scholarly journals Comprehensive micro-scaled proteome and phosphoproteome characterization of archived retrospective cancer repositories

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Corinna Friedrich ◽  
Simon Schallenberg ◽  
Marieluise Kirchner ◽  
Matthias Ziehm ◽  
Sylvia Niquet ◽  
...  
Keyword(s):  
Clinical Studies ◽  
Protein Extraction ◽  
Quantitative Information ◽  
Fixation Time ◽  
Label Free ◽  
Tandem Mass Tag ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues

AbstractFormalin-fixed paraffin-embedded (FFPE) tissues are a valuable resource for retrospective clinical studies. Here, we evaluate the feasibility of (phospho-)proteomics on FFPE lung tissue regarding protein extraction, quantification, pre-analytics, and sample size. After comparing protein extraction protocols, we use the best-performing protocol for the acquisition of deep (phospho-)proteomes from lung squamous cell and adenocarcinoma with >8,000 quantified proteins and >14,000 phosphosites with a tandem mass tag (TMT) approach. With a microscaled approach, we quantify 7,000 phosphosites, enabling the analysis of FFPE biopsies with limited tissue amounts. We also investigate the influence of pre-analytical variables including fixation time and heat-assisted de-crosslinking on protein extraction efficiency and proteome coverage. Our improved workflows provide quantitative information on protein abundance and phosphosite regulation for the most relevant oncogenes, tumor suppressors, and signaling pathways in lung cancer. Finally, we present general guidelines to which methods are best suited for different applications, highlighting TMT methods for comprehensive (phospho-)proteome profiling for focused clinical studies and label-free methods for large cohorts.

scholarly journals Characterization of epididymal and testicular histologic lesions and use of immunohistochemistry and PCR on formalin-fixed tissues to detect Brucella canis in male dogs

2021 ◽  
pp. 104063872098688
Author(s):  
Andrea M. Camargo-Castañeda ◽  
Lauren W. Stranahan ◽  
John F. Edwards ◽  
Daniel G. Garcia-Gonzalez ◽  
Leonardo Roa ◽  
...  
Keyword(s):  
Accurate Diagnosis ◽  
Testicular Atrophy ◽  
Detection Techniques ◽  
Formalin Fixed Paraffin ◽  
Brucella Canis ◽  
Formalin Fixed Paraffin Embedded ◽  
Variable Sensitivity ◽  
Ffpe Tissues ◽  
Formalin Fixed

In male dogs, Brucella canis frequently causes epididymitis, ultimately resulting in testicular atrophy and infertility. Although B. canis predominantly affects the epididymis, the misleading term “orchitis” is still commonly used by clinicians. Of additional concern, diagnosis in dogs remains challenging because of variable sensitivity and specificity of serologic assays and fluctuations in bacteremia levels in infected dogs, reducing the sensitivity of blood culture. We describe here the histologic lesions in the scrotal contents of 8 dogs suspected of being infected with B. canis and clinically diagnosed with orchitis. We explored the possibility of using immunohistochemistry (IHC) and real-time PCR (rtPCR) in formalin-fixed, paraffin-embedded (FFPE) tissues to detect the presence of B. canis. Epididymitis of variable chronicity was identified in all 8 dogs, with only 3 also exhibiting orchitis. Using rtPCR, the presence of B. canis was identified in 4 of 8 dogs, with 3 of these 4 dogs also positive by IHC. These results suggest that rtPCR and IHC are promising techniques that can be used in FFPE tissues to detect B. canis when other detection techniques are unavailable. Additionally, accurate recognition of epididymitis rather than orchitis in suspect cases could aid in accurate diagnosis.

scholarly journals Effect of polyethylene glycol 20 000 on protein extraction efficiency of formalin-fixed paraffin-embedded tissues in South Africa

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Sophia Rossouw ◽  
Hocine Bendou ◽  
Liam Bell ◽  
Jonathan Rigby ◽  
Alan Christoffels
Keyword(s):  
Polyethylene Glycol ◽  
Extraction Efficiency ◽  
Protein Extraction ◽  
Formalin Fixed Paraffin ◽  
Liquid Chromatography Tandem Mass ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Protein Selection ◽  
The Impact ◽  
Formalin Fixed

