| 1. |
- 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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| 2. |
- Oppermann, Madalina
(författare)
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Chemical and mass spectrometrical methods in protein analysis
- 2000
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Sensitive analytical methods are essential in protein studies because of frequently encountered limited sample availability. There is a constant need for optimization of sample preparation and analytical procedures for structural characterization of biomedically relevant proteins. Amino acid sequences can be derived from genomic data. However, gene processing and post-translational modifications alter the expressed protein product which can be further processed in tissues to a functional form. Therefore, both protein quantity and structure must be evaluated per se. A method for chemical deblocking of N-terminally acetylated proteins useful for identification via sequence analysis by Edman degradation was developed and optimized. The protocol was applied to the characterization of native and recombinant proteins. Deacetylation yields were up to 70%. The method is efficient with both proteins and peptides, and has a fairly low yield of unspecific cleavage of peptide bonds. Mass spectrometry is a different method to analyze protein structure. The technique was applied to verify the removal of an acetyl group and also to analyze the structure of gel separated proteins from brain, prostate and archaea. Two-dimensional gel electrophoresis is efficient to separate complex protein mixtures. Protein characterization can then be carried through by mass spectrometry after in-gel digestion of polypeptides. These techniques were employed to analyze foetal human brain tissue from individuals with or without Down's syndrome. Forty proteins were identified. Protein patterns were largely similar, although actin and tubulin were observed not only at the expected molecular weights and isoelectric points, but also as negatively charged fragments. Proteins in benign and malignant prostate glands were also analyzed. The protein patterns indicated a high degree of heterogeneity. Several proteins were identified and prostatic acid phosphatase and tropomyosin I were found to be downregulated in prostatic cancer, whereas heat shock protein 70 was found to be upregulated in the neoplastic state. The results indicate that integration of analytical methods provides strategies for the characterization of proteins expressed under different conditions in goals toward determination of protein function.
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| 3. |
- Pannee, Josef, et al.
(författare)
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A Selected Reaction Monitoring (SRM)-Based Method for Absolute Quantification of A beta(38), A beta(40), and A beta(42) in Cerebrospinal Fluid of Alzheimer's Disease Patients and Healthy Controls
- 2013
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Ingår i: Journal of Alzheimer's Disease. - 1387-2877. ; 33:4, s. 1021-1032
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Tidskriftsartikel (refereegranskat)abstract
- Cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) are increasingly used in research centers, clinical trials, and clinical settings. However, their broad-scale use is hampered by lack of standardization across analytical platforms and by interference from binding of amyloid-beta(A beta) to matrix proteins as well as self-aggregation. Here, we report on a matrix effect-resistant method for the measurement of the AD-associated 42 amino acid species of A beta(A beta(42)), together with A beta(40) and A beta(38) in human CSF based on mass spectrometric quantification using selected reaction monitoring (SRM). Samples were prepared by solid-phase extraction and quantification was performed using stable-isotope labeled A beta peptides as internal standards. The diagnostic performance of the method was evaluated on two independent clinical materials with research volunteers who were cognitively normal and AD patients with mild to moderate dementia. Analytical characteristics of the method include a lower limit of quantification of 62.5 pg/mL for A beta(42) and coefficients of variations below 10%. In a pilot study on AD patients and controls, we verified disease-association with decreased levels of A beta(42) similar to that obtained by ELISA and even better separation was obtained using the A beta(42)/A beta(40) ratio. The developed assay is sensitive and is not influenced by matrix effects, enabling absolute quantification of A beta(42), A beta(40), and A beta(38) in CSF, while it retains the ability to distinguish AD patients from controls. We suggest this SRM-based method for A beta peptide quantification in human CSF valuable for clinical research and trials.
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| 4. |
- Pannee, Josef, 1979, et al.
(författare)
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A selected reaction monitoring (SRM)-based method for absolute quantification of Aβ38, Aβ40, and Aβ42 in cerebrospinal fluid of Alzheimer's Disease patients and healthy controls.
- 2013
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Ingår i: Journal of Alzheimer's Disease. - 1387-2877. ; 33:4, s. 1021-32
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Tidskriftsartikel (refereegranskat)abstract
- Cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) are increasingly used in research centers, clinical trials, and clinical settings. However, their broad-scale use is hampered by lack of standardization across analytical platforms and by interference from binding of amyloid-β (Aβ) to matrix proteins as well as self-aggregation. Here, we report on a matrix effect-resistant method for the measurement of the AD-associated 42 amino acid species of Aβ (Aβ42), together with Aβ40 and Aβ38 in human CSF based on mass spectrometric quantification using selected reaction monitoring (SRM). Samples were prepared by solid-phase extraction and quantification was performed using stable-isotope labeled Aβ peptides as internal standards. The diagnostic performance of the method was evaluated on two independent clinical materials with research volunteers who were cognitively normal and AD patients with mild to moderate dementia. Analytical characteristics of the method include a lower limit of quantification of 62.5 pg/mL for Aβ42 and coefficients of variations below 10%. In a pilot study on AD patients and controls, we verified disease-association with decreased levels of Aβ42 similar to that obtained by ELISA and even better separation was obtained using the Aβ42/Aβ40 ratio. The developed assay is sensitive and is not influenced by matrix effects, enabling absolute quantification of Aβ42, Aβ40, and Aβ38 in CSF, while it retains the ability to distinguish AD patients from controls. We suggest this SRM-based method for Aβ peptide quantification in human CSF valuable for clinical research and trials.
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