Background and Objective: Mild hypospadias is a birth congenital condition characterized by the relocation of the male urethral meatus from its typical anatomical position near the tip of the glans penis, to a lower ventral position up to the brim of the glans corona, which can also be accompanied by foreskin ventral deficiency. For the most part, a limited number of cases have known etiology. We have followed a high-throughput proteomics approach to study the proteome in mild hypospadias patients. Methods: Foreskin samples from patients with mild hypospadias were collected during urethroplasty, while control samples were collected during elective circumcision (n = 5/group). A high-throughput, quantitative proteomics approach based on multiplexed peptide stable isotope labeling (SIL) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to ascertain protein abundance changes in hypospadias patients when compared to control samples. Results: A total of 4,815 proteins were quantitated (2,522 with at least two unique peptides). One hundred and thirty-three proteins from patients with mild hypospadias showed significant abundance changes with respect to control samples, where 38 proteins were increased, and 95 proteins were decreased. Unbiased functional biological analysis revealed that both mitochondrial energy production and apoptotic signaling pathways were enriched in mild hypospadias. Conclusions: This first comprehensive proteomics characterization of mild hypospadias shows molecular changes associated with essential cellular processes related to energy production and apoptosis. Further evaluation of the proteome may expand the search of novel candidates in the etiology of mild hypospadias and could also lead to the identification of biomarkers for this congenital urogenital condition.

Recent findings support potential roles for HDL in cardiovascular pathophysiology not related to lipid metabolism. We address whether HDL proteome is dynamically altered in atheroma plaque rupture. We used immunoaffinity purification of HDL samples from coronary artery disease patients before and after percutaneous transluminal coronary angioplasty (PTCA), a model of atheroma plaque disruption. Samples were analyzed by quantitative proteomics using stable isotope labeling and results were subjected to statistical analysis of protein variance using a novel algorithm. We observed high protein variability in HDL composition between individuals, indicating that HDL protein composition is highly patient-specific. However, intra-individual protein variances remained at low levels, confirming the reproducibility of the method used for HDL isolation and protein quantification. A systems biology analysis of HDL protein alterations induced by PTCA revealed an increase in two protein clusters that included several apolipoproteins, fibrinogen-like protein 1 and other intracellular proteins, and a decrease in antithrombin-III, annexin A1 and several immunoglobulins. Our results support the concept of HDL as dynamic platforms that donate and receive a variety of molecules and provide an improved methodology to use HDL proteome for the systematic analysis of differences among individuals and the search for cardiovascular biomarkers. Biological significance The HDL proteome is an interesting model of clinical relevance and has been previously described to be dynamically altered in response to pathophysiological conditions and cardiovascular diseases. Our study suggests that interindividual variability of HDL proteome is higher than previously thought and provided the detection of a set of proteins that changed their abundance in response to plaque rupture, supporting the concept of HDL as dynamic platforms that donate and receive a variety of molecules.

The combination of stable isotope labeling (SIL) with mass spectrometry (MS) allows comparison of the abundance of thousands of proteins in complex mixtures. However, interpretation of the large data sets generated by these techniques remains a challenge because appropriate statistical standards are lacking. Here, we present a generally applicable model that accurately explains the behavior of data obtained using current SIL approaches, including (18)O, iTRAQ, and SILAC labeling, and different MS instruments. The model decomposes the total technical variance into the spectral, peptide, and protein variance components, and its general validity was demonstrated by confronting 48 experimental distributions against 18 different null hypotheses. In addition to its general applicability, the performance of the algorithm was at least similar than that of other existing methods. The model also provides a general framework to integrate quantitative and error information fully, allowing a comparative analysis of the results obtained from different SIL experiments. The model was applied to the global analysis of protein alterations induced by low H₂O₂ concentrations in yeast, demonstrating the increased statistical power that may be achieved by rigorous data integration. Our results highlight the importance of establishing an adequate and validated statistical framework for the analysis of high-throughput data.

