Comprehensive Protocol to Simultaneously Study Protein Phosphorylation, Acetylation, and N-Linked Sialylated Glycosylation.

Posttranslational modifications (PTMs) such as phosphorylation, acetylation, and glycosylation are an essential regulatory mechanism of protein function and interaction, and they are associated with a wide range of biological processes. Since most PTMs alter the molecular mass of a protein, mass spectrometry (MS) is the ideal analytical tool for studying various PTMs. However, PTMs are often present in substoichiometric levels, and therefore their unmodified counterpart often suppresses their signal in MS. Consequently, PTM analysis by MS is a challenging task, requiring highly specialized and sensitive PTM-specific enrichment methods. Currently, several methods have been implemented for PTM enrichment, and each of them has its drawbacks and advantages as they differ in selectivity and specificity toward specific protein modifications. Unfortunately, for the vast majority of more than 400 known modifications, we have no or poor tools for selective enrichment.Here, we describe a comprehensive workflow to simultaneously study phosphorylation, acetylation, and N-linked sialylated glycosylation from the same biological sample. The protocol involves an initial titanium dioxide (TiO2) step to enrich for phosphopeptides and sialylated N-linked glycopeptides followed by glycan release and post-fractionation using sequential elution from immobilized metal affinity chromatography (SIMAC) to separate mono-phosphorylated and deglycosylated peptides from multi-phosphorylated ones. The IMAC flow-through and acidic elution are subsequently subjected to a next round of TiO2 enrichment for further separation of mono-phosphopeptides from deglycosylated peptides. Furthermore, the lysine-acetylated peptides present in the first TiO2 flow-through fraction are enriched by immunoprecipitation (IP) after peptide cleanup. Finally, the samples are fractionated by high pH reversed phase chromatography (HpH) or hydrophilic interaction liquid chromatography (HILIC ) to reduce sample complexity and increase the coverage in the subsequent LC-MS /MS analysis. This allows the analysis of multiple types of modifications from the same highly complex biological sample without decreasing the quality of each individual PTM study.

Evaluation of quantitative performance of sequential immobilized metal affinity chromatographic enrichment for phosphopeptides.

We evaluated a sequential elution protocol from immobilized metal affinity chromatography (SIMAC) employing gallium-based immobilized metal affinity chromatography (IMAC) in conjunction with titanium dioxide-based metal oxide affinity chromatography (MOAC). The quantitative performance of this SIMAC enrichment approach, assessed in terms of repeatability, dynamic range, and linearity, was evaluated using a mixture composed of tryptic peptides from caseins, bovine serum albumin, and phosphopeptide standards. Although our data demonstrate the overall consistent performance of the SIMAC approach under various loading conditions, the results also revealed that the method had limited repeatability and linearity for most phosphopeptides tested, and different phosphopeptides were found to have different linear ranges. These data suggest that, unless additional strategies are used, SIMAC should be regarded as a semiquantitative method when used in large-scale phosphoproteomics studies in complex backgrounds.

Candida albicans induces pro-inflammatory and anti-apoptotic signals in macrophages as revealed by quantitative proteomics and phosphoproteomics.

Macrophages play a pivotal role in the prevention of Candida albicans infections. Yeast recognition and phagocytosis by macrophages is mediated by Pattern Recognition Receptors (PRRs) that initiate downstream signal transduction cascades by protein phosphorylation and dephosphorylation. We exposed RAW 264.7 macrophages to C. albicans for 3h and used SILAC to quantify macrophage proteins and phosphoproteins by mass spectrometry to study the effects of infection. We identified 53 macrophage up-regulated proteins and 15 less abundant in the presence of C. albicans out of a total of 2071 identified proteins. 922 unique protein phosphorylation sites were identified by phosphopeptide enrichment and mass spectrometry, including 327 previously unidentified mouse protein phosphorylation sites. 126 peptides showed an increase and 70 a decrease in their phosphorylation level. The majority of the differentially expressed and phosphorylated proteins are receptors, mitochondrial ribosomal proteins, cytoskeletal proteins, and transcription factor activators involved in inflammatory and oxidative responses. In addition, we identified 22 proteins and phosphoproteins related to apoptosis. The analysis of apoptotic markers revealed that anti-apoptotic signals prevailed during the interaction of the yeast. Our proteomics study suggests that besides inflammation, apoptosis is a central pathway in the immune defense against C. albicans infection. BIOLOGICAL SIGNIFICANCE: This work uses SILAC and SIMAC methodology combined with CPP (+ TiO2) to study protein and phosphopeptide changes in RAW 264.7 macrophages in response to coincubation with Candida albicans for 3h. We show that the presence of C. albicans induces inflammatory responses and inhibits apoptosis in the macrophages. Our phosphoproteomic analysis identified 327 new mouse protein phosphorylation sites.

