Metaproteomic assessment of gut microbial and host functional perturbations in Helicobacter pylori-infected patients subjected to an antimicrobial protocol.

The impact of therapeutic interventions on the human gut microbiota (GM) is a clinical issue of paramount interest given the strong interconnection between microbial dynamics and human health. Orally administered antibiotics are known to reduce GM biomass and modify GM taxonomic profile. However, the impact of antimicrobial therapies on GM functions and biochemical pathways has scarcely been studied. Here, we characterized the fecal metaproteome of 10 Helicobacter pylori-infected patients before (T0) and after 10 days (T1) of a successful quadruple therapy (bismuth, tetracycline, metronidazole, and rabeprazole) and 30 days after therapy cessation (T2), to investigate how GM and host functions change during the eradication and healing processes. At T1, the abundance ratio between microbial and host proteins was reversed compared with that at T0 and T2. Several pathobionts (including Klebsiella, Proteus, Enterococcus, Muribaculum, and Enterocloster) were increased at T1. Therapy reshaped the relative contributions of the functions required to produce acetate, propionate, and butyrate. Proteins related to the uptake and processing of complex glycans were increased. Microbial cross-feeding with sialic acid, fucose, and rhamnose was enhanced, whereas hydrogen sulfide production was reduced. Finally, microbial proteins involved in antibiotic resistance and inflammation were more abundant after therapy. Moreover, a reduction in host proteins with known roles in inflammation and H. pylori-mediated carcinogenesis was observed. In conclusion, our results support the use of metaproteomics to monitor drug-induced remodeling of GM and host functions, opening the way for investigating new antimicrobial therapies aimed at preserving gut environmental homeostasis.

Vaccination and Antiviral Treatment Reduce the Time to Negative SARS-CoV-2 Swab: A Real-Life Study.

Clinical trials demonstrated the role of vaccines and antiviral treatments against SARS-CoV-2 in reducing the likelihood of disease progression and death. However, there are limited data available regarding the time to negativity of people who received these treatments. Further, several comorbidities and risk factors might affect the impact of vaccines and antiviral treatments. To this end, we aimed to evaluate and disentangle the impact of anti-SARS-CoV-2 treatments and that of underlying clinical factors associated with a shortened length of SARS-CoV-2 infection. Hence, we recorded the timeframe of positive nasopharyngeal swab in people infected while being hospitalized for reasons other than SARS-CoV-2 infection. All patients who died or were discharged with a positive swab were excluded from the study. A total of 175 patients were included in this study. Clinical conditions encompass malignancies, immunological disorders, cardiovascular, metabolic, neurodegenerative, and chronic kidney disease. Most of the participants (91.4%) were vaccinated before admission to the hospital, and 65.1% received antiviral treatment within three days after the symptom's onset. Unvaccinated patients had a longer median time to negativity than people who received at least two doses of vaccine (18 vs. 10 days). Concerning the clinical conditions of all patients, multivariate analysis highlighted a lower probability of 14-day conversion of antigenic test positivity in patients with hematological malignancy, including those vaccinated and those exposed to antiviral therapies. In conclusion, our data showed that prompt administration of antiviral treatments accelerates the clearance of SARS-CoV-2. Further, in the elderly patients under study, previous vaccination and antiviral treatment synergize to reduce time to negativity. This translates into a shorter hospitalization time and a lower risk of transmission through patients and connected healthcare workers in a hospital ward setting, with considerable improvement in cost-effective care management.

Meta4P: A User-Friendly Tool to Parse Label-Free Quantitative Metaproteomic Data and Taxonomic/Functional Annotations.

We present Meta4P (MetaProteins-Peptides-PSMs Parser), an easy-to-use bioinformatic application designed to integrate label-free quantitative metaproteomic data with taxonomic and functional annotations. Meta4P can retrieve, filter, and process identification and quantification data from three levels of inputs (proteins, peptides, PSMs) in different file formats. Abundance data can be combined with taxonomic and functional information and aggregated at different and customizable levels, including taxon-specific functions and pathways. Meta4P output tables, available in various formats, are ready to be used as inputs for downstream statistical analyses. This user-friendly tool is expected to provide a useful contribution to the field of metaproteomic data analysis, helping make it more manageable and straightforward.

