scholarly journals Accelerated Protein Biomarker Discovery from FFPE Tissue Samples Using Single-Shot, Short Gradient Microflow SWATH MS

2020 ◽  
Vol 19 (7) ◽  
pp. 2732-2741 ◽  
Author(s):  
Rui Sun ◽  
Christie Hunter ◽  
Chen Chen ◽  
Weigang Ge ◽  
Nick Morrice ◽  
...  
Keyword(s):  
Biomarker Discovery ◽  
Ffpe Tissue ◽  
Single Shot ◽  
Protein Biomarker ◽  
Tissue Samples

scholarly journals Accelerated Protein Biomarker Discovery from FFPE tissue samples using Single-shot, Short Gradient Microflow SWATH MS

2019 ◽  
Author(s):  
Rui Sun ◽  
Christie Hunter ◽  
Chen Chen ◽  
Weigang Ge ◽  
Nick Morrice ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Biomarker Discovery ◽  
Single Shot ◽  
Protein Biomarkers ◽  
Differential Abundance ◽  
Tissue Samples ◽  
Peptide Precursors ◽  
High Consistency ◽  
Method R ◽  
Acquisition Method

ABSTRACTWe report and evaluated a microflow, single-shot, short gradient SWATH MS method intended to accelerate the discovery and verification of protein biomarkers in clinical specimens. The method uses 15-min gradient microflow-LC peptide separation, an optimized SWATH MS window configuration and OpenSWATH software for data analysis.We applied the method to a cohort 204 of FFPE prostate tissue samples from 58 prostate cancer patients and 10 prostatic hyperplasia patients. Altogether we identified 27,976 proteotypic peptides and 4,043 SwissProt proteins from these 204 samples. Compared to a reference SWATH method with 2-hour gradient the accelerated method consumed only 27% instrument time, quantified 80% proteins and showed reduced batch effects. 3,800 proteins were quantified by both methods in two different instruments with relatively high consistency (r = 0.77). 75 proteins detected by the accelerated method with differential abundance between clinical groups were selected for further validation. A shortlist of 134 selected peptide precursors from the 75 proteins were analyzed using MRM-HR, exhibiting high quantitative consistency with the 15-min SWATH method (r = 0.89) in the same sample set. We further verified the capacity of these 75 proteins in separating benign and malignant tissues (AUC = 0.99) in an independent prostate cancer cohort (n=154).Overall our data show that the single-shot short gradient microflow-LC SWATH MS method achieved about 4-fold acceleration of data acquisition with reduced batch effect and a moderate level of protein attrition compared to a standard SWATH acquisition method. Finally, the results showed comparable ability to separate clinical groups.

scholarly journals High-throughput proteomic analysis of FFPE tissue samples facilitates tumor stratification

2019 ◽  
Author(s):  
Yi Zhu ◽  
Tobias Weiss ◽  
Qiushi Zhang ◽  
Rui Sun ◽  
Bo Wang ◽  
...  
Keyword(s):  
Biomarker Discovery ◽  
Temporal Stability ◽  
B Cell Lymphoma ◽  
Ffpe Tissue ◽  
Tissue Samples ◽  
Pattern Similarity ◽  
Ffpe Samples ◽  
Fresh Frozen ◽  
Peroxidase Enzyme ◽  
High Degree

AbstractFormalin-fixed, paraffin-embedded (FFPE), biobanked tissue samples offer an invaluable resource for clinical and biomarker research. Here we developed a pressure cycling technology (PCT)-SWATH mass spectrometry workflow to analyze FFPE tissue proteomes and applied it to the stratification of prostate cancer (PCa) and diffuse large B-cell lymphoma (DLBCL) samples. We show that the proteome patterns of FFPE PCa tissue samples and their analogous fresh frozen (FF) counterparts have a high degree of similarity and we confirmed multiple proteins consistently regulated in PCa tissues in an independent sample cohort. We further demonstrate temporal stability of proteome patterns from FFPE samples that were stored between one to 15 years in a biobank and show a high degree of the proteome pattern similarity between two types histological region of small FFPE samples, i.e. punched tissue biopsies and thin tissue sections of micrometer thickness, despite the existence of certain degree of biological variations. Applying the method to two independent DLBCL cohorts we identified myeloperoxidase (MPO), a peroxidase enzyme, as a novel prognostic marker. In summary, this study presents a robust proteomic method to analyze bulk and biopsy FFPE tissues and reports the first systematic comparison of proteome maps generated from FFPE and FF samples. Our data demonstrate the practicality and superiority of FFPE over FF samples for proteome in biomarker discovery. Promising biomarker candidates for PCa and DLBCL have been discovered.