SIMAC: Development and implementation of a coral reef monitoring network in Colombia

Significant coral reef decline has been observed in Colombia during the last three decades. However, due to the lack of monitoring activities, most of the information about health and changes was fragmentary or inadequate. To develop an expanded nation-wide reef-monitoring program, in 1998 INVEMAR (Instituto de Investigaciones Marinas y Costeras: "Colombian Institute of Marine and Coastal Research") designed and implemented SIMAC (Sistema Nacional de Monitoreo de Arrecifes Coralinos en Colombia: "National Monitoring System of Coral Reefs in Colombia") with the participation of other institutions. By the end of 2003 the SIMAC network reached more than twice its initial size, covering ten reef areas (seven in the Caribbean and three in the Pacific), 63 reef sites and 263 permanent transects. SIMAC monitoring continued without interruption until 2008 and should persist in the long-term. The SIMAC has a large database and consists basically of water quality measurements (temperature, salinity, turbidity) and a yearly estimation of benthic reef cover, coral disease prevalence, gorgonian density, abundance of important mobile invertebrates, fish diversity and abundance of important fish species. A methods manual is available in the internet. Data and results of SIMAC have been widely circulated through a summary report published annually since 2000 for the Colombian environmental agencies and the general public, as well as numerous national and international scientific papers and presentations at meetings. SIMAC information has contributed to support regional and global reef monitoring networks and databases (i.e. CARICOMP, GCRMN, ReefBase). Rev. Biol. Trop. 58 (Suppl. 1): 67-80. Epub 2010 May 01.

En respuesta al proceso de deterioro de los arrecifes coralinos colombianos en las últimas tres décadas, y con el propósito de establecer un sistema de vigilancia para el manejo apropiado de estos valiosos ecosistemas, el Instituto de Investigaciones Marinas y Costeras (INVEMAR) desde 1998 ha impulsando y puesto en marcha el Sistema Nacional de Monitoreo de Arrecies Coralinos en Colombia (SIMAC), con el apoyo de varias instituciones colombianas. El SIMAC ha operado sin interrupción por más de ocho años, periodo durante el cual se han establecido 63 parcelas permanentes de observación y 267 transectos fijos de evaluación, en diez de las principales áreas geográficas con arrecifes de Colombia (siete en le Caribe y tres en el Pacífico). Los procedimientos metodológicos del SIMAC se hallan descritos detalladamente en el manual de métodos e incluyen: cobertura del bentos arrecifal, densidad de gorgonáceos, prevalencia de enfermedades coralinas, abundancia de invertebrados móviles selectos, riqueza ictiológica y densidad de familias selectas de peces, así como algunas variables indicadoras de la calidad del agua. Los resultados del SIMAC han sido circulados a través de reportes resumen para las agencias del gobierno y público en general y han apoyado iniciativas regionales y globales de monitoreo (i.e. CARICOMP, GCRMN, ReefBase). En este artículo se describe el desarrollo de este sistema y en otros artículos dentro de este número especial se presentan los principales resultados del monitoreo.

