Mass Spectrometric Proteomics

As suggested by the title of this Special Issue, liquid chromatography-mass spectrometry plays a pivotal role in the field of proteomics. Indeed, the research and review articles  published in the Issue clearly evidence how the data produced by this sophisticated methodology may promote impressive advancements in this area. From among the topics discussed in the Issue, a few point to the development of  new procedures for the  optimization of the experimental conditions that should be applied  for the identification of proteins present in complex mixtures.  Other applications  described in these articles show  the huge potential of  these strategies in the protein profiling of organs and  range from  to the study of post-translational tissue modifications to the investigation of the molecular mechanisms behind human disorders and the identification of potential biomarkers of these diseases

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

differences in amino acid loss between high efficiency hemodialysis and postdilution and predilution hemodiafiltration using high convection volume exchange a new metabolic scenario a pilot study

Objective The objective of the study was to quantify the loss of total amino acids (TAAs), nonessential amino acids, essential amino acids, and branched chain amino acids (BCAAs) produced by high-efficiency hemodialysis (HEHD), postdilution hemodiafiltration (HDFpost), and predilution hemodiafiltration (HDFpre) using high ultrafiltration volumes; and to define the specific AA losses registered in HEHD, HDFpost, and HDFpre; to identify a potential metabolic and nutritional decline into protein energy wasting; to compare AA analysis of arterial blood samples taken from healthy controls and patients with end-stage renal disease undergoing hemodialysis. Design and Methods Identical dialysis monitors, membranes, and dialysate/infusate were used to homogenize extracorporeal body influence. Ten patients were recruited and randomized to receive treatment with HEHD, HDFpost, and HDFpre it was used on-line dialytic water methodologies (OL); patients' AA arterial concentrations were measured at the start and on completion of dialysis; TAA from the dialyzer filter was calculated, and baseline levels were subsequently compared with findings obtained 1 year later. Finally, the results obtained were compared with the data from a study of 8 healthy volunteers conducted using bioimpedance analysis and laboratory blood tests to assess nutritional status. Results A higher convective dose results in a higher weekly loss of TAA, nonessential AAs, essential AAs, and BCAAs (HEHD: 15.7 g; HDFpost-OL: 16.1 g; HDFpre-OL: 16.3 g, P P Conclusion The study shows that the AA losses in dialytic liquid are greater after high exchange volume HDF techniques, especially HDFpre. The AA losses are not metabolically compensated, so these increase the derangements of predialytic arterial plasma AA levels. Both AA losses and arterial AA perturbations further worsened body composition already after 12 months of additional dialysis

Identification of Novel Short C-Terminal Transcripts of Human SERPINA1 Gene

Human SERPINA1 gene is located on chromosome 14q31-32.3 and is organized into three (IA, IB, and IC) non-coding and four (II, III, IV, V) coding exons. This gene produces α1-antitrypsin (A1AT), a prototypical member of the serpin superfamily of proteins. We demonstrate that human peripheral blood leukocytes express not only a product corresponding to the transcript coding for the full-length A1AT protein but also two short transcripts (ST1C4 and ST1C5) of A1AT. In silico sequence analysis revealed that the last exon of the short transcripts contains an Open Reading Frame (ORF) and thus putatively can produce peptides. We found ST1C4 expression across different human tissues whereas ST1C5 was mainly restricted to leukocytes, specifically neutrophils. A high up-regulation (10-fold) of short transcripts was observed in isolated human blood neutrophils after activation with lipopolysaccharide. Parallel analyses by liquid chromatography-mass spectrometry identified peptides corresponding to C-terminal region of A1AT in supernatants of activated but not naïve neutrophils. Herein we report for the first time a tissue specific expression and regulation of short transcripts of SERPINA1 gene, and the presence of C-terminal peptides in supernatants from activated neutrophils, in vitro. This gives a novel insight into the studies on the transcription of SERPINA1 gene.This work has been partially funded by the Instituto de Salud Carlos III (www.isciii.es) grant PI14CIII/00070 (BMD) and SEPAR (Sociedad Española de Neumología y Cirugía Torácica, www.separ.es) grant 92/2014. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

An isoform of the giant protein titin is a master regulator of human T lymphocyte trafficking

Response to multiple microenvironmental cues and resilience to mechanical stress are essential features of trafficking leukocytes. Here, we describe unexpected role of titin (TTN), the largest protein encoded by the human genome, in the regulation of mechanisms of lymphocyte trafficking. Human T and B lymphocytes express five TTN isoforms, exhibiting cell-specific expression, distinct localization to plasma membrane microdomains, and different distribution to cytosolic versus nuclear compartments. In T lymphocytes, the LTTN1 isoform governs the morphogenesis of plasma membrane microvilli independently of ERM protein phosphorylation status, thus allowing selectin-mediated capturing and rolling adhesions. Likewise, LTTN1 controls chemokine-triggered integrin activation. Accordingly, LTTN1 mediates rho and rap small GTPases activation, but not actin polymerization. In contrast, chemotaxis is facilitated by LTTN1 degradation. Finally, LTTN1 controls resilience to passive cell deformation and ensures T lymphocyte survival in the blood stream. LTTN1 is, thus, a critical and versatile housekeeping regulator of T lymphocyte trafficking

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