregulated in many human malignancies. Phosphopeptide identification of protein-kinases and site determination are major challenges in biomedical mass spectrometry (MS). P38 and HuR protein kinases have been reported extensively in the general principles of signalling pathways modulated by phosphorylation, mainly by molecular biology and western blotting techniques. Thus, although it has been demonstrated they are phosphorylated in different stress/stimuli conditions, the phosphopeptides and specific amino acids in which the phosphate groups are located in those protein kinases have not been shown completely.MethodsWe have combined different resins: (a) IMAC (Immobilized Metal Affinity Capture), (b) TiO2 (Titanium dioxide) and (c) SIMAC (Sequential Elution from IMAC) to isolate phosphopeptides from p38 and HuR protein kinases in vitro.Different phosphopeptide MS strategies were carried out by the LTQ ion Trap mass spectrometer (Thermo): (a) Multistage activation (MSA) and (b) Neutral loss MS3 (DDNLMS3).In addition, Molecular Dynamics (MD) bioinformatic simulation has been applied in order to simulate, over a period of time, the effects of the presence of the extra phosphate group (and the associated negative charge) in the overall structure and behaviour of the protein HuR.This study is supported by the Declaration of Helsinki and subsequent ethical guidelines.ResultsThe combination of these techniques allowed for:(1) The identification of 6 unknown phosphopeptides of these protein kinases. (2) Amino acid site assignments of the phosphate groups from each identified phosphopeptide, including manual validation by inspection of all the spectra. (3) The analyses of the phosphopeptides discovered were carried out in four triplicate experiments to avoid false positives getting high reproducibility in all the isolated phosphopeptides recovered from both protein kinases. (4) Computer simulation using MD techniques allowed us to get functional models of both structure and interactions of the previously mentioned phosphorylated kinases and the differences between their phosphorylated and un-phosphorylated forms.ConclusionMany research studies are necessary to unfold the whole signalling network (human proteome), which is so important to advance in clinical research, especially in the cases of malignant diseases.

Project description:BackgroundGastric cancer (GC) is one of the most commonly occuring gastrointestinal tumor types, and early diagnosis and operation have a notable effect on the prognosis of patients. Although certain markers, including HER2, VEGER-2, ERCC1 and Bcl-2, have been utilized in clinical practise to screen out new patients with GC, the results of using these markers remains poor. The role of olfactomedin-like 2B (OLFML2B) in GC, as a member of the olfactomedin domain-containing proteins family, is remain unclear.MethodsIn the present study, we assessed the expression of OLFML2B, including mRNA and protein level, by using The Cancer Genome Atlas (TCGA) database and 13 pairs of clinical samples between GC and NG tissues. The correlation between expression of OLFML2B and prognosis of GC was evaculated by the Kaplan-Meier plotter and OncoLnc online tools. In addition, mechanism analysis of OLFML2B in GC was explored thought bioinformatic tools, including cBioPortal and FunRich software.ResultsIn our study, the mRNA expression of OLFML2B in GC both TCGA database and clinical samples was consistently revealed to be significantly higher compared with that in NG tissues (P < 0.0001 for TCGA database and P = 0.0034 for clinical samples), and high OLFML2B expression was found in 9 (69.23%) of 13 clinical GC by immunohistochemistry and was positively correlated with the depth of tumor invasion and clinical stage (TNM). In addition, the AUC for a ROC of 0.867 indicated a moderate diagnostic ability of OLFML2B for GC. Survival analysis from the Kaplan-Meier plotter (P = 2.6 × 10- 6) and OncoLnc (P = 0.00276) revealed that the high expression of OLFML2B was associated with a short overall survival. Futhermore, 5% (24/478) alterations of OLFML2B were identified from cBioPortal, and among them, missense mutation (14/478) was the primary type. The results from FunRich revealed that OLFML2B participated in mediating multiple biological processes including cell growth and maintenance, regulation of the cell cycle, apoptosis and cell communication through multiple signaling pathways including the M/G1 transition pathway, post-translational protein modification and DNA replication pre-initiation.ConclusionsTaken together, it could be deduced that OLFML2B may act as an oncogene in the development of GC and the overexpression of OLFML2B in GC may be used as a novel diagnostic and prognostic target for GC.

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Technical phosphoproteomic and bioinformatic tools useful in cancer research.

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Technical phosphoproteomic and bioinformatic tools useful in cancer research.

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