scholarly journals Comparison of different digestion methods for proteomic analysis of isolated cells and FFPE tissue samples

Talanta ◽  
2021 ◽  
pp. 122568
Author(s):  
Artur Pirog ◽  
Jakub Faktor ◽  
Zuzanna Urban-Wojciuk ◽  
Sachin Kote ◽  
Elżbieta Chruściel ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Ffpe Tissue ◽  
Isolated Cells ◽  
Tissue Samples ◽  
Digestion Methods

scholarly journals The Dutch National TissueArchive Portal enables efficient, consistent, and transparent procurement of diagnostic tissue samples for scientific use

2021 ◽  
Author(s):  
Robin Verjans ◽  
Annette H. Bruggink ◽  
Robby Kibbelaar ◽  
Jos Bart ◽  
Aletta Debernardi ◽  
...  
Keyword(s):  
The Netherlands ◽  
Tissue Sample ◽  
Ffpe Tissue ◽  
Tissue Samples ◽  
Internet Portal ◽  
Formalin Fixed Paraffin ◽  
Practical Support ◽  
Formalin Fixed Paraffin Embedded ◽  
The Rich ◽  
Formalin Fixed

AbstractBiobanks play a crucial role in enabling biomedical research by facilitating scientific use of valuable human biomaterials. The PALGA foundation—a nationwide network and registry of histo- and cytopathology in the Netherlands—was established to promote the provision of data within and between pathology departments, and to make the resulting knowledge available for healthcare. Apart from the pathology data, we aimed to utilize PALGA’s nationwide network to find and access the rich wealth of Formalin-Fixed Paraffin-Embedded (FFPE) tissue samples for scientific use.  We implemented the Dutch National TissueArchive Portal (DNTP) to utilize PALGA’s nationwide network for requesting FFPE tissue samples. The DNTP consists of (1) a centrally organized internet portal to improve the assessing, processing, harmonization, and monitoring of the procurement process, while (2) dedicated HUB-employees provide practical support at peripheral pathology departments. Since incorporation of the DNTP, both the number of filed requests for FFPE tissue samples and the amount of HUB-mediated support increased 55 and 29% respectively. In line, the sample procurement duration time decreased significantly (− 47%). These findings indicate that implementation of the DNTP improved the frequency, efficiency, and transparency of FFPE tissue sample procurement for research in the Netherlands. To conclude, the need for biological resources is growing persistently to enable precision medicine. Here, we access PALGA’s national, pathology network by implementation of the DNTP to allow for efficient, consistent, and transparent exchange of FFPE tissue samples for research across the Netherlands.

scholarly journals Cerebrospinal fluid extracellular vesicle enrichment for protein biomarker discovery in neurological disease; multiple sclerosis

2017 ◽  
Vol 6 (1) ◽  
pp. 1369805 ◽  
Author(s):  
Joanne L. Welton ◽  
Samantha Loveless ◽  
Timothy Stone ◽  
Chris von Ruhland ◽  
Neil P. Robertson ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Cerebrospinal Fluid ◽  
Neurological Disease ◽  
Biomarker Discovery ◽  
Extracellular Vesicle ◽  
Protein Biomarker

scholarly journals Tutorial: best practices and considerations for mass-spectrometry-based protein biomarker discovery and validation

2021 ◽  
Author(s):  
Ernesto S. Nakayasu ◽  
Marina Gritsenko ◽  
Paul D. Piehowski ◽  
Yuqian Gao ◽  
Daniel J. Orton ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Best Practices ◽  
Biomarker Discovery ◽  
Protein Biomarker

scholarly journals High sensitivity sanger sequencing detection of BRAF mutations in metastatic melanoma FFPE tissue specimens

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lauren Y. Cheng ◽  
Lauren E. Haydu ◽  
Ping Song ◽  
Jianyi Nie ◽  
Michael T. Tetzlaff ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Limit Of Detection ◽  
False Negative ◽  
High Sensitivity ◽  
Ffpe Tissue ◽  
Braf Mutations ◽  
Tissue Samples ◽  
Ffpe Samples ◽  
Droplet Pcr ◽  
Tissue Specimens