Metaproteomic Profile of the Colonic Luminal Microbiota From Patients With Colon Cancer.

Recent studies have provided evidence of interactions among the gut microbiota (GM), local host immune cells, and intestinal tissues in colon carcinogenesis. However, little is known regarding the functions exerted by the GM in colon cancer (CC), particularly with respect to tumor clinical classification and lymphocyte infiltration. In addition, stool, usually employed as a proxy of the GM, cannot fully represent the original complexity of CC microenvironment. Here, we present a pilot study aimed at characterizing the metaproteome of CC-associated colonic luminal contents and identifying its possible associations with CC clinicopathological features. Colonic luminal contents were collected from 24 CC tissue specimens immediately after surgery. Samples were analyzed by shotgun metaproteomics. Almost 30,000 microbial peptides were quantified in the samples, enabling the achievement of the taxonomic and functional profile of the tumor-associated colonic luminal metaproteome. Upon sample aggregation based on tumor stage, grade, or tumor-infiltrating lymphocytes (TILs), peptide sets enabling discrimination of sample groups were identified through discriminant analysis (DA). As a result, Bifidobacterium and Bacteroides fragilis were significantly enriched in high-stage and high-grade CC, respectively. Among metabolic functions, formate-tetrahydrofolate ligase was significantly associated with high-stage CC. Finally, based on the results of this pilot study, we assessed the optimal sample size for differential metaproteomic studies analyzing colonic luminal contents. In conclusion, we provide a detailed picture of the microbial and host components of the colonic luminal proteome and propose promising associations between GM taxonomic/functional features and CC clinicopathological features. Future studies will be needed to verify the prognostic value of these data and to fully exploit the potential of metaproteomics in enhancing our knowledge concerning CC progression.

Proteomic characterization of Echinococcus granulosus sensu stricto, Taenia hydatigena and Taenia multiceps metacestode cyst fluids.

Cystic echinococcosis (CE) diagnosis by means of serological assays is hampered by the presence of parasites closely related to Echinococcus granulosus sensu lato (s.l.), responsible of the zoonotic disease and with which share cross-reacting antigens. Thus, improvements on the characterization of Echinococcus specific antigens expressed in the larval stage are required, in order to provide useful information for the development of immunological assays for the serodiagnosis of CE in sheep. Here, the proteome of the hydatid cyst fluids (HFs) of Echinococcus granulosus (hydatid fluid, EgHF) and other ovine parasites cyst fluids (CFs), Taenia hydatigena (ThCF) and Taenia multiceps (TmCF) were analyzed by a shotgun proteomic approach. Parasite and host protein profiles in the three types of cyst fluids were characterized and compared. Among the identified proteins, differential parasitic markers with serodiagnostic potential, due to their well-known immunoreactivity in human, included Ag5, AgB proteins, 8-kDa glycoproteins, hydatid disease diagnostic antigen P29 and major egg antigen P40. In particular, seven proteoforms of AgB and 8-kDa glycoprotein resulted to be the most promising diagnostic biomarkers, as they might predict CE in ovine and discriminate between different types of parasites.

Effect of Different Disease-Modifying Therapies on Humoral Response to BNT162b2 Vaccine in Sardinian Multiple Sclerosis Patients.

