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The current report describes the use of CapLC-ESI-Q/TOF-MS for investigating the proteome profiles of hypertonic saline-induced sputum samples from 56 smokers. The severity of their lung disease ranged from normal (healthy smokers) to chronic bronchitis, chronic obstructive pulmonary disease (COPD), and COPD with emphysema. This pilot study examined the hypothesis that there were distinct differences in protein expression profiles that were related to the phenotype and cigarette smoking illness severity. A total of 203 unique proteins were identified. These may represent the most highly expressed proteins in induced sputum. Our results provide evidence that different proteins are expressed, as the disease progresses from health to more advanced stages, and support our contention that a proteomic approach would be beneficial in discovering selective molecules linked to specific COPD stages.

Desmosines are crosslinking amino acids unique to mature elastin in humans. Owing to this unicity, they have been discussed as potentially attractive indicators of connective tissue disorders whose clinical manifestations are mostly the result of elastin degradation. This review covers advances in immunochemical, chromatographic, and electrophoretic procedures applied in the last 25 years to detect and quantitate these crosslinksin a variety of biological samples. Recent applications of CE with LIF detection (CE-LIF) for investigating the content of desmosines in different fluids will also be discussed.

James N Baraniuk,1 Begona Casado,1 Lewis K Pannell,2 Peter B McGarvey,3 Piera Boschetto,4 Maurizio Luisetti,5,† Paolo Iadarola6 1Division of Rheumatology, Immunology and Allergy, Georgetown University, Washington, DC, 2Proteomics and Mass Spectrometry Laboratory, Mitchell Cancer Center, University of South Alabama, Mobile, AL, 3Innovation Center for Biomedical Informatics, Georgetown University, Washington, DC, USA; 4Department of Medical Sciences, University of Ferrara, Ferrara, 5SC Pneumologia, Dipartimento Medicina Molecolare, Fondazione IRCCS Policlinico San Matteo, 6Lazzaro Spallanzani Department of Biology and Biotechnology, University of Pavia, Pavia, Italy †Maurizio Luisetti passed away on October 20, 2014 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. Keywords: cigarette smokers, chronic bronchitis, emphysema, proteomics, mucous hypersecretion, mucin 5AC, neutrophil extracellular nets

The aim of the present article is to review the efforts performed in the past two decades by numerous research groups for the development of methods that allow a correct diagnosis of prolidase deficiency (PD), a rare autosomal recessive disorder and for the rationalization of a possible therapeutic intervention on these patients. In particular, the interest of the reader is focused on the application of capillary electrophoresis (i) for the detection of biological markers that reflect the pathological feature of the disease and (ii) for the determination of the efficiency of a carrier system in delivering prolidase inside cells in a possible therapy based on enzyme replacement.

Human urine plays a central role in clinical diagnostic being one of the most-frequently used body fluid for detection of biological markers. Samples from patients with different diseases display patterns of biomarkers that differ significantly from those obtained from healthy subjects. The availability of fast, reproducible, and easy-to-apply analytical techniques that would allow identification of a large number of these analytes is thus highly desiderable since they may provide detailed information about the progression of a pathological process. From among the variety of methods so far applied for the determination of urinary metabolites, capillary electrophoresis, both in the capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) modes, represents a robust and reliable analytical tool widely used in this area. The aim of the present article is to focus the interest of the reader on recent applications of MEKC and CZE in the field of urinary biomarkers and to discuss advantages and/or limitations of each mode.

Prolidase deficiency (PD) is a rare autosomal recessive disorder caused by inadequate levels of the cytosolic exopeptidase prolidase (E.C. 3.4.13.9), for which there is not, as yet, a resolutive cure. We have investigated whether biodegradable microspheres loaded with prolidase could release active enzyme inside cells, to consider this system as a possible therapeutic approach for prolidase deficiency. Poly(lactide-co-glycolide) microspheres were prepared, modifying the classical double emulsion solvent evaporation method to mitigate the burst effect of the enzyme from the microspheres. Ex-vivo experiments were performed, by incubating microencapsulated prolidase with cultured fibroblasts from PD patients and from controls, to determine the amount of active enzyme delivered to the cells. The microparticulate drug delivery system described carried small amounts of active prolidase inside fibroblasts, ensuring a response to the intracellular accumulation of X-Pro dipeptides, the mechanism that is supposed to be responsible for the development of clinical manifestations of this disorder in man. A positive result of the presence of active enzyme inside cells was an improvement in fibroblast shape.

