| 1. |
- Kamiie, Junichi, et al.
(författare)
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Amyloid-specific extraction using organic solvents
- 2020
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Ingår i: MethodsX. - : Elsevier BV. - 2215-0161. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Typing of amyloidosis by mass spectrometry (MS) based proteomic analysis contribute to the diagnosis of amyloidosis. For MS analysis, laser microdissection (LMD) is used for amyloid specific sampling. This study aimed to establish a method for selectively extracting amyloids from formalin-fixed, paraffin-embedded (FFPE) specimens by organic solvent instead of LMD. The extracts using dimethyl sulfoxide (DMSO), dimethylformamide (DMF), methanol, trifluoroethanol (TFE) or hexafluoro-2-propanol from FFPE brain of alzheimer's disease mouse model generated protein bands on SDS-PAGE, and Aβ was identified in the extract of DMF using mass spectrometry. The extract using DMSO from the kidney of a AA amyloidosis patient produced a protein band in SDS-PAGE. This protein band was identified to be serum amyloid A (SAA) by Western blotting and mass spectrometry. Circular dichroism spectrometry revealed that the secondary structures of Aβ and transthyretin were converted to α-helices from β-sheets in TFE. Our results suggest that organic solvents can extract amyloids from FFPE specimens by converting their secondary structure. This method could eliminate the LMD step and simplified amyloid typing by MS analysis. • DMSO, DMF, methanol, TFE and HFIP can extract Aβ specifically from the FFPE brain of a Alzheimer’ disease mouse model. • DMSO can extract SAA specifically from a FFPE section of AA amyloidosis. • Secondary structures of Aβ and transthyretin converted from β-sheet to α-helix in TFE.
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| 2. |
- Betancourt, Lazaro Hiram, et al.
(författare)
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Improved survival prognostication of node-positive malignant melanoma patients utilizing shotgun proteomics guided by histopathological characterization and genomic data
- 2019
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- Metastatic melanoma is one of the most common deadly cancers, and robust biomarkers are still needed, e.g. to predict survival and treatment efficiency. Here, protein expression analysis of one hundred eleven melanoma lymph node metastases using high resolution mass spectrometry is coupled with in-depth histopathology analysis, clinical data and genomics profiles. This broad view of protein expression allowed to identify novel candidate protein markers that improved prediction of survival in melanoma patients. Some of the prognostic proteins have not been reported in the context of melanoma before, and few of them exhibit unexpected relationship to survival, which likely reflects the limitations of current knowledge on melanoma and shows the potential of proteomics in clinical cancer research.
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| 3. |
- Betancourt, Lazaro Hiram, et al.
(författare)
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The hidden story of heterogeneous B-raf V600E mutation quantitative protein expression in metastatic melanoma—association with clinical outcome and tumor phenotypes
- 2019
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Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 11:12
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Tidskriftsartikel (refereegranskat)abstract
- In comparison to other human cancer types, malignant melanoma exhibits the greatest amount of heterogeneity. After DNA-based detection of the BRAF V600E mutation in melanoma patients, targeted inhibitor treatment is the current recommendation. This approach, however, does not take the abundance of the therapeutic target, i.e., the B-raf V600E protein, into consideration. As shown by immunohistochemistry, the protein expression profiles of metastatic melanomas clearly reveal the existence of inter-and intra-tumor variability. Nevertheless, the technique is only semi-quantitative. To quantitate the mutant protein there is a fundamental need for more precise techniques that are aimed at defining the currently non-existent link between the levels of the target protein and subsequent drug efficacy. Using cutting-edge mass spectrometry combined with DNA and mRNA sequencing, the mutated B-raf protein within metastatic tumors was quantitated for the first time. B-raf V600E protein analysis revealed a subjacent layer of heterogeneity for mutation-positive metastatic melanomas. These were characterized into two distinct groups with different tumor morphologies, protein profiles and patient clinical outcomes. This study provides evidence that a higher level of expression in the mutated protein is associated with a more aggressive tumor progression. Our study design, comprised of surgical isolation of tumors, histopathological characterization, tissue biobanking, and protein analysis, may enable the eventual delineation of patient responders/non-responders and subsequent therapy for malignant melanoma.
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| 4. |
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| 5. |
- Betancourt, Lazaro Hiram, et al.