Objectives: Vaccination against COVID-19 is highly recommended to patients affected by multiple sclerosis (MS); however, the impact of MS disease-modifying therapies (DMTs) on the immune response following vaccination has been only partially investigated. Here, we aimed to elucidate the effect of DMTs on the humoral immune response to mRNA-based anti-SARS-CoV-2 vaccines in MS patients. Methods: We obtained sera from 912 Sardinian MS patients and 63 healthy controls 30 days after the second dose of BNT162b2 vaccine and tested them for SARS-CoV-2 response using anti-Spike (S) protein-based serology. Previous SARS-CoV-2 infection was assessed by anti-Nucleocapsid (N) serology. Patients were either untreated or undergoing treatment with a total of 13 different DMTs. Differences between treatment groups comprised of at least 10 patients were assessed by generalized linear mixed-effects model. Demographic and clinical data and smoking status were analyzed as additional factors potentially influencing humoral immunity from COVID-19 vaccine. Results: MS patients treated with natalizumab, teriflunomide, azathioprine, fingolimod, ocrelizumab, and rituximab showed significantly lower humoral responses compared to untreated patients. We did not observe a statistically significant difference in response between patients treated with the other drugs (dimethyl fumarate, interferon, alemtuzumab and glatiramer acetate) and untreated patients. In addition, older age, male sex and active smoking were significantly associated with lower antibody titers against SARS-CoV-2. MS patients previously infected with SARS-CoV-2 had significantly higher humoral responses to vaccine than uninfected patients. Conclusion: Humoral response to BNT162b2 is significantly influenced by the specific DMTs followed by patients, as well as by other factors such as previous SARS-CoV-2 infection, age, sex, and smoking status. These results are important to inform targeted strategies to prevent clinically relevant COVID-19 in MS patients.

Critical Assessment of MetaProteome Investigation (CAMPI): a multi-laboratory comparison of established workflows.

Metaproteomics has matured into a powerful tool to assess functional interactions in microbial communities. While many metaproteomic workflows are available, the impact of method choice on results remains unclear. Here, we carry out a community-driven, multi-laboratory comparison in metaproteomics: the critical assessment of metaproteome investigation study (CAMPI). Based on well-established workflows, we evaluate the effect of sample preparation, mass spectrometry, and bioinformatic analysis using two samples: a simplified, laboratory-assembled human intestinal model and a human fecal sample. We observe that variability at the peptide level is predominantly due to sample processing workflows, with a smaller contribution of bioinformatic pipelines. These peptide-level differences largely disappear at the protein group level. While differences are observed for predicted community composition, similar functional profiles are obtained across workflows. CAMPI demonstrates the robustness of present-day metaproteomics research, serves as a template for multi-laboratory studies in metaproteomics, and provides publicly available data sets for benchmarking future developments.

Inappropriate Nasopharyngeal Sampling for SARS-CoV-2 Detection Is a Relevant Cause of False-Negative Reports.

Reverse transcriptase polymerase chain reaction (RT-PCR) detection of SARS-CoV-2 mRNA on nasopharyngeal swab is the standard for diagnosing active COVID-19 disease in asymptomatic cases and in symptomatic patients without the typical radiologic findings. For the present COVID-19 outbreak in Italy, we describe 4 symptomatic patients with negative RT-PCR results at the first nasopharyngeal swab, which became positive when collected a few hours later by an otolaryngologist. All the patients showed nasal obstruction. The present report suggests that inadequate nasopharyngeal sampling performed by untrained operators in the presence of nasal obstruction can be a relevant case of false-negative findings at RT-PCR, with a clear negative impact on the efforts to contain the current outbreak.

Fecal Metaproteomic Analysis Reveals Unique Changes of the Gut Microbiome Functions After Consumption of Sourdough Carasau Bread.

Sourdough-leavened bread (SB) is acknowledged for its great variety of valuable effects on consumer's metabolism and health, including a low glycemic index and a reduced content of the possible carcinogen acrylamide. Here, we aimed to investigate how these effects influence the gut microbiota composition and functions. Therefore, we subjected rats to a diet supplemented with SB, baker's yeast leavened bread (BB), or unsupplemented diet (chow), and, after 4 weeks of treatment, their gut microbiota was analyzed using a metaproteogenomic approach. As a result, diet supplementation with SB led to a reduction of specific members of the intestinal microbiota previously associated to low protein diets, namely Alistipes and Mucispirillum, or known as intestinal pathobionts, i.e., Mycoplasma. Concerning functions, asparaginases expressed by Bacteroides were observed as more abundant in SB-fed rats, leading to hypothesize that in their colonic microbiota the enzyme substrate, asparagine, was available in higher amounts than in BB- and chow-fed rats. Another group of protein families, expressed by Clostridium, was detected as more abundant in animal fed SB-supplemented diet. Of these, manganese catalase, small acid-soluble proteins (SASP), Ser/Thr kinase PrkA, and V-ATPase proteolipid subunit have been all reported to take part in Clostridium sporulation, strongly suggesting that the diet supplementation with SB might promote environmental conditions inducing metabolic dormancy of Clostridium spp. within the gut microbiota. In conclusion, our data describe the effects of SB consumption on the intestinal microbiota taxonomy and functions in rats. Moreover, our results suggest that a metaproteogenomic approach can provide evidence of the interplay between metabolites deriving from bread digestion and microbial metabolism.