Nitroxidative stress in cells occurs mainly through the action of reactive nitrogen and oxygen species (RNOS) on protein thiol groups. Reactive nitrogen and oxygen species-mediated protein modifications are associated with pathophysiological states, but can also convey physiological signals. Identification of Cys residues that are modified by oxidative stimuli still poses technical challenges and these changes have never been statistically analyzed from a proteome-wide perspective. Here we show that GELSILOX, a method that combines a robust proteomics protocol with a new computational approach that analyzes variance at the peptide level, allows a simultaneous analysis of dynamic alterations in the redox state of Cys sites and of protein abundance. GELSILOX permits the characterization of the major endothelial redox targets of hydrogen peroxide in endothelial cells and reveals that hypoxia induces a significant increase in the status of oxidized thiols. GELSILOX also detected thiols that are redox-modified by ischemia-reperfusion in heart mitochondria and demonstrated that these alterations are abolished in ischemia-preconditioned animals. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.016469, 800-813, 2012.

El dano isquemico se produce como resultado de una disminucion del flujo sanguineo al corazon, produciendo cambios metabolicos y estructurales que conducen a un deterioro irreversible. Se ha propuesto que las condiciones de estres oxidativo producido durante el proceso de isquemia-reperfusion (IR) comprometen el balance energetico celular, e inician vias de senalizacion que convergen en la mitocondria, reflejandose en modificaciones en los grupos tiol de las cisteinas. El precondicionamiento isquemico (IPC), proceso que consiste en varios ciclos breves de IR que preceden a periodos prolongados de isquemia, se ha descrito como posible mecanismo protector capaz de atenuar el dano. Con el fin de entender los mecanismos responsables de la proteccion adquirida durante IPC asi como los posibles cambios en el estado de oxidacion del proteoma mitocondrial de corazones de rata sometidos a IR, se realizo un analisis masivo mediante la tecnica GELSILOX, desarrollada en nuestro laboratorio, que permite la identificacion y cuantificacion simultaneas del estado redox de las Cys asi como del conjunto del proteoma. Mediante analisis de biologia de sistemas basados en un nuevo modelo estadistico, hemos observado que la IR produce una disminucion en las proteinas que pertenecen al grupo de la fosforilacion oxidativa (OXPHOS), y un aumento en la abundancia de peptidos que contienen residuos de cisteina oxidada, y que pertenecen a proteinas funcionalmente relevantes. Ambos tipos de cambios se atenuan cuando los corazones son sometidos previamente a IPC. Ademas, los cambios producidos por IR en la abundancia de proteinas de OXPHOS y de cisteinas oxidadas, asi como su reversion por IPC, se reproducen en un modelo in vitro en mitocondrias aisladas. Por tanto, los resultados obtenidos parecen indicar que el IPC atenua los cambios inducidos por IR en la abundancia de las proteinas de la OXPHOS y en el estado de oxidacion de cisteinas mediante mecanismos independientes de la senalizacion citosolica.

It has been demonstrated that heart can survive a short period of ischaemia and recover upon reperfusion (IR), but reperfusion can exacerbate the damage that takes place during the ischemic period. There is increasing evidence that mitochondria are implicated in the mechanisms that mediate reperfusion injury, which can be prevented or attenuated by ischemic preconditioning (IP). It has also been described that connexin 43 (Cx43) is located in the cardiomyocyte mitochondria and is important for the cardioprotection mediated by IP. Moreover, IP does not occur in transgenic mice Cx43KI32, which lacks Cx43.

In a previous work, we presented a random effects hierarchical model for the analysis of protein expression changes in 18 O-labelling/LIT experiments. This statistical model for the null hypothesis splits the total variance into three sources (spectrum, peptide and protein) and has been tested on several proteomes of different nature. The model has been implemented into QuiXoT, a software platform developed in our laboratory. Furthermore, in a collaborative, large-scale project using H2O2-treated Saccharomyces cerevisiae as a model system, we demonstrated this model is also valid for other SIL techniques, including SILAC, iTRAQ and different MS instruments.