Begona Casado

A chronic fatigue syndrome – related proteome in human cerebrospinal fluid

BackgroundChronic Fatigue Syndrome (CFS), Persian Gulf War Illness (PGI), and fibromyalgia are overlapping symptom complexes without objective markers or known pathophysiology. Neurological dysfunction is common. We assessed cerebrospinal fluid to find proteins that were differentially expressed in this CFS-spectrum of illnesses compared to control subjects.MethodsCerebrospinal fluid specimens from 10 CFS, 10 PGI, and 10 control subjects (50 μl/subject) were pooled into one sample per group (cohort 1). Cohort 2 of 12 control and 9 CFS subjects had their fluids (200 μl/subject) assessed individually. After trypsin digestion, peptides were analyzed by capillary chromatography, quadrupole-time-of-flight mass spectrometry, peptide sequencing, bioinformatic protein identification, and statistical analysis.ResultsPooled CFS and PGI samples shared 20 proteins that were not detectable in the pooled control sample (cohort 1 CFS-related proteome). Multilogistic regression analysis (GLM) of cohort 2 detected 10 proteins that were shared by CFS individuals and the cohort 1 CFS-related proteome, but were not detected in control samples. Detection of ≥1 of a select set of 5 CFS-related proteins predicted CFS status with 80% concordance (logistic model). The proteins were α-1-macroglobulin, amyloid precursor-like protein 1, keratin 16, orosomucoid 2 and pigment epithelium-derived factor. Overall, 62 of 115 proteins were newly described.ConclusionThis pilot study detected an identical set of central nervous system, innate immune and amyloidogenic proteins in cerebrospinal fluids from two independent cohorts of subjects with overlapping CFS, PGI and fibromyalgia. Although syndrome names and definitions were different, the proteome and presumed pathological mechanism(s) may be shared.

Therapeutic apheresis exchange in two patients with prolidase deficiency

Summary BackgroundProlidase deficiency is a rare genetic disorder for which a cure has not yet been found.

Human neuroglobin protein in cerebrospinal fluid

BackgroundNeuroglobin is a hexacoordinated member of the globin family of proteins. It is predominantly localized to various brain regions and retina where it may play a role in protection against ischemia and nitric oxide-induced neural injury. Cerebrospinal fluid was collected from 12 chronic regional or systemic pain and 5 control subjects. Proteins were precipitated by addition of 50% 0.2 N acetic acid, 50% ethanol, 0.02% sodium bisulfite. The pellet was extensively digested with trypsin. Peptides were separated by capillary liquid chromatography using a gradient from 95% water to 95% acetonitrile in 0.2% formic acid, and eluted through a nanoelectrospray ionization interface into a quadrapole – time-of-flight dual mass spectrometer (QToF2, Waters, Milford, MA). Peptides were sequenced (PepSeq, MassLynx v3.5) and proteins identified using MASCOT ®.ResultsSix different neuroglobin peptides were identified in various combinations in 3 of 9 female pain subjects, but none in male pain, or female or male control subjects.ConclusionThis is the first description of neuroglobin in cerebrospinal fluid. The mechanism(s) leading to its release in chronic pain states remain to be defined.

Protein networks in induced sputum from smokers and COPD patients.