Structural and Immunodiagnostic Characterization of Synthetic Antigen B Subunits From Echinococcus granulosus and Their Evaluation as Target Antigens for Cyst Viability Assessment.

Background: Several tools have been proposed for serodiagnosis of cystic echinococcosis (CE), but none seems promising for cyst viability assessment. Antigens with stage-specific diagnostic value have been described, but few studies with well-characterized antigens and human serum samples have been performed. Antigen B (AgB) proteoforms hold promise as markers of viability, due to their differential stage-related expression and immunoreactivity. Methods: Four AgB subunits (AgB1, AgB2, AgB3, AgB4) were synthesized and structurally characterized. Based on the preliminary evaluation of the subunits by western immunoblotting and enzyme-linked immunosorbent assay (ELISA), AgB1 and AgB2 were further tested in two ELISA setups and extensively validated on 422 human serum samples. Results: All subunits showed a high degree of spontaneous oligomerization. Interacting residues within oligomers were identified, showing that both the N-terminal and C-terminal of each subunit are involved in homo-oligomer contact interfaces. No hetero-oligomer was identified. AgB1 and AgB2 ELISAs revealed different sensitivities relative to cyst stage. Of note, besides high specificity (97.2%), AgB1 revealed a higher sensitivity for active-transitional cysts (100% for CE1, 77.8% for CE2, 81.5% for CE3a, and 86.3% for CE3b) than for inactive cysts (41.7% for CE4 and 11.1% for CE5) and postsurgical patients (44%). Interestingly, 19 of 20 patients with spontaneously inactive cysts and 6 of 9 treated with albendazole >5 years earlier were negative on the AgB1 assay. Conclusions: The structural characterization of subunits provides insights into the synthetic antigen conformation. The stage-related sensitivity of synthetic AgB1 holds promise as part of a multiantigen setting and deserves further longitudinal evaluation as marker of cyst viability.

Lack of NLRP3-inflammasome leads to gut-liver axis derangement, gut dysbiosis and a worsened phenotype in a mouse model of NAFLD.

Non-Alcoholic Fatty Liver Disease (NAFLD) represents the most common form of chronic liver injury and can progress to cirrhosis and hepatocellular carcinoma. A "multi-hit" theory, involving high fat diet and signals from the gut-liver axis, has been hypothesized. The role of the NLRP3-inflammasome, which senses dangerous signals, is controversial. Nlrp3-/- and wild-type mice were fed a Western-lifestyle diet with fructose in drinking water (HFHC) or a chow diet. Nlrp3-/--HFHC showed higher hepatic expression of PPAR γ2 (that regulates lipid uptake and storage) and triglyceride content, histological score of liver injury and greater adipose tissue inflammation. In Nlrp3-/--HFHC, dysregulation of gut immune response with impaired antimicrobial peptides expression, increased intestinal permeability and the occurrence of a dysbiotic microbiota led to bacterial translocation, associated with higher hepatic expression of TLR4 (an LPS receptor) and TLR9 (a receptor for double-stranded bacterial DNA). After antibiotic treatment, gram-negative species and bacterial translocation were reduced, and adverse effects restored both in liver and adipose tissue. In conclusion, the combination of a Western-lifestyle diet with innate immune dysfunction leads to NAFLD progression, mediated at least in part by dysbiosis and bacterial translocation, thus identifying new specific targets for NAFLD therapy.

Cathelicidin production and release by mammary epithelial cells during infectious mastitis.