The nasal lavage fluids (NLFs) from four subjects with acute sinusitis were analyzed to investigate the amount of proteins expressed in this pathology at the beginning of the event (day 1) and after 6 days of treatment with antibiotics and a nasal steroid spray. The protein identification was performed with capillary liquid chromatography-electrospray-quadrupole time of flight-(LC-ESI-Q-TOF)-mass spectrometry. The samples collected on the first day contained high-abundant plasma proteins, such as albumin and immunoglobulins, glandular serous cell proteins (lysozyme, lactoferrin, and polymeric immunoglobulin receptor), epithelial keratins, and inflammatory cell proteins (myeloperoxidase, IL-16, and IL-17E). After six days of therapy, the complexity of the proteome was reduced to plasma proteins and lysozyme with no inflammatory markers. The presence of hemoglobin, however, suggested that significant squamous metaplasia with breaches in the epithelial barrier, or nasal steroid-related bleeding, had occurred. The proteomic approach presented here allowed us to identify, in the high complexity of acute sinusitis nasal secretions, the proteins that respond to a pharmacological treatment and that could be suitable as markers of this pathology.

Among various biomarkers believed to behave as descriptors of the disease process in chronic obstructive pulmonary disease (COPD), urinary desmosines are commonly used for monitoring elastin degradation. Given the low concentrations of urinary desmosines, their quantitative determination in this biological matrix often requires preconcentration steps. To minimize both solute losses and effects of sample matrix, and to decrease data variability related to the above-mentioned manipulation processes, we have developed a capillary electrophoresis approach combined with laser-induced fluorescence (CE-LIF) detection system using urine samples not sub' mitted to any pretreatment procedure other than filtering the sample. Urines were hydrolyzed, derivatized with fluoresceine isothiocyanate (FITC) and endogenous desmosines were identified by addition of standard analytes and submitting to mass spectrometry (MS) analysis the material collected from micropreparative runs. The assay showed good linearity, reproducibility and precision, allowing to detect amounts of desmosines as low as 10 - 8 M (equivalent to 0.1 fmol on column). We conclude that CE-LIF technique is a highly sensitive method for detecting urinary desmosines.

(2003). IDENTIFICATION OF LIPOCALIN FAMILY PROTEINS IN NASAL SECRETIONS. Experimental Lung Research: Vol. 29, No. 1, pp. 93-121.

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 advan...

Microfungi were isolated from different moss species in Victoria Land. Twenty-eight taxa belonging to 18 genera were identified. New records for continental Antarctica were: Arthrobotrys superba, Conidiobolus sp., Penicillium minioluteum, Verticillium psalliotae and V. lamellicola. The most frequently isolated fungal species were: Cladosporium cladosporioides, Cryptococcus albidus, Cryptococcus laurentii, Geomyces pannorum var. pannorum, G. pannorum var. vinaceus, Mortierella antarctica, Cadophora malorum, Phoma herbarum and V. lecanii. Bryum pseudotriquetrum was the moss richest in fungal species. Within the Antarctic environment, moss is one of the microhabitats richest in microfungi, particularly in psychrophilic indigenous species.

Prolidase is a naturally occurring enzyme involved in the final stage of protein catabolism. Deficient enzyme activity causes prolidase deficiency (PD), a rare autosomal recessive inherited disorder whose main manifestations are chronic, intractable ulcerations of the skin, particularly of lower limbs. Although several attempts have been made towards the treatment of this pathology, a cure for this disease has yet to be found. The purpose of this work is to evaluate the possibility of enzyme replacement therapy through prolidase microencapsulation in biodegradable microspheres. The poly( d,l -lactide-co-glycolide) (PLGA) prolidase loaded microparticulate systems have been prepared utilizing the w–o–w double emulsion solvent evaporation method. They have been characterized “in vitro” by morphological analysis, total protein content and an in vitro dissolution test of active protein. “Ex vivo” evaluation of prolidase activity from the microspheres has been performed on cellular extracts of cultured skin fibroblasts from healthy subjects (controls) and from patients affected by PD. The results reported in this work on prolidase from pig kidney (available on the market) demonstrate the positive role of microencapsulation as a process of enzymatic activity stabilization inside PLGA microspheres achieving both in vitro and ex vivo active enzyme release. This formulation can be proposed as a parenteral depot drug delivery system.

Batten disease, or human late-infantile neuronal ceroid lipofuscinosis (LINCL) is a familiar progressive degenerative disease affecting children, caused by a deficiency of a lysosomal proteinase (tripeptidyl peptidase I, TPP-I) and characterized by the accumulation of autofluorescent storage bodies in the brain and other tissues of the body. Current methodology used to diagnose this disease needs to be improved in order to have less invasive techniques with higher resolution and shorter assay time. In this report, we discuss the potential merits of micellar electrokinetic chromatography as an excellent tool that requires minute samples but offers high resolution and a short running time for monitoring TPP-I activity in human and animal specimens.

