| 1. |
- Antberg, Linn, et al.
(författare)
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Critical Comparison of Multidimensional Separation Methods for Increasing Protein Expression Coverage
- 2012
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 11:5, s. 2644-2652
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Tidskriftsartikel (refereegranskat)abstract
- We present a comparison of two-dimensional separation methods and how they affect the degree of coverage of protein expression in complex mixtures. We investigated the relative merits of various protein and peptide separations prior to acidic reversed-phase chromatography directly coupled to an ion trap mass spectrometer. The first dimensions investigated were density gradient organelle fractionation of cell extracts, 1D SDS-PAGE protein separation followed by digestion by trypsin or GluC proteases, strong cation exchange chromatography, and off-gel isoelectric focusing of tryptic peptides. The number of fractions from each first dimension and the total data accumulation RP-HPLC-MS/MS time was kept constant and the experiments were run in triplicate. We find that the most critical parameters are the data accumulation time, which defines the level of under-sampling and the avoidance of peptides from high expression level proteins eluting over the entire gradient.
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| 2. |
- Cifani, Paolo, et al.
(författare)
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An efficient geometric method for incompressible hydrodynamics on the sphere
- 2023
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Ingår i: Journal of Computational Physics. - : Elsevier BV. - 1090-2716 .- 0021-9991. ; 473
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Tidskriftsartikel (refereegranskat)abstract
- We present an efficient and highly scalable geometric numerical method for two-dimensional ideal fluid dynamics on the sphere. The starting point is Zeitlin's finite-dimensional model of hydrodynamics. The efficiency stems from exploiting a tridiagonal splitting of the discrete spherical Laplacian combined with highly optimized, scalable numerical algorithms. For time-stepping, we adopt a recently developed isospectral integrator able to preserve the geometric structure of Euler's equations, in particular conservation of the Casimir functions. To overcome previous computational bottlenecks, we formulate the matrix Lie algebra basis through a sequence of tridiagonal eigenvalue problems, efficiently solved by well-established linear algebra libraries. The same tridiagonal splitting allows for computation of the stream matrix, involving the inverse Laplacian, for which we design an efficient parallel implementation on distributed memory systems. The resulting overall computational complexity is O(N3) per time-step for N2 spatial degrees of freedom. The dominating computational cost is matrix-matrix multiplication, carried out via the parallel library ScaLAPACK. Scaling tests show approximately linear scaling up to around 2500 cores for the matrix size N=4096 with a computational time per time-step of about 0.55 seconds. These results allow for long-time simulations and the gathering of statistical quantities while simultaneously conserving the Casimir functions. We illustrate the developed algorithm for Euler's equations at the resolution N=2048.
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| 3. |
- Cifani, Paolo
(författare)
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Building a map of the breast cancer proteome - Strategies to increase coverage
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Amongst the various –omics sciences, proteomics has the highest potential for functional characterization and consequently can contribute significantly to the field of cancer research. In particular, the focus of this thesis is on breast cancer. Alas, since state-of-the-art technologies cannot meet the complexity of upper eukaryotic proteomes, a complete resolution of clinical samples is still unachievable. Comprehensive mapping of proteins involved in cancer and of their PTMs is proposed in this thesis as a general strategy to increase the output of mass-spectrometry based proteomics. Different approaches to improve the coverage of this map are proposed: optimization of sample fractionation, focusing on difficult sub-proteomes, targeting of specific biological processes and optimization of data analysis. A combination of these approaches will provide a growing collection of empirical MS-spectra, which will enhance the detection by shotgun proteomics and facilitate the transition towards the development of targeted assays.
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| 4. |
- Cifani, Paolo, et al.
(författare)
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Hunting for Protein Markers of Hypoxia by Combining Plasma Membrane Enrichment with a New Approach to Membrane Protein Analysis
- 2011
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 10:4, s. 1645-1656
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Tidskriftsartikel (refereegranskat)abstract
- Nontransient hypoxia is strongly associated with malignant lesions, resulting in aggressive behavior and resistance to treatment. We present an analysis of mRNA and protein expression changes in neuroblastoma cell lines occurring upon the transition from normoxia to hypoxia. The correlation between mRNA and protein level changes was poor, although some known hypoxia-driven genes and proteins correlated well. We present previously undescribed membrane proteins expressed under hypoxic conditions that are candidates for evaluation as biomarkers.
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| 5. |
- Cifani, Paolo, et al.
(författare)
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Molecular Portrait of Breast-Cancer-Derived Cell Lines Reveals Poor Similarity with Tumors.
- 2015
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 14:7, s. 2819-2827
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Tidskriftsartikel (refereegranskat)abstract
- Breast-cancer-derived cell lines are an important sample source for cancer proteomics and can be classified on the basis of transcriptomic analysis into subgroups corresponding to the molecular subtypes observed in mammary tumors. This study describes a tridimensional fractionation method that allows high sequence coverage and proteome-wide estimation of protein expression levels. This workflow has been used to conduct an in-depth quantitative proteomic survey of five breast cancer cell lines matching all major cancer subgroups and shows that despite their different classification, these cell lines display a very high level of similarity. A proteome-wide comparison with the RNA levels observed in the same samples showed very little to no correlation. Finally, we demonstrate that the proteomes of in vitro models of breast cancer display surprisingly little overlap with those of clinical samples.