Cathelicidins are well-characterized antimicrobial peptides (AMPs) that are present in significant amounts in mastitic milk. Neutrophils are believed to be the main producers of these AMPs, while the role of mammary epithelial cells (MECs) in their production and release is still unclear. In this work, cathelicidin production patterns were investigated in mammary tissues of ewes infected by Staphylococcus aureus, Streptococcus uberis, or Mycoplasma agalactiae, with a combined approach including immunohistochemistry, immune-colocalization, and fluorescent in situ hybridization. Our results confirm that MECs produce and release cathelicidins in response to different mastitis pathogens. As opposed to neutrophils, however, MECs do not seem to store the preformed protein precursor in their cytoplasm, but appear to synthesize and release it only upon exposure to the microorganisms. Cathelicidin production by MECs appears to occur before leukocyte influx in the milk, suggesting a role for these cells in the initial response of the mammary epithelium to microbial infection. Once in the milk, infiltrating neutrophils release massive amounts of cathelicidin by degranulation and production of neutrophil extracellular traps, acting as the main contributor for cathelicidin abundance in mastitic milk. Taken together, our results support the active contribution of MECs to cathelicidin production and release, and reinforce the value of cathelicidins as sensitive and pathogen-independent mastitis markers.

Protein expression changes induced in a malignant melanoma cell line by the curcumin analogue compound D6.

BACKGROUND: We have previously demonstrated that the hydroxylated biphenyl compound D6 (3E,3'E)-4,4'-(5,5',6,6'-tetramethoxy-[1,1'-biphenyl]-3,3'-diyl)bis(but-3-en-2-one), a structural analogue of curcumin, exerts a strong antitumor activity on melanoma cells both in vitro and in vivo. Although the mechanism of action of D6 is yet to be clarified, this compound is thought to inhibit cancer cell growth by arresting the cell cycle in G2/M phase, and to induce apoptosis through the mitochondrial intrinsic pathway. To investigate the changes in protein expression induced by exposure of melanoma cells to D6, a differential proteomic study was carried out on D6-treated and untreated primary melanoma LB24Dagi cells. METHODS: Proteins were fractionated by SDS-PAGE and subjected to in gel digestion. The peptide mixtures were analyzed by liquid chromatography coupled with tandem mass spectrometry. Proteins were identified and quantified using database search and spectral counting. Proteomic data were finally uploaded into the Ingenuity Pathway Analysis software to find significantly modulated networks and pathways. RESULTS: Analysis of the differentially expressed protein profiles revealed the activation of a strong cellular stress response, with overexpression of several HSPs and stimulation of ubiquitin-proteasome pathways. These were accompanied by a decrease of protein synthesis, evidenced by downregulation of proteins involved in mRNA processing and translation. These findings are consistent with our previous results on gene expression profiling in melanoma cells treated with D6. CONCLUSIONS: Our findings confirm that the curcumin analogue D6 triggers a strong stress response in melanoma cells, turning down majority of cell functions and finally driving cells to apoptosis.

Atypical carcinoid and large cell neuroendocrine carcinoma of the lung: a proteomic dataset from formalin-fixed archival samples.

Here we present a dataset generated using formalin-fixed paraffin-embedded archival samples from two rare lung neuroendocrine tumor subtypes (namely, two atypical carcinoids, ACs, and two large-cell neuroendocrine carcinomas, LCNECs). Samples were subjected to a shotgun proteomics pipeline, comprising full-length protein extraction, SDS removal through spin columns, in solution trypsin digestion, long gradient liquid chromatography peptide separation and LTQ-Orbitrap mass spectrometry analysis. A total of 1260 and 2436 proteins were identified in the AC and LCNEC samples, respectively, with FDR <1%. MS data are available in the PeptideAtlas repository at http://www.peptideatlas.org/PASS/PASS00375.

Diagnostic Accuracy of Antigen 5-Based ELISAs for Human Cystic Echinococcosis.

