| 1. |
- Almeida, Natália, et al.
(författare)
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Mapping the melanoma plasma proteome (MPP) using single-shot proteomics interfaced with the WiMT database
- 2021
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Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 13:24
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Tidskriftsartikel (refereegranskat)abstract
- Plasma analysis by mass spectrometry-based proteomics remains a challenge due to its large dynamic range of 10 orders in magnitude. We created a methodology for protein identification known as Wise MS Transfer (WiMT). Melanoma plasma samples from biobank archives were directly analyzed using simple sample preparation. WiMT is based on MS1 features between several MS runs together with custom protein databases for ID generation. This entails a multi-level dynamic protein database with different immunodepletion strategies by applying single-shot proteomics. The highest number of melanoma plasma proteins from undepleted and unfractionated plasma was reported, mapping >1200 proteins from >10,000 protein sequences with confirmed significance scoring. Of these, more than 660 proteins were annotated by WiMT from the resulting ~5800 protein sequences. We could verify 4000 proteins by MS1t analysis from HeLA extracts. The WiMT platform provided an output in which 12 previously well-known candidate markers were identified. We also identified low-abundant proteins with functions related to (i) cell signaling, (ii) immune system regulators, and (iii) proteins regulating folding, sorting, and degradation, as well as (iv) vesicular transport proteins. WiMT holds the potential for use in large-scale screening studies with simple sample preparation, and can lead to the discovery of novel proteins with key melanoma disease functions.
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| 2. |
- Barbara Sahlin, K., et al.
(författare)
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Short-term effect of induced alterations in testosterone levels on fasting plasma amino acid levels in healthy young men
- 2021
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Ingår i: Life. - : MDPI AG. - 0024-3019 .- 2075-1729. ; 11:11
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Tidskriftsartikel (refereegranskat)abstract
- Long term effect of testosterone (T) deficiency impairs metabolism and is associated with muscle degradation and metabolic disease. The association seems to have a bidirectional nature and is not well understood. The present study aims to investigate the early and unidirectional metabolic effect of induced T changes by measuring fasting amino acid (AA) levels in a human model, in which short-term T alterations were induced. We designed a human model of 30 healthy young males with pharmacologically induced T changes, which resulted in three time points for blood collection: (A) baseline, (B) low T (3 weeks post administration of gonadotropin releasing hormone antagonist) and (C) restored T (2 weeks after injection of T undecanoate). The influence of T on AAs was analyzed by spectrophotometry on plasma samples. Levels of 9 out of 23 AAs, of which 7 were essential AAs, were significantly increased at low T and are restored upon T supplementation. Levels of tyrosine and phenylalanine were most strongly associated to T changes. Short-term effect of T changes suggests an increased protein breakdown that is restored upon T supplementation. Fasting AA levels are able to monitor the early metabolic changes induced by the T fluctuations.
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| 3. |
- 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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| 4. |
- 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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| 5. |
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| 6. |
- 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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| 7. |
- Betancourt, Lazaro, et al.
(författare)
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Quantitative Assessment of Urea In-Solution Lys-C/Trypsin Digestions Reveals Superior Performance at Room Temperature over Traditional Proteolysis at 37 °C.
- 2018
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 17:7, s. 2556-2561
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Tidskriftsartikel (refereegranskat)abstract
- Urea-containing buffer solutions are generally used in proteomic studies to aid protein denaturation and solubilization during cell and tissue lysis. It is well-known, however, that urea can lead to carbamylation of peptides and proteins and, subsequently, incomplete digestion of proteins. By the use of cells and tissues that had been lysed with urea, different solution digestion strategies were quantitatively assessed. In comparison with traditional proteolysis at 37 °C, urea in-solution digestion performed at room temperature improved peptide and protein identification and quantitation and had a minimum impact on miscleavage rates. Furthermore, the signal intensities and the number of carbamylated and pyroglutamic acid-modified peptides decreased. Overall, this led to a reduction in the negative effects often observed for such modifications. Data are available via ProteomeXchange with identifier PXD009426.
