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.

BACKGROUND:The 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.METHODS:We 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.RESULTS:The 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.CONCLUSION:Many 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.

7 paginas, 1 figura.-- Informe de la 2a Reunion Anual de la SICAM celebrada en Madrid (Espana) el 15 de Octubre de 2010.

La Garantía de Calidad es un elemento central para el funcionamiento de todo laboratorio, y va más allá de únicamente disponer de controles de calidad internos y externos en la determinación analítica. La Sociedad Española de Inmunología (SEI) promueve un proceso para la selección de un centro responsable (CR) donde desarrollar un programa concreto para la de Garantía Externa de Calidad para Laboratorios de Inmunología Diagnóstica asociado a la Sociedad Española de Inmunología (GECLID-SEI). Este escrito presenta los aspectos principales de este proceso que debería permitir la estructuración del GECLID-SEI.

Despite their clinical utility and the importance that laboratory tests have in APS diagnosis, probably the most important drawback of such tests is the elevated intra- and inter-laboratory variation. The aim of the present work was to assess the multilaboratory performance of aCL and anti-β the multilaboratory performance of anti-cardiolipin (aCL) and antibeta 2 glycoprotein I antibodies (anti-β2GPI) assays and to assess the interlaboratory and inter-assay variability. Here, we report the most significant results from the Autoimmunity Workshop of the Spanish Society of Immunology (AWSEI). Seventeen sera from patients with antiphospholipid syndrome (APS) and/or probable APS were collected after written informed consent. Thirty-three laboratories participated and measured aCL and anti-anti-β2GPI. 61 and 49 results/serum for IgG/IgM aCL and anti- anti-β2GPI, respectively, were informed with 20 different assays. A high interlaboratory variation was found in quantitative results regardless the method used. Coefficient of variation ranged from 50% to 128% for aCL and from 9% to 200% for anti-anti-β2GPI. A limited consensus (defined as >90% agreement) was observed in semiquantitative results for IgG/IgM aCL and anti-β2GPI: 47%, 65%, 47% and 70%, respectively. In general, there was concordance between aCL and anti-β2GPI, yet 2 of the 17 sera were positive for anti-β2GPI only. In conclusion, interpretation of aCL and anti-β2GPI results from different laboratories may be done only in semiquantitative terms and its real value for clinical diagnosis of APS is still limited. Cut off values must be set in each laboratory.