(författare)
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The human melanoma proteome atlas-Defining the molecular pathology
- 2021
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Ingår i: Clinical and Translational Medicine. - : Wiley. - 2001-1326. ; 11:7, s. 1-20
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Tidskriftsartikel (refereegranskat)abstract
- The MM500 study is an initiative to map the protein levels in malignant melanoma tumor samples, focused on in-depth histopathology coupled to proteome characterization. The protein levels and localization were determined for a broad spectrum of diverse, surgically isolated melanoma tumors originating from multiple body locations. More than 15,500 proteoforms were identified by mass spectrometry, from which chromosomal and subcellular localization was annotated within both primary and metastatic melanoma. The data generated by global proteomic experiments covered 72% of the proteins identified in the recently reported high stringency blueprint of the human proteome. This study contributes to the NIH Cancer Moonshot initiative combining detailed histopathological presentation with the molecular characterization for 505 melanoma tumor samples, localized in 26 organs from 232 patients.
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| 6. |
- Gil, Jeovanis, et al.
(författare)
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An observational study on the molecular profiling of primary melanomas reveals a progression dependence on mitochondrial activation
- 2021
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Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 13:23
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Tidskriftsartikel (refereegranskat)abstract
- Melanoma in advanced stages is one of the most aggressive tumors and the deadliest of skin cancers. To date, the histopathological staging focuses on tumor thickness, and clinical staging is a major estimate of the clinical behavior of primary melanoma. Here we report on an observational study with in‐depth molecular profiling at the protein level including post-translational modifications (PTMs) on eleven primary tumors from melanoma patients. Global proteomics, phosphoproteomics, and acetylomics were performed on each sample. We observed an up‐regulation of key mitochondrial functions, including the mitochondrial translation machinery and the down‐regulation of structural proteins involved in cell adhesion, the cytoskeleton organization, and epidermis development, which dictates the progression of the disease. Additionally, the PTM level pathways related to RNA processing and transport, as well as chromatin organization, were dysregulated in relation to the progression of melanoma. Most of the pathways dysregulated in this cohort were enriched in genes differentially expressed at the transcript level when similar groups are compared or metastasis to primary melanomas. At the genome level, we found significant differences in the mutation profiles between metastatic and primary melanomas. Our findings also highlighted sex‐related differences in the molecular profiles. Remarkably, primary melanomas in women showed higher levels of antigen processing and presentation, and activation of the immune system response. Our results provide novel insights, relevant for developing personalized precision treatments for melanoma patients.
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| 7. |
- Gil, Jeovanis, et al.
(författare)
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Clinical protein science in translational medicine targeting malignant melanoma
- 2019
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Ingår i: Cell Biology and Toxicology. - : Springer Science and Business Media LLC. - 0742-2091 .- 1573-6822. ; 35:4, s. 293-332
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Tidskriftsartikel (refereegranskat)abstract
- Melanoma of the skin is the sixth most common type of cancer in Europe and accounts for 3.4% of all diagnosed cancers. More alarming is the degree of recurrence that occurs with approximately 20% of patients lethally relapsing following treatment. Malignant melanoma is a highly aggressive skin cancer and metastases rapidly extend to the regional lymph nodes (stage 3) and to distal organs (stage 4). Targeted oncotherapy is one of the standard treatment for progressive stage 4 melanoma, and BRAF inhibitors (e.g. vemurafenib, dabrafenib) combined with MEK inhibitor (e.g. trametinib) can effectively counter BRAFV600E-mutated melanomas. Compared to conventional chemotherapy, targeted BRAFV600E inhibition achieves a significantly higher response rate. After a period of cancer control, however, most responsive patients develop resistance to the therapy and lethal progression. The many underlying factors potentially causing resistance to BRAF inhibitors have been extensively studied. Nevertheless, the remaining unsolved clinical questions necessitate alternative research approaches to address the molecular mechanisms underlying metastatic and treatment-resistant melanoma. In broader terms, proteomics can address clinical questions far beyond the reach of genomics, by measuring, i.e. the relative abundance of protein products, post-translational modifications (PTMs), protein localisation, turnover, protein interactions and protein function. More specifically, proteomic analysis of body fluids and tissues in a given medical and clinical setting can aid in the identification of cancer biomarkers and novel therapeutic targets. Achieving this goal requires the development of a robust and reproducible clinical proteomic platform that encompasses automated biobanking of patient samples, tissue sectioning and histological examination, efficient protein extraction, enzymatic digestion, mass spectrometry–based quantitative protein analysis by label-free or labelling technologies and/or enrichment of peptides with specific PTMs. By combining data from, e.g. phosphoproteomics and acetylomics, the protein expression profiles of different melanoma stages can provide a solid framework for understanding the biology and progression of the disease. When complemented by proteogenomics, customised protein sequence databases generated from patient-specific genomic and transcriptomic data aid in interpreting clinical proteomic biomarker data to provide a deeper and more comprehensive molecular characterisation of cellular functions underlying disease progression. In parallel to a streamlined, patient-centric, clinical proteomic pipeline, mass spectrometry–based imaging can aid in interrogating the spatial distribution of drugs and drug metabolites within tissues at single-cell resolution. These developments are an important advancement in studying drug action and efficacy in vivo and will aid in the development of more effective and safer strategies for the treatment of melanoma. A collaborative effort of gargantuan proportions between academia and healthcare professionals has led to the initiation, establishment and development of a cutting-edge cancer research centre with a specialisation in melanoma and lung cancer. The primary research focus of the European Cancer Moonshot Lund Center is to understand the impact that drugs have on cancer at an individualised and personalised level. Simultaneously, the centre increases awareness of the relentless battle against cancer and attracts global interest in the exceptional research performed at the centre.