BACKGROUND: Clinical diagnosis and follow up of cystic echinococcosis (CE) are based on imaging complemented by serology. Several immunodiagnostic tests are commercially available, but the development of new tools is still needed to overcome the lack of standardization of the target antigen, generally consisting of a crude extract of Echinococcus granulosus hydatid cyst fluid. In a previous work, we described a chromatographic method for the preparation of a highly enriched Antigen 5 fraction from hydatid cyst fluid. The high reactivity of patient sera against this preparation prompted us to evaluate further this antigen for the serodiagnosis of CE on a larger cohort of samples. METHODOLOGY/PRINCIPAL FINDINGS: A total of 327 sera from CE patients with heterogeneous conditions for cyst stage, cyst number, organ localization, drug therapy, and surgical intervention, together with 253 sera from healthy controls, were first analyzed by an ELISA based on the Ag5 preparation in two different experimental setups and, in parallel, by a commercial ELISA routinely used in clinical laboratories for CE serodiagnosis. The Ag5 ELISAs revealed different sensitivity (88.3% vs 95.3%) without significant differences in specificity (94.1% vs 92.5%), for the two setups, respectively. Moreover, possible relationships between the Ag5 ELISA absorbance results and clinical variables were investigated. Chi squared test, bivariate logistic regression and multiple regression analyses highlighted differences in the serology reactivity according to pharmacological treatment, cyst activity, and cyst number. CONCLUSIONS/SIGNIFICANCE: The two Ag5 ELISAs revealed different performances depending on the setup. The good diagnostic sensitivity and the high reliability of the Ag5 preparation method make this antigen a promising candidate for the serodiagnosis of CE. Further studies will be needed to evaluate the ability of our test to provide useful information on specific CE clinical traits.

An easy and efficient method for native and immunoreactive Echinococcus granulosus antigen 5 enrichment from hydatid cyst fluid.

BACKGROUND: Currently, the serodiagnosis of cystic echinococcosis relies mostly on crude Echinococcus granulosus hydatid cyst fluid as the antigen. Consequently, available immunodiagnostic tests lack standardization of the target antigen and, in turn, this is reflected on poor sensitivity and specificity of the serological diagnosis. METHODOLOGY/PRINCIPAL FINDINGS: Here, a chromatographic method enabling the generation of highly enriched Antigen 5 (Ag5) is described. The procedure is very easy, efficient and reproducible, since different hydatid cyst fluid (HCF) sources produced very similar chromatograms, notwithstanding the clearly evident and extreme heterogeneity of the starting material. In addition, the performance of the antigen preparation in immunological assays was preliminarily assessed by western immunoblotting and ELISA on a limited panel of cystic echinococcosis patients and healthy controls. Following western immunoblotting and ELISA experiments, a high reactivity of patient sera was seen, with unambiguous and highly specific results. CONCLUSIONS/SIGNIFICANCE: The methods and results reported open interesting perspectives for the development of sensitive diagnostic tools to enable the timely and unambiguous detection of cystic echinococcosis antibodies in patient sera.

Production and release of antimicrobial and immune defense proteins by mammary epithelial cells following Streptococcus uberis infection of sheep.

Investigating the innate immune response mediators released in milk has manifold implications, spanning from elucidation of the role played by mammary epithelial cells (MECs) in fighting microbial infections to the discovery of novel diagnostic markers for monitoring udder health in dairy animals. Here, we investigated the mammary gland response following a two-step experimental infection of lactating sheep with the mastitis-associated bacterium Streptococcus uberis. The establishment of infection was confirmed both clinically and by molecular methods, including PCR and fluorescent in situ hybridization of mammary tissues. Proteomic investigation of the milk fat globule (MFG), a complex vesicle released by lactating MECs, enabled detection of enrichment of several proteins involved in inflammation, chemotaxis of immune cells, and antimicrobial defense, including cathelicidins and calprotectin (S100A8/S100A9), in infected animals, suggesting the consistent involvement of MECs in the innate immune response to pathogens. The ability of MECs to produce and release antimicrobial and immune defense proteins was then demonstrated by immunohistochemistry and confocal immunomicroscopy of cathelicidin and the calprotectin subunit S100A9 on mammary tissues. The time course of their release in milk was also assessed by Western immunoblotting along the course of the experimental infection, revealing the rapid increase of these proteins in the MFG fraction in response to the presence of bacteria. Our results support an active role of MECs in the innate immune response of the mammary gland and provide new potential for the development of novel and more sensitive tools for monitoring mastitis in dairy animals.