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| 8. |
- Byrling, Johannes, et al.
(författare)
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Mass spectrometry-based analysis of formalin-fixed, paraffin-embedded distal cholangiocarcinoma identifies stromal thrombospondin-2 as a potential prognostic marker
- 2020
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Ingår i: Journal of Translational Medicine. - : Springer Science and Business Media LLC. - 1479-5876. ; 18:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Distal cholangiocarcinoma is an aggressive malignancy with a dismal prognosis. Diagnostic and prognostic biomarkers for distal cholangiocarcinoma are lacking. The aim of the present study was to identify differentially expressed proteins between distal cholangiocarcinoma and normal bile duct samples. Methods: A workflow utilizing discovery mass spectrometry and verification by parallel reaction monitoring was used to analyze surgically resected formalin-fixed, paraffin-embedded samples from distal cholangiocarcinoma patients and normal bile duct samples. Bioinformatic analysis was used for functional annotation and pathway analysis. Immunohistochemistry was performed to validate the expression of thrombospondin-2 and investigate its association with survival. Results: In the discovery study, a total of 3057 proteins were identified. Eighty-seven proteins were found to be differentially expressed (q < 0.05 and fold change ≥ 2 or ≤ 0.5); 31 proteins were upregulated and 56 were downregulated in the distal cholangiocarcinoma samples compared to controls. Bioinformatic analysis revealed an abundance of differentially expressed proteins associated with the tumor reactive stroma. Parallel reaction monitoring verified 28 proteins as upregulated and 18 as downregulated in distal cholangiocarcinoma samples compared to controls. Immunohistochemical validation revealed thrombospondin-2 to be upregulated in distal cholangiocarcinoma epithelial and stromal compartments. In paired lymph node metastases samples, thrombospondin-2 expression was significantly lower; however, stromal thrombospondin-2 expression was still frequent (72%). Stromal thrombospondin-2 was an independent predictor of poor disease-free survival (HR 3.95, 95% CI 1.09-14.3; P = 0.037). Conclusion: Several proteins without prior association with distal cholangiocarcinoma biology were identified and verified as differentially expressed between distal cholangiocarcinoma and normal bile duct samples. These proteins can be further evaluated to elucidate their biomarker potential and role in distal cholangiocarcinoma carcinogenesis. Stromal thrombospondin-2 is a potential prognostic marker in distal cholangiocarcinoma.
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| 9. |
- de Siqueira Guedes, Jéssica, et al.
(författare)
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Plasma metabolome study reveals metabolic changes induced by pharmacological castration and testosterone supplementation in healthy young men
- 2022
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Testosterone is a hormone that plays a key role in carbohydrate, fat, and protein metabolism. Testosterone deficiency is associated with multiple comorbidities, e.g., metabolic syndrome and type 2 diabetes. Despite its importance in many metabolic pathways, the mechanisms by which it controls metabolism are not fully understood. The present study investigated the short-term metabolic changes of pharmacologically induced castration and, subsequently, testosterone supplementation in healthy young males. Thirty subjects were submitted to testosterone depletion (TD) followed by testosterone supplementation (TS). Plasma samples were collected three times corresponding to basal, low, and restored testosterone levels. An untargeted metabolomics study was performed by liquid chromatography–high resolution mass spectrometry (UHPLC–HRMS) to monitor the metabolic changes induced by the altered hormone levels. Our results demonstrated that TD was associated with major metabolic changes partially restored by TS. Carnitine and amino acid metabolism were the metabolic pathways most impacted by variations in testosterone. Furthermore, our results also indicated that LH and FSH might strongly alter the plasma levels of indoles and lipids, especially glycerophospholipids and sphingolipids. Our results demonstrated major metabolic changes induced by low testosterone that may be important for understanding the mechanisms behind the association of testosterone deficiency and its comorbidities.
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| 10. |
- 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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