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| 8. |
- James, Connell, et al.
(författare)
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Localization of Sunitinib in in vivo Animal and in vitro Experimental Models by MALDI Mass Spectrometry Imaging
- 2015
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Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 407:8, s. 2245-2253
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Tidskriftsartikel (refereegranskat)abstract
- The spatial distribution of an anti-cancer drug and its intended target within a tumor plays a major role on determining how effective the drug can be at tackling the tumor. This study provides data regarding the lateral distribution of sunitinib, an oral antiangiogenic receptor tyrosine kinase inhibitor using an in vitro animal model as well as an in vitro experimental model that involved deposition of a solution of sunitinib onto tissue sections. All tumor sections were analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging and compared with subsequent histology staining. Six tumors at four different time points after commencement of in vivo sunitinib treatment were examined to observe the patterns of drug uptake. The levels of sunitinib present in in vivo treated tumor sections increased continuously until day seven but a decrease was observed at day 10. Furthermore, the in vitro experimental model was adjustable to produce a drug level similar to that obtained in the in vivo model experiments. The distribution of sunitinib in tissue sections treated in vitro appeared to agree with the histological structure of tumors suggesting that this approach may be useful for testing drug update.
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| 9. |
- Jankovskaja, Skaidre, et al.
(författare)
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Optimization of sample preparation for transporter protein quantification in tissues by LC–MS/MS
- 2019
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Ingår i: Journal of Pharmaceutical and Biomedical Analysis. - : Elsevier BV. - 0731-7085 .- 1873-264X. ; 164, s. 9-15
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Tidskriftsartikel (refereegranskat)abstract
- Background: Reproducible quantification of drug transporter protein expression in tissues is important for predicting transporter mediated drug disposition. Many mass-spectrometry based transporter protein quantification methods result in high variability of the estimated transporter quantities. Therefore, we aimed to evaluate and optimize mass spectrometry-based quantification method for drug transporter proteins in tissues. Materials and methods: Plasma membrane (PM) proteins from mouse tissues were isolated by applying three extraction protocols: commercial plasma membrane extraction kit, tissue homogenization by Potter-Elvehjem homogenizer in combination with sucrose-cushion ultracentrifugation, and PM enrichment with Tween 40. Moreover, five different protein digestion protocols were applied on the same PM fraction. PM isolation and digestion protocols were evaluated by measuring the amount of transporter proteins by liquid chromatography-tandem mass spectrometry in selected reaction monitoring mode. Results: Mouse liver homogenization by Potter-Elvehjem homogenizer in combination with sucrose-cushion ultracentrifugation and PM enrichment with Tween 40 resulted in two times higher transporter protein quantity (Breast cancer resistance protein (Bcrp) 18.0 fmol/μg protein) in comparison with the PM samples isolated by extraction kit (Bcrp 9.8 fmol/μg protein). The evaluation of protein digestion protocols revealed that the most optimal protocol for PM protein digestion is with Lys-C and trypsin, in combination with trypsin enhancer and heat denaturation. Overall, quantities of Bcrp and Na+/K + ATPase proteins evaluated in mouse liver and kidney cortex by using our optimized PM isolation method, as well as, established digestion protocol were two to three times higher than previously reported and coefficient of variation (CV) for technical replicates was below 10%. Conclusion: We have established an improved transporter protein quantification methodology by optimizing PM isolation and protein digestion procedures. The optimized procedure resulted in a higher transporter protein yield and improved precision.
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| 10. |
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