Maria Filippa Addis

Generation of high-quality protein extracts from formalin-fixed, paraffin-embedded tissues

A wealth of information on proteins involved in many aspects of disease is encased within formalin‐fixed paraffin‐embedded (FFPE) tissue repositories stored in hospitals worldwide. Recently, access to this “hidden treasure” is being actively pursued by the application of two main extraction strategies: digestion of the entangled protein matrix with generation of tryptic peptides, or decrosslinking and extraction of full‐length proteins. Here, we describe an optimised method for extraction of full‐length proteins from FFPE tissues. This method builds on the classical “antigen retrieval” technique used for immunohistochemistry, and allows generation of protein extracts with elevated and reproducible yields. In model animal tissues, average yields of 16.3 μg and 86.8 μg of proteins were obtained per 80 mm2 tissue slice of formalin‐fixed paraffin‐embedded skeletal muscle and liver, respectively. Protein extracts generated with this method can be used for the reproducible investigation of the proteome with a wide array of techniques. The results obtained by SDS‐PAGE, western immunoblotting, protein arrays, ELISA, and, most importantly, nanoHPLC‐nanoESI‐Q‐TOF MS of FFPE proteins resolved by SDS‐PAGE, are presented and discussed. An evaluation of the extent of modifications introduced on proteins by formalin fixation and crosslink reversal, and their impact on quality of MS results, is also reported.

Equine and canine Anaplasma phagocytophilum strains isolated on the island of Sardinia (Italy) are phylogenetically related to pathogenic strains from the United States

ABSTRACT The presence of Anaplasma phagocytophilum, a tick-transmitted zoonotic pathogen, was investigated in Sardinia using a molecular approach. Phylogenetic analysis revealed that Sardinian strains are genetically distinct from the two lineages previously described in Europe and are closely related to strains isolated in different areas of the United States.

The sheep milk fat globule membrane proteome

Milk fat globule membranes (MFGM) are three-layered structures that enclose fat droplets, and are composed by an internal monolayer of endoplasmic reticulum origin, surrounded by a bilayer derived from the apical membrane of the lactating cell. In this work, an optimized protein extraction method was applied to sheep MFGM, and extracts were subjected to SDS-PAGE separation followed by shotgun LC tandem mass spectrometry (GeLC-MS/MS) for identification and characterization. In total, 140 unique sheep MFGM proteins (MFGMPs) were identified. All protein identification data were subjected to Gene Ontology (GO) classification for localization and function. Moreover, the relative abundance of all identified MFGMPs was estimated by means of the normalized spectral abundance factor (NSAF) approach, and GO abundance classes were obtained. The data gathered in this work provide a detailed picture of the proteome expressed in healthy sheep MFGs, and lay the foundations for future studies on sheep lactation physiology and on its alterations in pathological conditions.

The flagellated parasite Trichomonas vaginalis: new insights into cytopathogenicity mechanisms.

Our knowledge concerning cytopathogenicity of Trichomonas vaginalis has been enriched in the past by numerous findings. In this paper, we review the latest advances in the field and discuss the different mechanisms and molecules responsible for the parasites virulence.

Comparison of detergent-based sample preparation workflows for LTQ-Orbitrap analysis of the Escherichia coli proteome.

This work presents a comparative evaluation of several detergent‐based sample preparation workflows for the MS‐based analysis of bacterial proteomes, performed using the model organism Escherichia coli. Initially, RapiGest‐ and SDS‐based buffers were compared for their protein extraction efficiency and quality of the MS data generated. As a result, SDS performed best in terms of total protein yields and overall number of MS identifications, mainly due to a higher efficiency in extracting high molecular weight (MW) and membrane proteins, while RapiGest led to an enrichment in periplasmic and fimbrial proteins. Then, SDS extracts underwent five different MS sample preparation workflows, including: detergent removal by spin columns followed by in‐solution digestion (SC), protein precipitation followed by in‐solution digestion in ammonium bicarbonate or urea buffer, filter‐aided sample preparation (FASP), and 1DE separation followed by in‐gel digestion. On the whole, about 1000 proteins were identified upon LC‐MS/MS analysis of all preparations (>1100 with the SC workflow), with FASP producing more identified peptides and a higher mean sequence coverage. Each protocol exhibited specific behaviors in terms of MW, hydrophobicity, and subcellular localization distribution of the identified proteins; a comparative assessment of the different outputs is presented.

2-D PAGE and MS analysis of proteins from formalin-fixed, paraffin-embedded tissues

In the past decade, encouraging results have been obtained in extraction and analysis of proteins from formalin‐fixed, paraffin‐embedded (FFPE) tissues. However, 2‐D PAGE protein maps with satisfactory proteomic information and comparability to fresh tissues have never been described to date. In the present study, we report 2‐D PAGE separation and MS identification of full‐length proteins extracted from FFPE skeletal muscle tissue. The 2‐D protein profiles obtained from FFPE tissues could be matched to those achieved from frozen tissues replicates. Up to 250 spots were clearly detected in 2‐D maps of proteins from FFPE tissue following standard mass‐compatible silver staining. Protein spots from both FFPE and frozen tissue 2‐D gels were excised, subjected to in situ hydrolysis, and identified by MS analysis. Matched spots produced matched protein identifications. Moreover, 2‐D protein maps from FFPE tissues were successfully subjected to Western immunoblotting, producing comparable results to fresh‐frozen tissues. In conclusion, this study provides evidence that, when adequately extracted, full‐length proteins from FFPE tissues might be suitable to 2‐D PAGE‐MS analysis, allowing differential proteomic studies on the vast existing archives of healthy and pathological‐fixed tissues.