Coral diseases and bleaching on Colombian Caribbean coral reefs

Since 1998 the National Monitoring System for the Coral Reefs of Colombia (SIMAC) has monitored the occurrence of coral bleaching and diseases in some Colombian coral reefs (permanent stations at San Andres Island, Rosario Islands, Tayrona, San Bernardo Islands and Urabá). The main purpose is to evaluate their health status and to understand the factors that have been contributing to their decline. To estimate these occurrences, annual surveys in 126 permanent belt transects (10x2m) with different depth intervals (3-6 meters, 9-12 meters and 15-18 meters) are performed at all reef sites. Data from the 1998-2004 period, revealed that San Andrés Island had many colonies with diseases (38.9 colonies/m2), and Urabá had high numbers with bleaching (54.4 colonies/m2). Of the seven reported coral diseases studied, Dark Spots Disease (DSD), and White Plague Disease (WPD) were noteworthy because they occurred in all Caribbean monitored sites, and because of their high interannual infection incidence. Thirty five species of scleractinian corals were affected by at least one disease and a high incidence of coral diseases on the main reef builders is documented. Bleaching was present in 34 species. During the whole monitoring period, Agaricia agaricites and Siderastrea siderea were the species most severely affected by DSD and bleaching, respectively. Diseases on species such as Agaricia fragilis, A.grahamae, A. humilis, Diploria clivosa, Eusmilia fastigiata, Millepora complanata, and Mycetophyllia aliciae are recorded for first time in Colombia. We present bleaching and disease incidences, kinds of diseases, coral species affected, reef localities studied, depth intervals of surveys, and temporal (years) variation for each geographic area. This variation makes difficult to clearly determine defined patterns or general trends for monitored reefs. This is the first long-term study of coral diseases and bleaching in the Southwestern Caribbean, and one of the few long term monitoring studies on coral diseases worldwide. Rev. Biol. Trop. 58 (Suppl. 1): 95-106. Epub 2010 May 01.

Desde 1998 el "Sistema Nacional de Monitoreo de Arrecifes Coralinos de Colombia" SIMAC, ha observado la ocurrencia de enfermedades coralinas y blanqueamiento en arrecifes colombianos (estaciones fijas en la Isla de San Tayrona, Islas del Rosario, Islas de San Bernardo y Urabá Chocoano). Para estimar la ocurrencia se ha examinado anualmente un total de 126 bandas permanentes (10x2m), dispuestas en diferentes rangos de profundidad en las áreas arrecifales objeto de estudio. El análisis de la información obtenida entre 1998 y el 2004 revela que San Andrés presenta altos promedios de colonias enfermas (38.9 colonias/100m2) y que Urabá exhibe ésta condición para el blanqueamiento (54.4 colonias/100m2). Del total de siete enfermedades detectadas, se destacan por su presencia en todas las áreas y la ocurrencia interanual, los Lunares Oscuros (DSD) y la Plaga Blanca (WPD). Un total de 35 especies de corales pétreos fueron registradas con al menos una enfermedad y se encontró una alta ocurrencia de enfermedades en las principales especies formadoras de arrecifes. El blanqueamiento se halló presente en 34 especies. Mientras WPD se halló en más especies (33), la DSD se registró un mayor número de veces. Agaricia agaricites fue la especie con mayor número de registros de DSD; por otra parte Siderastrea siderea fue vista un mayor número de veces con signos de blanqueamiento. Especies como Agaricia fragilis, A.grahamae, A. humilis, Diploria clivosa, Eusmilia fastigiata, Millepora complanata, Mycetophyllia aliciae y Siderastrea radians son registradas por primera vez con presencia de enfermedades en Colombia. Cada área geográfica presentó variaciones espaciales (localidades, rangos de profundidad) y temporales (años) en cuanto a las prevalencias, tipos de enfermedades y especies de corales afectadas, que dificultan determinar patrones claramente definidos o tendencias generales para los arrecifes evaluados.

Recent dynamics and condition of coral reefs in the Colombian Caribbean

Long-term monitoring data provide a basis to recognize changes in coral reef communities and to implement appropriate management strategies. Unfortunately, coral reef dynamics have been poorly documented at any temporal scale in the Southern Caribbean. Through the "National Monitoring System of Coral Reefs in Colombia" (Spanish acronym: SIMAC), we assessed 32 permanent plots at different depth levels in six reefs areas of the Colombian Caribbean from 1998 to 2004. Temporal trends in coral and algal cover were evaluated by repeated measures ANOVA. The model included the effect of depth levels (a fixed effect), monitoring plots (a random effect) as a nested factor within depths, and time (repeated factor). We found high spatial variability in major benthic components. Overall means indicated that algae were the most abundant biotic component in nearly all areas, ranging from 30.3% at Rosario to 53.3% at San Andrés. Live coral cover varied considerably from 10.1% at Santa Marta up to 43.5% at Urabá. Coral and algae cover per se are not always accurate reef indicators and therefore they need supplementary information. Temporal analyses suggested relative stability of coral and algal cover along the study but the causes for the observed trends were rarely identified. A significant decrease (p=0.042) in coral cover was only identified for some monitoring plots in Tayrona-time x plot (depth level) interaction, and importantly, few coral species explained this trend. Significant increase (p=0.005) in algal cover was observed over time for most plots in Rosario. Temporal trajectories in algal cover were influenced by depth-significant time x depth interaction-in San Andrés (increase, p=0.004) and Urabá (decrease, p=0.027). Algae trends were mainly explained by changes in algal turfs. Monitoring programs must focus on the mechanisms mediating the changes, in particular those concerning coral recovery and reef resilience in the current context of climate change. Rev. Biol. Trop. 58 (Suppl. 1): 107-131. Epub 2010 May 01.