Background: Optimal protocols for efficient and reproducible protein extraction from formalin-fixed paraffin-embedded (FFPE) tissues are not yet standardised and new techniques are continually developed and improved. The effect of polyethylene glycol (PEG) 20 000 on protein extraction efficiency has not been evaluated using human FFPE colorectal cancer tissues and there is no consensus on the protein extraction solution required for efficient, reproducible extraction.Objective: The impact of PEG 20 000 on protein extraction efficiency, reproducibility and protein selection bias was evaluated using FFPE colonic tissue via liquid chromatography tandem mass spectrometry analysis.Methods: This study was conducted from August 2017 to July 2019 using human FFPE colorectal carcinoma tissues from the Anatomical Pathology department at Tygerberg Hospital in South Africa. Samples were analysed via label-free liquid chromatography tandem mass spectrometry to determine the impact of using PEG 20 000 in the protein extraction solution. Data were assessed regarding peptide and protein identifications, method efficiency, reproducibility, protein characteristics and organisation relating to gene ontology categories.Results: Polyethylene glycol 20 000 exclusion increased peptides and proteins identifications and the method was more reproducible compared to the samples processed with PEG 20 000. However, no differences were observed with regard to protein selection bias. We found that higher protein concentrations ( 10 µg) compromised the function of PEG.Conclusion: This study indicates that protocols generating high protein yields from human FFPE tissues would benefit from the exclusion of PEG 20 000 in the protein extraction solution.

Sarcoma Subgrouping by Detection of Fusion Transcripts Using NanoString nCounter Technology

2018 ◽  
Vol 22 (3) ◽  
pp. 205-213 ◽  
Author(s):  
Javal Sheth ◽  
Anthony Arnoldo ◽  
Yunan Zhong ◽  
Paula Marrano ◽  
Carlos Pereira ◽  
...  
Keyword(s):  
Rt Pcr ◽  
Fusion Transcripts ◽  
Pediatric Sarcoma ◽  
Formalin Fixed Paraffin ◽  
Pediatric Sarcomas ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Nanostring Technology ◽  
Future Work ◽  
Formalin Fixed

Background NanoString technology is an innovative barcode-based system that requires less tissue than traditional techniques and can test for multiple fusion transcripts in a single reaction. The objective of this study was to determine the utility of NanoString technology in the detection of sarcoma-specific fusion transcripts in pediatric sarcomas. Design Probe pairs for the most common pediatric sarcoma fusion transcripts were designed for the assay. The NanoString assay was used to test 22 specific fusion transcripts in 45 sarcoma samples that had exhibited one of these fusion genes previously by reverse transcription polymerase chain reaction (RT-PCR). A mixture of frozen (n = 18), formalin-fixed, paraffin-embedded (FFPE) tissue (n = 23), and rapid extract template (n = 4) were used for testing. Results Each of the 22 transcripts tested was detected in at least one of the 45 tumor samples. The results of the NanoString assay were 100% concordant with the previous RT-PCR results for the tumor samples, and the technique was successful using both FFPE and rapid extract method. Conclusion Multiplexed interrogation for sarcoma-specific fusion transcripts using NanoString technology is a reliable approach for molecular diagnosis of pediatric sarcomas and works well with FFPE tissues. Future work will involve validating additional sarcoma fusion transcripts as well as determining the optimal workflow for diagnostic purposes.

scholarly journals PTM-Shepherd: analysis and summarization of post-translational and chemical modifications from open search results

2020 ◽  
pp. mcp.TIR120.002216
Author(s):  
Daniel J. Geiszler ◽  
Andy T. Kong ◽  
Dmitry M Avtonomov ◽  
Fengchao Yu ◽  
Felipe da Veiga Leprevost ◽  
...  
Keyword(s):  
Shotgun Proteomics ◽  
Chemical Modifications ◽  
Bioinformatics Tool ◽  
Site Specific ◽  
Search Results ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Small Set ◽  
Formalin Fixed

Open searching has proven to be an effective strategy for identifying both known and unknown modifications in shotgun proteomics experiments. Rather than being limited to a small set of user-specified modifications, open searches identify peptides with any mass shift that may correspond to a single modification or a combination of several modifications. Here we present PTM-Shepherd, a bioinformatics tool that automates characterization of PTM profiles detected in open searches based on attributes such as amino acid localization, fragmentation spectra similarity, retention time shifts, and relative modification rates. PTM-Shepherd can also perform multi-experiment comparisons for studying changes in modification profiles, e.g. in data generated in different laboratories or under different conditions. We demonstrate how PTM-Shepherd improves the analysis of data from formalin-fixed paraffin-embedded samples, detects extreme underalkylation of cysteine in some datasets, discovers an artefactual modification introduced during peptide synthesis, and uncovers site-specific biases in sample preparation artifacts in a multi-center proteomics profiling study.

scholarly journals Molecular Markers for Prostate Cancer in Formalin-Fixed Paraffin-Embedded Tissues

2013 ◽  
Vol 2013 ◽  
pp. 1-15 ◽  
Author(s):  
Tamara Sequeiros ◽  
Marta García ◽  
Melania Montes ◽  
Mireia Oliván ◽  
Marina Rigau ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Molecular Markers ◽  
Tumor Grade ◽  
Developed Countries ◽  
Response To Therapy ◽  
Tissue Samples ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Formalin Fixed

