Optimization of a MALDI-Imaging protocol for studying adipose tissue-associated disorders.

Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is increasingly recognized for its potential in the discovery of novel biomarkers directly from tissue sections. However, there are no MALDI IMS studies as yet on the adipose tissue, a lipid-enriched tissue that plays a pivotal role in the development of obesity-associated disorders. Herein, we aimed at developing an optimized method for analyzing adipose tissue lipid composition under both physiological and pathological conditions by MALDI IMS. Our studies showed an exacerbated lipid delocalization from adipose tissue sections when conventional strategies were applied. However, our optimized method using conductive-tape sampling and 2,5-dihydroxybenzoic acid (DHB) as a matrix, preserved the anatomical organization and minimized lipid diffusion from sample sections. This method enabled the identification of a total of 625 down-regulated and 328 up-regulated m/z values in the adipose tissue from a rat model of extreme obesity as compared to lean animals. Combination of MALDI IMS and liquid chromatography (LC)-MS/MS data identified 44 differentially expressed lipid species between lean and obese animals, including phospholipids and sphingomyelins. Among the lipids identified, SM(d18:0_18:2), PE(P-16:0_20:0), and PC(O-16:0_16:1) showed a differential spatial distribution in the adipose tissue of lean vs. obese animals. In sum, our method provides a valuable new tool for research on adipose tissue that may pave the way for the identification of novel biomarkers of obesity and metabolic disease.

Desarrollo y validación de un protocolo para el análisis proteómico de muestras de broncoaspirado de pacientes con adenocarcinoma de pulmón / Development and validation of a protocol for the proteomic analysis of bronchoaspiration samples in patients with lung adenocarcinoma

INTRODUCCIÓN: la identificación de marcadores en el broncoaspirado puede representar un hallazgo relevante, complementario al estudio citohistológico. OBJETIVO: determinar si el procedimiento aplicado es válido para obtener péptidos y proteínas suficientes, normalizar el protocolo y realizar un análisis shotgun por espectrometría de masas de los péptidos resultantes. MATERIAL Y MÉTODOS: se incluyeron 4 muestras de broncoaspirado de pacientes con adenocarcinoma de pulmón, aplicando el siguiente flujo de trabajo (I) recolección del broncoaspirado; (II) purificación de su componente proteica; (III) digestión proteica en solución; y (IV) análisis shotgun por espectrometría de masas de los péptidos resultantes. Para clasificar las proteínas según componente celular, proceso biológico y función molecular, se realizó un análisis Gene Onthology. RESULTADOS: en la muestra de 2,1 ml de broncoaspirado medio por paciente, fijando una tasa de falso descubrimiento (FDR, False Discovery Rate) restrictiva del 1%, se identificaron un número elevado de proteínas, con una media de 625, rango entre 529 a 708, y 6.290 péptidos únicos, rango entre 5.585 a 6.759. La composición proteica de las muestras fue homogénea, sin diferencias en la clasificación en relación a los componentes celulares, procesos biológicos y funciones moleculares. La eficacia de la digestión enzimática fue mayor del 90% en todos los casos, lo que asegura la reproducibilidad de la metodología utilizada. CONCLUSIONES: la metodología desarrollada para el análisis proteómico es altamente eficaz y reproducible, identificando un número elevado de proteínas en el broncoaspirado y permitirá realizar, de manera robusta, un estudio cuantitativo en una muestra amplia de pacientes y grupo control

INTRODUCTION: identifying markers in aspirated bronchial samples could represent a relevant discovery, complementary to cytohistological studies, in patients with lung adenocarcinoma. OBJECTIVE: determine whether the procedure applied is valid to obtain sufficient peptides and proteins to establish a protocol and perform mass spectrometry-based shotgun proteomic analysis for the resulting peptides. Material and METHOD: 4 aspirated bronchial samples were included from patients with pulmonary adenocarcinoma, applying the following work flow (I) recollection of aspirated bronchial samples; (II) purification of its protein component; (III) protein digestion in solution; and (IV) mass spectrometry-based shotgun proteomic analysis for the resulting peptides. To classify proteins based on cellular component, biological process and molecular function, gene ontology analysis was performed. RESULTS: in the 2.1 mlmeanbronchial sample per patient, a 1% restrictive false discovery rate (FDR)was established;an elevated number of proteins were identified, with an average of 625, range between 529 to 708 and 6.290 single peptides, range between 5.585 to 6.759. The proteincomposition of the samples was uniform, without differences in the classification with regards to cellular components, biological processes and molecular functions. The efficacy of enzymatic digestion was greater than 90% in all cases, which guarantees the reproducibility of the methodology used. CONCLUSIONS: themethodology developed for the proteomic analysis is highly effective and reproducible; it identifies a high number of proteins in the bronchial samples and allows us to perform, in a robust manner, a quantitative study in a wide range of patients and control group

Discovery of potential protein biomarkers of lung adenocarcinoma in bronchoalveolar lavage fluid by SWATH MS data-independent acquisition and targeted data extraction.