AbstractMutations in the BRAF gene at or near the p. V600 locus are informative for therapy selection, but current methods for analyzing FFPE tissue DNA generally have a limit of detection of 5% variant allele frequency (VAF), or are limited to the single variant (V600E). These can result in false negatives for samples with low VAFs due to low tumor content or subclonal heterogeneity, or harbor non-V600 mutations. Here, we show that Sanger sequencing using the NuProbe VarTrace BRAF assay, based on the Blocker Displacement Amplification (BDA) technology, is capable of detecting BRAF V600 mutations down to 0.20% VAF from FFPE lymph node tissue samples. Comparison experiments on adjacent tissue sections using BDA Sanger, immunohistochemistry (IHC), digital droplet PCR (ddPCR), and NGS showed 100% concordance among all 4 methods for samples with BRAF mutations at ≥ 1% VAF, though ddPCR did not distinguish the V600K mutation from the V600E mutation. BDA Sanger, ddPCR, and NGS (with orthogonal confirmation) were also pairwise concordant for lower VAF mutations down to 0.26% VAF, but IHC produced a false negative. Thus, we have shown that Sanger sequencing can be effective for rapid detection and quantitation of multiple low VAF BRAF mutations from FFPE samples. BDA Sanger method also enabled detection and quantitation of less frequent, potentially actionable non-V600 mutations as demonstrated by synthetic samples.

Rapid Somatic Mutation Testing in Colorectal Cancer by Use of a Fully Automated System and Single-Use Cartridge: A Comparison with Next-Generation Sequencing

2018 ◽  
Vol 3 (2) ◽  
pp. 178-184 ◽  
Author(s):  
M Rabie Al-Turkmani ◽  
Kelley N Godwin ◽  
Jason D Peterson ◽  
Gregory J Tsongalis
Keyword(s):  
Colorectal Cancer ◽  
Next Generation Sequencing ◽  
Ffpe Tissue ◽  
Braf V600e ◽  
Automated System ◽  
Next Generation ◽  
Tissue Samples ◽  
Tissue Sections ◽  
Single Use ◽  
Generation Sequencing

AbstractBackgroundMolecular tests have been increasingly used in the management of various cancers as more targeted therapies are becoming available as treatment options. The Idylla™ system is a fully integrated, cartridge-based platform that provides automated sample processing (deparaffinization, tissue digestion, and DNA extraction) and real-time PCR-based mutation detection with all reagents included in a single-use cartridge. This retrospective study aimed at evaluating both the Idylla KRAS and NRAS-BRAF-EGFR492 Mutation Assay cartridges (research use only) against next-generation sequencing (NGS) by using colorectal cancer (CRC) tissue samples.MethodsForty-four archived formalin-fixed paraffin-embedded (FFPE) CRC tissue samples previously analyzed by targeted NGS were tested on the Idylla system. Among these samples, 17 had a mutation in KRAS proto-oncogene, GTPase (KRAS), 5 in NRAS proto-oncogene, GTPase (NRAS), and 12 in B-Raf proto-oncogene, serine/threonine kinase (BRAF) as determined using the Ion AmpliSeq 50-gene Cancer Hotspot Panel v2. The remaining 10 samples were wild-type for KRAS, NRAS, and BRAF. Two 10-μm FFPE tissue sections were used for each Idylla run, 1 for the KRAS cartridge, and 1 for the NRAS-BRAF-EGFR492 cartridge. All cases met the Idylla minimum tumor content requirement for KRAS, NRAS, and BRAF (≥10%). Assay reproducibility was evaluated by testing commercial controls derived from human cell lines, which had an allelic frequency of 50% and were run in triplicate.ResultsThe Idylla system successfully detected all mutations previously identified by NGS in KRAS (G12C, G12D, G12V, G13D, Q61K, Q61R, A146T), NRAS (G12V, G13R, Q61H), and BRAF (V600E). Compared with NGS, Idylla had a sensitivity of 100%. Analysis of the mutated commercial controls demonstrated agreement with the expected result for all samples and 100% reproducibility. The Idylla system produced results quickly with a turnaround time of approximately 2 h.ConclusionThe Idylla system offers reliable and sensitive testing of clinically actionable mutations in KRAS, NRAS, and BRAF directly from FFPE tissue sections.

scholarly journals Functional Proteomic Profiling of Phosphodiesterases Using SeraFILE Separations Platform

