Lumican is overexpressed in lung adenocarcinoma pleural effusions.

Adenocarcinoma (AdC) is the most common lung cancer subtype and is often associated with pleural effusion (PE). Its poor prognosis is attributable to diagnostic delay and lack of effective treatments and there is a pressing need in discovering new biomarkers for early diagnosis or targeted therapies. To date, little is known about lung AdC proteome. We investigated protein expression of lung AdC in PE using the isobaric Tags for Relative and Absolute Quantification (iTRAQ) approach to identify possible novel diagnostic/prognostic biomarkers. This provided the identification of 109 of lung AdC-related proteins. We further analyzed lumican, one of the overexpressed proteins, in 88 resected lung AdCs and in 23 malignant PE cell-blocks (13 lung AdCs and 10 non-lung cancers) using immunohistochemistry. In AdC surgical samples, lumican expression was low in cancer cells, whereas it was strong and diffuse in the stroma surrounding the tumor. However, lumican expression was not associated with tumor grade, stage, and vascular/pleural invasion. None of the lung cancer cell-blocks showed lumican immunoreaction, whereas those of all the other tumors were strongly positive. Finally, immunoblotting analysis showed lumican expression in both cell lysate and conditioned medium of a fibroblast culture but not in those of A549 lung cancer cell line. PE is a valid source of information for proteomic analysis without many of the restrictions of plasma. The high lumican levels characterizing AdC PEs are probably due to its release by the fibroblasts surrounding the tumor. Despite the role of lumican in lung AdC is still elusive, it could be of diagnostic value

Polar Electrophoresis: Shape of Two-Dimensional Maps Is as Important as Size

The performance of two-dimensional electrophoresis in conventional gels in Cartesian coordinates (2-DE) vs. polar coordinates (2-PE) is here evaluated. Although 2-DE is performed in much longer Immobiline gels in the first dimension (17 cm) vs. barely 7-cm in 2-PE, an equivalent resolving power is found. Moreover, due to the possibility of running up to seven Immobiline strips in the radial gel format, the reproducibility of spot position is seen to be higher, this resulting in a 20% higher matching efficiency. As an extra bonus, strings of “isobaric” spots (i.e. polypeptides of identical mass with different pI values) are more resolved in the radial gel format, especially in the 10 to 30 kDa region, where the gel area fans out leaving extra space for spot resolution. In conclusion, this novel gel format in the second dimension of 2D gels is seen as an important improvement of this technique, still one of the most popular in proteome analysis

High Abundance Proteins Depletion vs Low Abundance Proteins Enrichment: Comparison of Methods to Reduce the Plasma Proteome Complexity

BACKGROUND: To date, the complexity of the plasma proteome exceeds the analytical capacity of conventional approaches to isolate lower abundance proteins that may prove to be informative biomarkers. Only complex multistep separation strategies have been able to detect a substantial number of low abundance proteins (<100 ng/ml). The first step of these protocols is generally the depletion of high abundance proteins by the use of immunoaffinity columns or, alternatively, the enrichment of by the use of solid phase hexapeptides ligand libraries. METHODOLOGY/PRINCIPAL FINDINGS: Here we present a direct comparison of these two approaches. Following either approach, the plasma sample was further fractionated by SCX chromatography and analyzed by RP-LC-MS/MS with a Q-TOF mass spectrometer. The depletion of the 20 most abundant plasma proteins allowed the identification of about 25% more proteins than those detectable following low abundance proteins enrichment. The two datasets are partially overlapping and the identified proteins belong to the same order of magnitude in terms of plasma concentration. CONCLUSIONS/SIGNIFICANCE: Our results show that the two approaches give complementary results. However, the enrichment of low abundance proteins has the great advantage of obtaining much larger amount of material that can be used for further fractionations and analyses and emerges also as a cheaper and technically simpler approach. Collectively, these data indicate that the enrichment approach seems more suitable as the first stage of a complex multi-step fractionation protocol

Proteomica di fibroblasti cutanei coltivati in vitro, ottenuti da biopsie di soggetti normali e di pazienti diabetici di tipo 1, caratterizzati dalla presenza o assenza di complicanze microvascolari renali

The target of this research project was to identify protein markers of diabetic nephropathy (DN), a microvascular complication which develops in about 30% of subject with type 1 diabetes and which is often associated with an increase risk of developing cardiovascular diseases and premature death in comparison with non nephropatic subjects. Many studies indicate that hyperglycemia is a necessary although not sufficient condition to determine the diabetes associated renal damage. Indeed, the incidence of nephropathy reaches a maximum after 15-20 years of disease and thereafter it decreases. Such a behaviour is compatible with the existence of an individual susceptibility (genetic) to renal damage, induced by factors other than hyperglycaemia, thus highlighting the need for an early detection of the type 1 diabetic subjects with high risk of developing DN, and to clarify the pathophysiological mechanisms Human fibroblasts represent an ideal experimental model, also because easily accessible, for an ample investigation on the genetic predisposition to diabetic disease, as well as to study of the mechanisms leading to cellular and metabolic damages occurring in diabetic complications. We have established an experimental protocol for the extraction of proteins from cultured cells and we have defined the optimal experimental conditions to obtain reproducible 2-D gels. So we compared protein profiles of cultured fibroblasts, harvested from skin biopsies of type 1 diabetic patients with long disease duration and with or without DN, and normal healthy subjects. This research has allowed, firstly, a more and deeper knowledge about human fibloblast proteome, useful data as a reference for subsequent investigations applied to the study of various diseases including non-diabetic. Secondly, the comparison among groups showed significant changes in the amounts of some proteins including cytoskeletal proteins and proteins involved in energy metabolism and protein turnover. Some of these results have been validated with more depth analysis as western blot and enzymatic activity test. These data, entirely original at the international level, represent a starting point for further investigations for the early identification of subjects with a genetic predisposition to DN and revealed new clues to understand physiopathological mechanisms that lead to the development of this pathology. Finally we made the comparison of protein levels after exposure to high levels of glucose, within each group and among the three groups, obtaining information about proteins involved in cellular response to hyperglycemia