The exact knowledge of the qualitative and quantitative protein components of rice bran is an essential aspect to be considered for a better understanding of the functional properties of this resource. Aim of the present investigation was to extract the largest number of rice bran proteins and to obtain their qualitative characterization. For this purpose, three different extraction protocols have been applied either on full-fat or on defatted rice bran. Likewise, to identify the highest number of proteins, MS data collected from 1-DE, 2-DE and gel-free procedures have been combined. These approaches allowed to unambiguously identify 43 proteins that were classified as signalling/regulation proteins (30%), proteins with enzymatic activity (30%), storage proteins (30%), transfer (5%) and structural (5%) proteins. The fact that all extraction and identification procedures have been performed in triplicate with an excellent reproducibility provides a rationale for considering the platform of proteins shown in this study as the potential proteome profile of rice bran. It also represents a source of information to evaluate better the qualities of rice bran as food resource.

The determination of protein patterns in nasal secretions of healthy subjects can help in the early diagnosis of diseases such as acute sinusitis. The comparison of nasal lavage fluid collected from subjects with acute sinusitis before and after pharmacological treatment gives information about the drug effects on glandular secretions. Nasal secretions were stimulated with 1x NS (0.9% Normal Saline) and 24x NS in healthy subjects and in sinusitis subjects before and after pharmacological treatment. The nasal lavage fluid (NLF) proteins are precipitated with a solution of "acid-ethanol." Using this solution, the high molecular weight proteins precipitate and separate from the low molecular weight proteins. The proteins are digested and the peptides are separated using a capillary liquid chromatographic system. Eluted peptides are analyzed on ESI-Q-TOF mass spectrometry instrument.

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.

The determination of protein patterns in nasal secretions of healthy subjects can help in the early diagnosis of diseases such as acute sinusitis. The comparison of nasal lavage fluid collected from subjects with acute sinusitis before and after pharmacological treatment gives information about the drug effects on glandular secretions. Nasal secretions were stimulated with 1x NS (0.9% Normal Saline) and 24x NS in healthy subjects and in sinusitis subjects before and after pharmacological treatment. The nasal lavage fluid (NLF) proteins are precipitated with a solution of "acid-ethanol." Using this solution, the high molecular weight proteins precipitate and separate from the low molecular weight proteins. The proteins are digested and the peptides are separated using a capillary liquid chromatographic system. Eluted peptides are analyzed on ESI-Q-TOF mass spectrometry instrument.

The determination of possible biomarkers in nasal secretion of healthy subjects can have a role in early diagnosis of diseases such as rhinosinusitis. For this purpose, nasal lavage fluids (NLFs) from ten volunteers, collected before and after they had been submitted to nasal provocations, were investigated. Separation and analysis of proteins present in this complex matrix was performed using a capillary liquid chromatography-electrospray-quadrupole-time of flight mass spectrometry equipment. From among a total of 111 proteins found (89 known and two unknown proteins), 42 of which had never been previously described in this fluid, such as Deleted in Malignant Brain Tumors 1 isoform a precursors, and cytoskeletal proteins were identified with high statistical score. Three proteins of palate lung nasal epithelial clone (PLUNC) family: SPLUNC1, LPLUNC1, and LPLUNC2 were identified. Proteins involved in innate (27%) and acquired immunity (21%) systems were major components of NLF. Cellular (52% of all proteins identified) such as cytoskeletal (33%), functional (15%), and regulatory (4%) proteins, normally present in the nasal cavity, have also been identified. The proteomic approach presented here allowed us to identify the proteins involved in acquired and innate immune response in the nose against microbial infections and unclean inhaled air.

On July 9, 2003, the National Institutes of Health (NIH) released a new program announcement entitled "Proteomics in Auditory and Developmental Disease Processes." This initiative makes it clear that proteomic analysis in otology is a multi-year research priority for the NIH. The goal of this article is to describe the mechanics of modern proteomic techniques and review their applications in otology to date.General articles from the proteomic literature were used to construct a review of modern proteomic techniques. For literature on proteomics in otology, MEDLINE and CRISP databases were searched by various topics in otology and cross-referenced with principle proteomic technologies.The criterion for selection was any study in otology that employs proteomic technology.Incredible progress has been made in proteomic technology. However, modern proteomic techniques are currently underutilized in otologic research. The NIH proteomics initiative referenced above, in combination with an understanding of the basic tools of modern proteomic science, should help motivate otologists to discover innovative ways to apply modern proteomic techniques to specific problems in otology.

Since the completion of the human genome, the interest of the scientific community has evolved toward understanding the human proteome. The genomic and proteomic data will facilitate our understanding of the functions of proteins in diseases and the discovery of novel drug target proteins and biomarkers of diseases. Highly sensitive analytic techniques are necessary to study the complexity of biologic samples. The key to any proteomics experiment is to reduce the complexity of the sample before mass spectrometry (MS) analysis. Numerous separation techniques have been used, including one- and two-dimensional gel electrophoresis, chromatography, and affinity techniques. MS has become a powerful method for analyzing biologic samples. This review does not cover all aspects of proteomics, but is intended to give an introductory explanation of the technology using the example of the proteomics of nasal secretions.

Background Chronic 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. Methods Cerebrospinal 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. Results Pooled 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. Conclusion This 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.

Background Neuroglobin 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 ®.