Prostate cancer (PCa) is the most frequently diagnosed type of cancer in developed countries. The decisive method of diagnosis is based on the results of biopsies, morphologically evaluated to determine the presence or absence of cancer. Although this approach leads to a confident diagnosis in most cases, it can be improved by using the molecular markers present in the tissue. Both miRNAs and proteins are considered excellent candidates for biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues, due to their stability over long periods of time. In the last few years, a concerted effort has been made to develop the necessary tools for their reliable measurement in these types of samples. Furthermore, the use of these kinds of markers may also help in establishing tumor grade and aggressiveness, as well as predicting the possible outcomes in each particular case for the different treatments available. This would aid clinicians in the decision-making process. In this review, we attempt to summarize and discuss the potential use of microRNA and protein profiles in FFPE tissue samples as markers to better predict PCa diagnosis, progression, and response to therapy.

scholarly journals Label-Free Semiquantitative Liquid Chromatography-Tandem Mass Spectrometry Proteomics Analysis of Laryngeal/Hypopharyngeal Squamous Cell Carcinoma on Formalin-Fixed, Paraffin-Embedded Tissue Samples - a Pilot Study

2020 ◽  
Vol 26 (4) ◽  
pp. 2801-2807
Author(s):  
Andras Burian ◽  
Laszlo Lujber ◽  
Imre Gerlinger ◽  
Tamas Jarai ◽  
Eva Orosz ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Squamous Cell Carcinoma ◽  
Liquid Chromatography ◽  
Cell Carcinoma ◽  
Tumor Markers ◽  
Squamous Cell ◽  
Label Free ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Abstract Squamous cell carcinoma (SCC) of the head and neck region is the sixth most frequent malignancy with high mortality rate. Due to its poor prognosis it is considered a growing public health problem worldwide inspite of existing treatment modalities. Thus, early diagnosis of new diseases and recurrences is emerging on one hand, but on the other hand troublesome in the lack of reliable tumor markers in this field. The rapid development of proteomics has opened new perspectives in tumor marker discovery. Liquid chromatography/mass spectrometry (LC/MS) as the gold standard in proteomics enables the semi-quantitative analysis of proteins within various tissues. Abundance differences between tumor and normal tissue also can be interpreted as tumor specific changes. The aim of this study was to identify potential tumor markers of laryngeal/hypopharyngeal SCC by revealing abundance changes between cancerous and the surrounding phenotypically healthy tissue. After separating the phenotypically cancerous and healthy parts of formalin-fixed paraffin-embedded tissues, each sample underwent protein recovery process and tryptic digestion for label-free semi-quantitative LC/MS analysis. Eight proteins showed significantly higher abundance in tumor including tenascin, transmembrane emp24 domain-containing protein 2, cytoplasmic dynein light chain 1, coactosin-like protein, small proline-rich protein 2D, nucleolin, U5 small nuclear RNP 200-kDa helicase and fatty aldehyde dehydrogenase. Desmoglein-1 and keratin type I cytoskeletal 9 were down-regulated in tumor. Using Ingenuity Pathway Analysis we mapped the signaling pathways these proteins play role in regarding other tumors. Based on these findings these proteins may serve as promising biomarkers in the fight against laryngeal/hypopharyngeal SCCs.

scholarly journals Clostridium perfringens–Associated Necrotic Enteritis-Like Disease in Coconut Lorikeets (Trichoglossus haematodus)

2020 ◽  
pp. 030098582097178
Author(s):  
Llorenç Grau-Roma ◽  
Mauricio Navarro ◽  
Sohvi Blatter ◽  
Christian Wenker ◽  
Sonja Kittl ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Toxin Gene ◽  
Necrotic Enteritis ◽  
Gene Encoding ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Polymerase Chain ◽  
Beta Toxin ◽  
Formalin Fixed

Several outbreaks of necrotic enteritis-like disease in lorikeets, from which Clostridium perfringens was consistently isolated, are described. All lorikeets had acute, segmental, or multifocal fibrinonecrotizing inflammatory lesions in the small and/or the large intestine, with intralesional gram-positive rods. The gene encoding C. perfringens alpha toxin was detected by PCR (polymerase chain reaction) on formalin-fixed, paraffin-embedded (FFPE) tissues in 20 out of 24 affected lorikeets (83%), but it was not amplified from samples of any of 10 control lorikeets ( P < .0001). The second most prevalent C. perfringens toxin gene detected was the beta toxin gene, which was found in FFPE from 7 out of 24 affected lorikeets (29%). The other toxin genes were detected inconsistently and in a relatively low number of samples. These cases seem to be associated with C. perfringens, although the specific type involved could not be determined.

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