Application of 2D-DIGE to formalin-fixed diseased tissue samples from hospital repositories: results from four case studies.

PURPOSE: In the recent past, the potential suitability of fixed samples to 2D-DIGE studies has been demonstrated on model tissues, but not on "real-world" archival tissues. Therefore, this study was aimed to assess the quality of the results delivered by 2D-DIGE on samples retrieved from hospital tissue repositories. EXPERIMENTAL DESIGN: Diseased and normal tissue samples (namely, human gastric adenocarcinoma and normal gastric tissue, human lung neuroendocrine tumors, canine mammary tubulo-papillary carcinoma and normal mammary tissue, sheep liver with cloudy swelling degeneration and normal liver tissue) were retrieved from human and veterinary biorepositories and subjected to full-length protein extraction, cyanine labeling, 2D-DIGE separation, image analysis, MS analysis, and protein identification. RESULTS: Archival samples could be successfully subjected to 2D-DIGE, providing maps of satisfactory resolution, although with varying pattern complexity (possibly influenced by preanalytical variables). Moreover, differentially expressed protein identities were consistent with the disease biology. CONCLUSIONS AND CLINICAL RELEVANCE: 2D-DIGE can support biomarker discovery and validation studies on large sample cohorts. In fact, although some information complexity is lost when compared to fresh-frozen tissues, their vast availability and the associated patient information can considerably boost studies suffering limited sample availability or involving long-distance exchange of samples.

Comparability of differential proteomics data generated from paired archival fresh-frozen and formalin-fixed samples by GeLC-MS/MS and spectral counting.

In this study, a Veterinary Department repository composed by paired formalin-fixed paraffin-embedded (FFPE) and fresh-frozen (FrFr) sets of the same tissues, routinely archived in the typical conditions of a clinical setting, was exploited to perform a comparative evaluation of the results generated by GeLC-MS/MS (1-DE followed by in-gel digestion and LC-MS/MS) and spectral counting with the two types of archival samples. Therefore, two parallel differential proteomic studies were performed using 3 canine mammary carcinomas and 3 normal controls in a paired fashion (6 FrFr and 6 FFPE in total). As a result, the FrFr and FFPE differential proteomic datasets exhibited fair consistency in differential expression trends, according to protein molecular function, cellular localization, networks, and pathways. However, FFPE samples were globally slightly less informative, especially concerning the high-MW subproteome. As a further investigation, new insights into the molecular aspects of protein fixation and retrieval were obtained. In conclusion, archival FFPE samples can be reliably used for differential proteomics studies employing a spectral counting GeLC-MS/MS approach, although some typical biases need to be taken into account, and FrFr specimens (when available) should still be considered as the gold standard for clinical proteomics.

Evaluation of the suitability of archival Bouin-fixed paraffin-embedded tissue specimens to proteomic investigation.

Bouin's solution has been used for over a century as a common fixative in several pathology laboratories worldwide. Therefore, a considerable number of Bouin-fixed paraffin-embedded (BFPE) tumor samples of various origin are available in hospital repositories as a powerful information mine for clinical investigations. To date, however, such archived tissues have not been subjected to a systematic study aimed to evaluate their potential use in proteomics. In this report, we investigated whether archival BFPE tissue specimens could be exploited for proteomic studies, upon application of protein extraction and proteomic analysis methods previously optimized for formalin-fixed samples. As a result, gastric BFPE protein extracts exhibited poor suitability for 2D-PAGE analysis, whereas over 300 unique proteins could be successfully detected when extracts were subjected to SDS-PAGE followed by LC-MS/MS (GeLC-MS/MS). Among these, several known markers for gastric cancer and normal gastric functionality were identified, indicative of biological and clinical significance of proteomic data mined from BFPE tissues. A quantitative and qualitative comparison of FFPE and BFPE tissue proteomes was also performed, and results are reported. In conclusion, we demonstrated that BFPE specimens can be analyzed by means of a proteomic approach such as GeLC-MS/MS. Although considerable molecular biases and technical constraints exist, BFPE tissue archives can be fruitfully exploited for gathering proteomic data from particularly precious samples.