The Role of One- and Two-Dimensional Electrophoretic Techniques in Proteomics of the Lung

The current chapter was designed to keep the reader informed about the present status of pulmonary proteome. Taken together, the results documented here demonstrate that, after a decade of activity, proteomics of pulmonary diseases is catching up with its promise. The constantly growing number of reports in this area supports the view of this approach as one of the decisive methodological tools for the identification/characterization of disease-associated proteins. In terms of experimental procedures, the basic options available for proteomic investigations consist in the identification of proteins through the use of gel-based or gel-free techniques followed by MS. Obviously, the question arises of whether sophisticated technologies (such as the non-gel-based proteomic procedures) may currently be more fruitful, in terms of candidate protein marker identification, than “conventional” (read electrokinetic) approaches. In light of the versatility and high degree of reproducibility shown by these new potent strategies, a positive answer is perhaps not surprising. Nevertheless, as documented in this chapter, despite being less sophisticated than competing ones, gel-based techniques still represent a widely used procedure able to generate a reliable protein “fingerprint” and to produce qualitative and quantitative information on the protein patterns of a variety of human fluids

The “History” of Desmosines: Forty Years of Debate on the Hypothesis That These Two Unnatural Amino Acids May Be Potential Biomarkers of Chronic Obstructive Pulmonary Disease

Desmosine and isodesmosine (collectively known as desmosines), two unnatural amino acids unique to mature elastin in humans, have been widely discussed as being potential biomarkers of disorders, which involve connective tissue and whose clinical manifestations result in elastin degradation. In particular, experimental data accumulated over the last 40 years have demonstrated that patients with chronic obstructive pulmonary disease (COPD) excrete higher amounts of urinary desmosines than healthy controls. Based on this evidence, it has been speculated by several authors that these cross-links may be potential biomarkers of COPD with clinical significance. Nevertheless, a strict correlation between the amount of these amino acids and the severity of the disease still has to be demonstrated. For this reason, the debate on the opportunity to consider desmosines as biomarkers of COPD is still open, and the development of sophisticated methods aimed at obtaining very precise measurement of their concentration is still considered technically challenging. The aim of this chapter is to trace the history of this debate through the presentation and discussion of a large number of articles dealing with the detection and quantification of desmosines in different biological fluids, from early years until the present

Wound Repair Capability in EDS Fibroblasts can be Retrieved by Exogenous Type V Collagen

Impaired wound healing is a typical clinical hallmark of Ehlers-Danlos Syndrome (EDS). Mutated fibroblasts from EDS patients, which deposit an abnormal extracellular matrix, showed defective migration resulting in a marked delay in wound repair. The migratory capability remarkably improved in the presence of exogenous type V collagen

Proteomic Analysis of Human Sputum for the Diagnosis of Lung Disorders: Where Are We Today?

The identification of markers of inflammatory activity at the early stages of pulmonary diseases which share common characteristics that prevent their clear differentiation is of great significance to avoid misdiagnosis, and to understand the intrinsic molecular mechanism of the disorder. The combination of electrophoretic/chromatographic methods with mass spectrometry is currently a promising approach for the identification of candidate biomarkers of a disease. Since the fluid phase of sputum is a rich source of proteins which could provide an early diagnosis of specific lung disorders, it is frequently used in these studies. This report focuses on the state-of-the-art of the application, over the last ten years (2011–2021), of sputum proteomics in the investigation of severe lung disorders such as COPD; asthma; cystic fibrosis; lung cancer and those caused by COVID-19 infection. Analysis of the complete set of proteins found in sputum of patients affected by these disorders has allowed the identification of proteins whose levels change in response to the organism’s condition. Understanding proteome dynamism may help in associating these proteins with alterations in the physiology or progression of diseases investigated

Could proteomics become a future useful tool to shed light on the mechanisms of rare neurodegenerative disorders?

Very often the clinical features of rare neurodegenerative disorders overlap with those of other, more common clinical disturbances. As a consequence, not only the true incidence of these disorders is underestimated, but many patients also experience a significant delay before a definitive diagnosis. Under this scenario, it appears clear that any accurate tool producing information about the pathological mechanisms of these disorders would offer a novel context for their precise identification by strongly enhancing the interpretation of symptoms. With the advent of proteomics, detection and identification of proteins in different organs/tissues, aimed at understanding whether they represent an attractive tool for monitoring alterations in these districts, has become an area of increasing interest. The aim of this report is to provide an overview of the most recent applications of proteomics as a new strategy for identifying biomarkers with a clinical utility for the investigation of rare neurodegenerative disorders

Determination of amino acids by micellar EKC: recent advances in method development and novel applications to different matrices.

The extensive use of CE for the analysis of amino acids has been well documented in a series of research articles and reviews. Aim of this report is to address the attention of the reader on the recent advances of micellar electrokinetic chromatography for the separation and determination of these analytes. Enhancements in selectivity of this technique through the use of pseudostationary phases containing mixed micelles, polymers, and chiral selectors are presented. Selected applications concerning separation and quantitation of even minute amounts of amino acids in: (i) biological fluids; (ii) microdialysates; (iii) plant cells; (iv) food stuff; and (v) pharmaceutical formulations have also been covered. Advantages of MEKC over other techniques for the amino acid analysis have been underlined

MEKC: a powerful tool for the determination of amino acids in a variety of biomatrices.

The continued publication from year to year of new MEKC formulations for the analysis of amino acids provides evidence that both CZE and MEKC still have a great power of attraction in this area. The present review intends to cover the literature on MEKC of amino acids from 2007 until present: it has been planned to follow the same format of our previous review (Electrophoresis 2008, 29, 224–236) representing its ideal continuation. In addition to methodological developments, the more recent practical applications of MEKC procedures for the determination of amino acids in different matrices will also be described here as an evidence, once again, of the suitability of this technique on samples of different origin

Conductivity in Exhaled Breath Condensate from Subjects with Emphysema and Type ZZ alpha-1-Antitrypsin Deficiency

The assessment of biomarkers in biological samples from the lung has long been employed. Upon cooling water vapor present in exhaled breath, variable amounts of droplets of condensate (EBC) containing volatile and non-volatile compounds may be easily and non-invasively obtained from patients of any age.Objective of the present study was to compare the level of EBC conductivity determined for cohorts of individuals with different inflammatory lung disorders with that of healthy never-smoking individuals.The conductivity in EBC of PiZZ-Alpha-1-antitrypsin deficiency patients with a diagnosis of emphysema (PiZZ-AATD) was 3 fold lower than in spouse controls (54.5 ± 11.6 vs 165.3 ± 10.7 μS/cm). Non-PiZZ emphysema patients had conductivity in EBC of 59.6 ± 5.8 μS/cm and patients with sarcoidosis without airflow obstruction had EBC conductivity of 178,8 ± 6,2 μS/cm, \u2028not significantly different (p = 0.5) from healthy controls. Conductivity in serial EBC samples from patients with PiZZ-AATD emphysema and healthy controls was stable in 6 different samples collected over a period of 14 months. We conclude that conductivity values in EBC can be used as a correction factor for dilution of non-volatile components in EBC