Informed sequential pooling approach to detect SARS-CoV-2 infection

The alarming spread of the pandemic coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus requires several measures to reduce the risk of contagion. Every successful strategy in controlling the SARS-CoV-2 infection depends on timely diagnosis, which should include testing of asymptomatic carriers. Consequently, increasing the throughput for clinical laboratories for the purposes of conducting large-scale diagnostic testing is urgently needed. Here we support the hypothesis that standard diagnostic protocol for SARS-CoV-2 virus could be conveniently applied to pooled samples obtained from different subjects. We suggest that a two-step sequential pooling procedure could identify positive subjects, ensuring at the same time significant benefits of cost and time. The simulation data presented herein were used to assess the efficiency, in terms of number of required tests, both for random assignment of the subjects to the pools and for situations in which epidemiological and clinical data are used to create "informed" pools. Different scenarios were simulated to measure the effect of different pool sizes and different values for virus frequency. Our results allow for a customization of the pooling strategy according to the specific characteristics of the cohort being teste

Test Groups, Not Individuals: A Review of the Pooling Approaches for SARS-CoV-2 Diagnosis

Massive molecular testing for SARS-CoV-2 diagnosis is mandatory to manage the spread of COVID-19. Diagnostic screening should be performed at a mass scale, extended to the asymptomatic population, and repeated over time. An accurate diagnostic pipeline for SARS-CoV-2 that could massively increase the laboratory efficiency, while being sustainable in terms of time and costs, should be based on a pooling strategy. In the past few months, researchers from different disciplines had this same idea: test groups, not individuals. This critical review intends to highlight both the general consents\u2014even if the results from different publications have been obtained with different protocols\u2014and the points of disagreement that are creating some interpretative/comprehension difficulties. Different pooling schemes and technical aspects associated to the type of pooling adopted are described and discussed. We hope that this review can consolidate information to support researchers in designing optimized COVID-19 testing protocols based on pooling

Proteomica e Diabete

La \u201cProteomica\u201d appartiene ad un gruppo di metodologie, le \u201c-omiche\u201d, che comprendono misurazioni estese di analiti (vedi genomica per i geni, trascrittomica per i trascritti dei geni, cio\ue8 gli mRNA, metabolomica, per le analisi di metaboliti, lipidomica, ecc.). Le \u201c-omiche\u201d hanno l\u2019ambizione di misurare la totalit\ue0 degli analiti di un certo gruppo presenti in un determinato campione. Tali misurazioni sono rese possibili da metodologie sviluppate e perfezionate negli anni pi\uf9 recenti, e presuppongono quindi procedure e piattaforme di analisi capaci, in teoria, di identificare (quali- e quantitativamente) tutti gli analiti presenti nel campione.Le \u201comiche\u201d (definite anche indagini \u201cwide-search\u201d, o \u201cunbiased\u201d) rappresentano quindi un approccio differente da quello della biochimica classica, in cui si parte da una definita ipotesi sperimentale, per provare la quale si disegnano esperimenti specifici e mirati. Nelle \u201comiche\u201d invece, si ambisce a misurare la totalit\ue0 degli analiti di un campione, spesso senza alcuna ipotesi sperimentale \u201ca priori\u201d, verificando eventuali differenze tra casi e controlli, o tra diverse condizioni sperimentali, in base alle quali vengono poi derivate ipotesi, costruiti meccanismi, predisposti ulteriori esperimenti per lo studio di relazioni causa-effetto, ecc. La Proteomica pu\uf2 quindi rappresentare una metodica innovativa, seppur complessa, nella ricerca di markers precoci (sia di tipo genetico che non) anche di nefropatia diabetica, e pu\uf2 contribuire ad una pi\uf9 precisa definizione della storia clinica della malattia e della sua eziopatogenesi. Per la sua complessit\ue0, tuttavia, richiede standardizzazione di metodiche e personale competente nell\u2019analisi sperimentale e nell\u2019elaborazione bioinformatica dei dati. La costante verifica e condivisione dei dati da parte dei ricercatori dovrebbe inoltre rappresentare una irrinunciabile metodologia per rendere plausibili e applicabili le evidenze sperimentali

No peptide left behind: the "out of range" recovery in IPG-IEF fractionation.

IEF is often used in multidimensional shotgun proteomics and the narrow range of 3.5-4.5 is the recommended pH interval for the fractionation of tryptic peptides. Usually, even if IEF is performed in IPG strip with a narrow range pH, the entire sample must be loaded onto the strip, including the "out of IPG range" peptides. We describe a simple protocol to recover at least a part of these missing peptides and show that this recovery significantly influences the overall fractionation result, increasing the number of the identified proteins and the protein coverage

Rapid, simple and effective technical procedure for the regeneration of IgG and HSA affinity columns for proteomic analysis.

In plasma and serum, the presence of high-abundance proteins can overwhelm the signals of low-abundance proteins, which then become undetectable either by two-dimensional gels or chromatographic techniques. Therefore, depletion of abundant proteins is a prerequisite to detect low-abundance components. Furthermore, the regeneration of pre-purification tools could be money-saving. We applied an affinity chromatography kit to remove albumin and the immunoglobulin chains from plasma and propose a simple and effective technical procedure for the regeneration of these affinity column