Evaluating the Impact of Different Sequence Databases on Metaproteome Analysis: Insights from a Lab-Assembled Microbial Mixture

Metaproteomics enables the investigation of the protein repertoire expressed by complex microbial communities. However, to unleash its full potential, refinements in bioinformatic approaches for data analysis are still needed. In this context, sequence databases selection represents a major challenge. This work assessed the impact of different databases in metaproteomic investigations by using a mock microbial mixture including nine diverse bacterial and eukaryotic species, which was subjected to shotgun metaproteomic analysis. Then, both the microbial mixture and the single microorganisms were subjected to next generation sequencing to obtain experimental metagenomic- and genomic-derived databases, which were used along with public databases (namely, NCBI, UniProtKB/SwissProt and UniProtKB/TrEMBL, parsed at different taxonomic levels) to analyze the metaproteomic dataset. First, a quantitative comparison in terms of number and overlap of peptide identifications was carried out among all databases. As a result, only 35% of peptides were common to all database classes; moreover, genus/species-specific databases provided up to 17% more identifications compared to databases with generic taxonomy, while the metagenomic database enabled a slight increment in respect to public databases. Then, database behavior in terms of false discovery rate and peptide degeneracy was critically evaluated. Public databases with generic taxonomy exhibited a markedly different trend compared to the counterparts. Finally, the reliability of taxonomic attribution according to the lowest common ancestor approach (using MEGAN and Unipept software) was assessed. The level of misassignments varied among the different databases, and specific thresholds based on the number of taxon-specific peptides were established to minimize false positives. This study confirms that database selection has a significant impact in metaproteomics, and provides critical indications for improving depth and reliability of metaproteomic results. Specifically, the use of iterative searches and of suitable filters for taxonomic assignments is proposed with the aim of increasing coverage and trustworthiness of metaproteomic data.

Setting proteins free: progresses and achievements in proteomics of formalin-fixed, paraffin-embedded tissues.

Formalin fixation, followed by paraffin embedding, is long established as the standard procedure for the stabilization and preservation of tissue architecture, essential for enabling microscopic examination and long‐term storage of samples. During the years, this has led to the generation of a worldwide repository of patient tissues with associated complete clinical records. As such, this represents a golden mine for all those attempting to identify proteomic signatures of disease, aimed to the understanding of pathological processes and to the identification of new biomarkers. However, access to this resource has been hampered by the stable cross‐linked network generated on tissue molecules during formalin fixation. Recently, researchers have been actively working to overcome this limitation, reaching unexpected achievements. This review aims to discuss and compare the various strategies devised for extracting full‐length proteins or peptides from fixed tissues, and to provide a general perspective on studies comparing matched fixed and fresh‐frozen tissue proteomes, applying proteomic techniques for biomarker discovery from archival tissues, and attempting to exploit gel‐based approaches. In addition, the concomitant progresses in understanding the impact of tissue processing variables and the extent and nature of formaldehyde‐induced modifications are presented. In conclusion, the future perspectives and open challenges in this field are discussed.

Mycoplasma hominis parasitism of Trichomonas vaginalis

Vaginal infections during pregnancy are a risk factor for intrauterine growth retardation, preterm birth, and perinatal mortality or morbidity. Studies of pregnant and non-pregnant women have shown an association between Trichomonas vaginalis and Mycoplasma hominis infections. In an African area with a high prevalence of trichomoniasis, we noticed anti- Mycoplasma antibodies in an unexpectedly high number of women with anti- Trichomonas antibodies. Because an electron-microscopy study showed Mycoplasma organisms in food vacuoles of protozoa, we investigated the cohabitation of the two micro-organisms in the vagina.

Spontaneous feline mammary intraepithelial lesions as a model for human estrogen receptor- and progesterone receptor-negative breast lesions

BackgroundBreast cancer is the most frequently diagnosed cancer in women. Intraepithelial lesions (IELs), such as usual ductal hyperplasia (UH), atypical ductal hyperplasia (ADH), and ductal carcinoma in situ (DCIS) are risk factors that predict a womans chance of developing invasive breast cancer. Therefore, a comparative study that establishes an animal model of pre-invasive lesions is needed for the development of preventative measures and effective treatment for both mammary IELs and tumors. The purpose of this study was to characterize the histologic and molecular features of feline mammary IELs and compare them with those in women.MethodsFormalin-fixed, paraffin-embedded specimens (n = 205) from 203 female cats with clinical mammary disease were retrieved from the archives of the Purdue University Animal Disease Diagnostic Laboratory and Veterinary Teaching Hospital (West Lafayette, IN), and the Department of Pathology and Veterinary Clinic, School of Veterinary Medicine (Sassari, Italy). Histologic sections, stained with hematoxylin and eosin (HE), were evaluated for the presence of IELs in tissue adjacent to excised mammary tumors. Lesions were compared to those of humans. Immunohistochemistry for estrogen receptor (ER-alpha), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2/neu) and Ki-67 was performed in IELs and adjacent tumor tissues.ResultsIntraepithelial lesions were found in 57 of 203 (28%) feline mammary specimens and were categorized as UH (27%), ADH (29%), and DCIS (44%). Most IELs with atypia (ADH and DCIS) were associated with mammary cancer (91%), whereas UH was associated with benign lesions in 53% of cases. Feline IELs were remarkably similar to human IELs. No ER or PR immunoreactivity was detected in intermediate-grade or high-grade DCIS or their associated malignant tumors. HER-2 protein overexpression was found in 27% of IELs.ConclusionThe remarkable similarity of feline mammary IELs to those of humans, with the tendency to lose hormone receptor expression in atypical IELs, supports the cat as a possible model to study ER- and PR-negative breast lesions.