2012 ◽  
Vol 2012 ◽  
pp. 1-8
Author(s):  
Amita R. Oka ◽  
Matthew P. Kuruc ◽  
Ketan M. Gujarathi ◽  
Swapan Roy
Keyword(s):  
Biomarker Discovery ◽  
Brain Homogenate ◽  
Disease Diagnosis ◽  
Bovine Brain ◽  
Tissue Cell ◽  
Kinetic Properties ◽  
Proteomic Profiling ◽  
Tissue Samples ◽  
Camp Hydrolysis ◽  
Few Data

Functional proteomic profiling can help identify targets for disease diagnosis and therapy. Available methods are limited by the inability to profile many functional properties measured by enzymes kinetics. The functional proteomic profiling approach proposed here seeks to overcome such limitations. It begins with surface-based proteome separations of tissue/cell-line extracts, using SeraFILE, a proprietary protein separations platform. Enzyme kinetic properties of resulting subproteomes are then characterized, and the data integrated into proteomic profiles. As a model, SeraFILE-derived subproteomes of cyclic nucleotide-hydrolyzing phosphodiesterases (PDEs) from bovine brain homogenate (BBH) and rat brain homogenate (RBH) were characterized for cAMP hydrolysis activity in the presence (challenge condition) and absence of cGMP. Functional profiles of RBH and BBH were compiled from the enzyme activity response to the challenge condition in each of the respective subproteomes. Intersample analysis showed that comparable profiles differed in only a few data points, and that distinctive subproteomes can be generated from comparable tissue samples from different animals. These results demonstrate that the proposed methods provide a means to simplify intersample differences, and to localize proteins attributable to sample-specific responses. It can be potentially applied for disease and nondisease sample comparison in biomarker discovery and drug discovery profiling.

scholarly journals Evaluation of the Xpert MTB/RIF Performance on Tissues: Potential Impact on Airborne Infection Isolation at a Tertiary Cancer Care Center

2018 ◽  
Vol 39 (4) ◽  
pp. 462-466 ◽  
Author(s):  
Tracy McMillen ◽  
Shauna C. Usiak ◽  
Liang Hua Chen ◽  
Luz Gomez ◽  
Peter Ntiamoah ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Limit Of Detection ◽  
Tertiary Care ◽  
Ffpe Tissue ◽  
Cancer Center ◽  
Oncology Patients ◽  
Tissue Samples ◽  
Airborne Infection ◽  
Potential Impact ◽  
Tissue Specimens

OBJECTIVESIn this study, we sought to evaluate the performance of the Xpert MTB/RIF (Cepheid) assay for the detection of Mycobacterium tuberculosis (MTB) complex DNA on fresh and formalin-fixed, paraffin-embedded (FFPE) tissue specimens from oncology patients in an area with a low prevalence of tuberculosis. We also aimed to retrospectively assess the potential impact of Xpert MTB/RIF on the duration of airborne infection isolation (AII).SETTINGA 473-bed, tertiary-care cancer center in New York City.DESIGNA total of 203 tissue samples (101 FFPE and 102 fresh) were tested using Xpert MTB/RIF, including 133 pulmonary tissue samples (65.5%) and 70 extrapulmonary tissue samples (34.5%). Acid-fast bacilli (AFB) culture was used as the diagnostic gold standard. The limit of detection (LOD) and reproducibility were also evaluated for both samples types using contrived specimens. The potential impact of the Xpert MTB PCR assay on tissue samples from AII patients on AII duration was retrospectively assessed.RESULTSUsing the Xpert MTB/RIF for fresh tissue specimens, the sensitivity was 50% (95% CI, 1.3%–98.7%) and the specificity was 99% (95% CI, 94.5%–99.9%). For FFPE tissue specimens, the sensitivity was 100% (95% CI, 63.1%–100%) and the specificity was 98.3% (95% CI, 95.5%–100%. The LOD was 103 colony-forming units (CFU)/mL for both fresh and FFPE tissue specimens, and the Xpert MTB/RIF was 100% reproducible at concentrations 10 times that of the LOD. With an expected turnaround time of 24 hours, the Xpert MTB PCR could decrease the duration of AII from a median of 8 days to a median of 1 day.CONCLUSIONSThe Xpert MTB/RIF assay offers a valid option for ruling out Mycobacterium tuberculosis complex (MTBC) on tissue samples from oncology patients and for minimizing AII resource utilization.Infect Control Hosp Epidemiol 2018;39:462–466

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