Rationale Subtypes of cigarette smoke-induced disease affect different lung structures and may have distinct pathophysiological mechanisms. Objective To determine if proteomic classification of the cellular and vascular origins of sputum proteins can characterize these mechanisms and phenotypes. Subjects and methods Individual sputum specimens from lifelong nonsmokers (n=7) and smokers with normal lung function (n=13), mucous hypersecretion with normal lung function (n=11), obstructed airflow without emphysema (n=15), and obstruction plus emphysema (n=10) were assessed with mass spectrometry. Data reduction, logarithmic transformation of spectral counts, and Cytoscape network-interaction analysis were performed. The original 203 proteins were reduced to the most informative 50. Sources were secretory dimeric IgA, submucosal gland serous and mucous cells, goblet and other epithelial cells, and vascular permeability. Results Epithelial proteins discriminated nonsmokers from smokers. Mucin 5AC was elevated in healthy smokers and chronic bronchitis, suggesting a continuum with the severity of hypersecretion determined by mechanisms of goblet-cell hyperplasia. Obstructed airflow was correlated with glandular proteins and lower levels of Ig joining chain compared to other groups. Emphysema subjects’ sputum was unique, with high plasma proteins and components of neutrophil extracellular traps, such as histones and defensins. In contrast, defensins were correlated with epithelial proteins in all other groups. Protein-network interactions were unique to each group. Conclusion The proteomes were interpreted as complex “biosignatures” that suggest distinct pathophysiological mechanisms for mucin 5AC hypersecretion, airflow obstruction, and inflammatory emphysema phenotypes. Proteomic phenotyping may improve genotyping studies by selecting more homogeneous study groups. Each phenotype may require its own mechanistically based diagnostic, risk-assessment, drug- and other treatment algorithms.

Proteomics of Sinusitis Nasal Lavage Fluid

This chapter provides a brief survey of the two principal techniques applied in the study of the nasal lavage fluid (NLF) proteome. Lindahl et al. (11) used 2D gel electrophoresis (2-DE) to analyze the proteome of NLFs from subjects exposed to methyltetrahydrophthalic anhydride (MHHPA) or dimethylbenzylamine (DMBA) and from healthy nonsmokers and smokers. Casado et al. (2) used liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to study the NLF proteome in normal subjects and individuals affected by sinusitis before and after pharmacological treatment. New proteins involved in the acquired and innate immune response in the nose against microbial infections were identified with both techniques. A comparison between the normal and sinusitis NLF proteome facilitates understanding of changes in the protective mechanisms of the nasal mucosa against pathogens and pollutants. Acute sinusitis was associated with a large increase in plasma, glandular, and cellular components. Treatment successfully reduced the complexity of the nasal proteome. Finally, the presence of carbohydrate sulfotransferase is an indication of the acidic mucin synthesis.

IDENTIFICATION OF LIPOCALIN FAMILY PROTEINS IN NASAL SECRETIONS

Nasal lavage was performed in 10 normal subjects. Lipids and peptides were removed by extraction with 50% ethanol, 0.2M acetic acid, 0.2% sodium bisulfite. The pellet was digested with trypsin (1 nmole per mg protein). Digest peptides were separated by reversed phase capillary liquid chromatography with a C-18 column and gradient of 95% water, 5% 0.2% formic acid to 95% acetonitrile, 5% 0.2% formic acid. Peptides were further separated by mass/charge (M/Z) in a quadrapole—time of flight (QToF1, Micromass) mass spectrometer—mass spectrometer. Peptides were sequenced with MassLynx software. Various bioinformatics programs were then used to identify members of the lipocalin superfamily.

CAPILLARY ELECTROPHORESIS AS A MODERN TOOL FOR DETERMINING PROTEOLYTIC ACTIVITIES IN PURIFIED SPECIMENS AND IN REAL SAMPLES

ABSTRACT Owing to a series of interesting advantages, capillary electrophoresis (CE) is being widely used as an analytical tool for detecting enzymatic activities. On account of their resolving power, high speed, and small amount of sample required, several assays based on CE techniques have supported and/or replaced, during the past decade, traditional spectrophotometric or HPLC methods. In particular, a series of assays of practical utility have been designed for monitoring “in vitro” and “ex vivo”, the hydrolytic activity of proteinases of different origin involved in the development of human diseases. The aim of the present article is to focus the interest of the reader on some recent applications of different CE modes (capillary zone electrophoresis—CZE; micellar electrokinetic chromatography—MEKC and capillary isoelectric focusing—cIEF) in the field of proteolytic enzymes to underline the wide applicability of this methodology. The article presents examples of particular interest for which the advantages and/or limitations of CE over established assays are discussed.