Este trabajo contiene el primer análisis temporal de la información obtenida por el Sistema Nacional de Monitoreo de Arrecifes Coralinos en Colombia (SIMAC). Entre 1998 y el 2004 se monitorearon un total de 32 parcelas permanentes ubicadas a diferentes niveles de profundidad en seis áreas arrecifales del Caribe colombiano. Los patrones temporales de algas y corales fueron evaluados mediante análisis de varianza de medidas repetidas. Los promedios generales indicaron que las algas dominaron en la mayoría de las áreas evaluadas, variando de 30.3% (Rosario) hasta 53.3% (San Andrés). La cobertura coralina fluctuó considerablemente entre 10.1% (Santa Marta) y 43.5% (Urabá). Los arrecifes estudiados han permanecido relativamente estables durante el periodo evaluado en términos de algas y corales. El único cambio significativo en la cobertura se detectó en algunas parcelas de monitoreo del Tayrona, y pocas especies coralinas explicaron la tendencia de disminución. En Rosario se detectó una tendencia significativa de incremento para las algas en la mayoría de las parcelas. En San Andrés y Urabá las tendencias temporales (aumento y disminución respectivamente) se presentaron en ciertos niveles de profundidad. En estas dos áreas las tendencias en la cobertura de las algas fueron explicadas principalmente por cambios en los tapetes algales. En general las causas de los patrones observados no pudieron identificarse. Los programas de monitoreo deben evaluar no solo las tendencias generales de algas y corales sino también las de sus componentes (especies de coral y grupos funcionales de algas). Así mismo, deben enfocarse en evaluar los mecanismos involucrados en los cambios, en especial aquellos relacionados con la recuperación coralina y la resiliencia arrecifal, de manera que se pueda enfrentar el deterioro arrecifal en el actual contexto de cambio climático.

Agents of coral mortality on reef formations of the Colombian Pacific

The National Monitoring System for Coral Reefs of Colombia (SIMAC) monitors the impact of some of the most important agents of coral tissue loss (bleaching and/or disease) in the Colombian Pacific coral formations since 1998. Physiological bleaching is among the main results of stress in the area. Signs of coral diseases resembling bacterial bleaching such as White Plague and White Band, were observed in Malpelo and Gorgona islands. Damage to the Pacific gorgonian Pacifigorgia spp., similar to those produced by Aspergillosis in Caribbean corals, was detected in Utría Bay. The presence of tumors in colonies of massive corals was also recorded. Even though coral diseases are globally widespread, their occurrence in American Pacific reefs has been poorly documented to date. Rev. Biol. Trop. 58 (Suppl. 1): 133-138. Epub 2010 May 01.

A través del Sistema Nacional de Monitoreo de Arrecifes Coralinos en Colombia-SIMAC se han evaluado algunos agentes de mortalidad coralina en el Pacifico Colombiano desde 1998. Uno de los principales factores que han contribuido a la pérdida de cobertura coralina han sido los eventos de blanqueamiento. No obstante, también se han observado signos que sugieren la presencia de enfermedades coralinas como el blanqueamiento bacteriano, la Plaga Blanca, la Banda Blanca, los tumores coralinos y la Aspergilosis en Pacifigorgia spp.. Aunque las enfermedades coralinas están globalmente distribuidas, su ocurrencia en el Pacifico tropical americano ha sido pobremente documentada. Esta nota incluye la ocurrencia de potenciales enfermedades coralinas en el Pacífico Colombiano.