UNLABELLED: Lung cancer currently ranks as the neoplasia with the highest global mortality rate. Although some improvements have been introduced in recent years, new advances in diagnosis are required in order to increase survival rates. New mildly invasive endoscopy-based diagnostic techniques include the collection of bronchoalveolar lavage fluid (BALF), which is discarded after using a portion of the fluid for standard pathological procedures. BALF proteomic analysis can contribute to clinical practice with more sensitive biomarkers, and can complement cytohistological studies by aiding in the diagnosis, prognosis, and subtyping of lung cancer, as well as the monitoring of treatment response. The range of quantitative proteomics methodologies used for biomarker discovery is currently being broadened with the introduction of data-independent acquisition (DIA) analysis-related approaches that address the massive quantitation of the components of a proteome. Here we report for the first time a DIA-based quantitative proteomics study using BALF as the source for the discovery of potential lung cancer biomarkers. The results have been encouraging in terms of the number of identified and quantified proteins. A panel of candidate protein biomarkers for adenocarcinoma in BALF is reported; this points to the activation of the complement network as being strongly over-represented and suggests this pathway as a potential target for lung cancer research. In addition, the results reported for haptoglobin, complement C4-A, and glutathione S-transferase pi are consistent with previous studies, which indicates that these proteins deserve further consideration as potential lung cancer biomarkers in BALF. Our study demonstrates that the analysis of BALF proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS), combining a simple sample pre-treatment and SWATH DIA MS, is a useful method for the discovery of potential lung cancer biomarkers. SIGNIFICANCE: Bronchoalveolar lavage fluid (BALF) analysis can contribute to clinical practice with more sensitive biomarkers, thus complementing cytohistological studies in order to aid in the diagnosis, prognosis, and subtyping of lung cancer, as well as the monitoring of treatment response. Here we report a panel of candidate protein biomarkers for adenocarcinoma in BALF. Forty-four proteins showed a fold-change higher than 3.75 among adenocarcinoma patients compared with controls. This report is the first DIA-based quantitative proteomics study to use bronchoalveolar lavage fluid (BALF) as a matrix for discovering potential biomarkers. The results are encouraging in terms of the number of identified and quantified proteins, demonstrating that the analysis of BALF proteins by a SWATH approach is a useful method for the discovery of potential biomarkers of pulmonary diseases.

Comparison of protein expression profiles between three Perkinsus spp., protozoan parasites of molluscs, through 2D electrophoresis and mass spectrometry.

The genus Perkinsus includes protozoan parasites of a wide range of marine molluscs worldwide, some of which have been responsible for heavy mollusc mortalities and dramatic economic losses. This study was performed with the aim of increasing the knowledge of Perkinsus spp. proteome. Proteins extracted from in vitro cultured cells of three species of this genus, P. marinus, P. olseni and P. chesapeaki, were analysed using 2D electrophoresis. Four gels from each species were produced. Qualitative and quantitative comparisons among gels were performed with Proteamweaver software. Cluster analysis grouped the four gels of each Perkinsus sp.; furthermore, P. marinus and P. olseni gels were grouped in a cluster different from P. chesapeaki. Around 2000 spots of each species were considered, from which 213 spots were common to the 3 species; P. chesapeaki and P. marinus shared 310 spots, P. chesapeaki and P. olseni shared 315 spots and P. marinus and P. olseni shared 242 spots. A number of spots were exclusive of each Perkinsus species: 1161 spots were exclusive of P. chesapeaki, 1124 of P. olseni and 895 of P. marinus. A total of 84 spots, including common and species-specific ones, were excised from the gels and analysed using MALDI-TOF and nESI-IT (MS/MS) techniques. Forty-two spots were successfully sequenced, from which 28 were annotated, most of them clustered into electron transport, oxidative stress and detoxification, protein synthesis, carbohydrate metabolism, signal transduction, metabolic process and proteolysis.

Alterations to proteome and tissue recovery responses in fish liver caused by a short-term combination treatment with cadmium and benzo[a]pyrene.

The livers of soles (Solea senegalensis) injected with subacute doses of cadmium (Cd), benzo[a]pyrene (B[a]P), or their combination, were screened for alterations to cytosolic protein expression patterns, complemented by cytological and histological analyses. Cadmium and B[a]P, but not combined, induced hepatocyte apoptosis and Kupfer cell hyperplasia. Proteomics, however, suggested that apoptosis was triggered through distinct pathways. Cadmium and B[a]P caused upregulation of different anti-oxidative enzymes (peroxiredoxin and glutathione peroxidase, respectively) although co-exposure impaired induction. Similarly, apoptosis was inhibited by co-exposure, to which may have contributed a synergistic upregulation of tissue metalloproteinase inhibitor, beta-actin and a lipid transport protein. The regulation factors of nine out of eleven identified proteins of different types revealed antagonistic or synergistic effects between Cd and B[a]P at the prospected doses after 24 h of exposure. The results indicate that co-exposure to Cd and B[a]P may enhance toxicity by impairing specific responses and not through cumulative damage.