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| 6. |
- Franchin, Cinzia, et al.
(författare)
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Quantitative analysis of a phosphoproteome readily altered by the protein kinase CK2 inhibitor quinalizarin in HEK-293T cells.
- 2015
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002. ; 1854:6, s. 609-623
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Tidskriftsartikel (refereegranskat)abstract
- CK2 is an extremely pleiotropic Ser/Thr protein kinase, responsible for the generation of a large proportion of the human phosphoproteome and implicated in a wide variety of biological functions. CK2 plays a global role as an anti-apoptotic agent, a property which is believed to partially account for the addiction of many cancer cells to high CK2 levels. To gain information about the CK2 targets whose phosphorylation is primarily implicated in its pro-survival signaling advantage has been taken of quinalizarin (QZ) a cell permeable fairly specific CK2 inhibitor, previously shown to be able to block endogenous CK2 triggering an apoptotic response. HEK-293T cells either treated or not for 3h with 50μM QZ were exploited to perform a quantitative SILAC phosphoproteomic analysis of phosphosites readily responsive to QZ treatment. Our analysis led to the identification of 4883 phosphosites, belonging to 1693 phosphoproteins. 71 phosphosites (belonging to 47 proteins) underwent a 50% or more decreased occupancy upon QZ treatment. Almost 50% of these fulfilled the typical consensus sequence recognized by CK2 (S/T-x-x-E/D/pS) and in several cases were validated as bona fide substrates of CK2 either based on data in the literature or by performing in vitro phosphorylation experiments with purified proteins. The majority of the remaining phosphosites drastically decreased upon QZ treatment display the pS/T-P motif typical of proline directed protein kinases and a web logo extracted from them differentiates from the web logo extracted from all the proline directed phosphosites quantified during our analysis (1151 altogether). A paradoxical outcome of our study was the detection of 116 phosphosites (belonging to 92 proteins altogether) whose occupancy is substantially increased (50% or more), rather than decreased by QZ treatment: 40% of these display the typical motif recognized by proline directed kinases, while about 25% fulfill the CK2 consensus. Collectively taken our data on one side have led to the disclosure of a subset of CK2 targets which are likely to be implicated in the early steps of CK2 signaling counteracting apoptosis, on the other they provide evidence for the existence of side and off-target effects of the CK2 inhibitor quinalizarin, paving the road toward the detection of other kinases susceptible to this compound. This article is part of a Special Issue entitled: Medical Proteomics.
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| 7. |
- Kirik, Ufuk, et al.
(författare)
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Multimodel Pathway Enrichment Methods for Functional Evaluation of Expression Regulation.
- 2012
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 11:5, s. 2955-2967
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Tidskriftsartikel (refereegranskat)abstract
- Functional analysis of quantitative expression data is becoming common practice within the proteomics and transcriptomics fields; however, a gold standard for this type of analysis has yet not emerged. To grasp the systemic changes in biological systems, efficient and robust methods are needed for data analysis following expression regulation experiments. We discuss several conceptual and practical challenges potentially hindering the emergence of such methods and present a novel method, called FEvER, that utilizes two enrichment models in parallel. We also present analysis of three disparate differential expression data sets using our method and compare our results to other established methods. With many useful features such as pathway hierarchy overview, we believe the FEvER method and its software implementation will provide a useful tool for peers in the field of proteomics. Furthermore, we show that the method is also applicable to other types of expression data.
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| 8. |
- Stella, Roberto, et al.
(författare)
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Relative Quantification of Membrane Proteins in Wild-Type and Prion Protein (PrP)-Knockout Cerebellar Granule Neurons
- 2012
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 11:2, s. 523-536
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Tidskriftsartikel (refereegranskat)abstract
- Approximately 25% of eukaryotic proteins possessing homology to at least two trans membrane domains are predicted to be embedded in biological membranes. Nevertheless, this group of proteins is not usually well represented in proteome-wide experiments due to their refractory nature. Here we present a quantitative mass spectrometry-based comparison of membrane protein expression in cerebellar granule neurons grown in primary culture that were isolated from wild-type mice and mice lacking the cellular prion protein. This protein is a cell-surface glycoprotein that is mainly expressed in the central nervous system and is involved in several neurodegenerative disorders, though its physiological role is unclear. We used a low specificity enzyme a-chymotrypsin to digest membrane proteins preparations that had been separated by SDS-PAGE. The resulting peptides were labeled with tandem mass tags and analyzed by MS. The differentially expressed proteins identified using this approach were further analyzed by multiple reaction monitoring to confirm the expression level changes.
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| 9. |
- Waldemarson, Sofia, et al.
(författare)
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Proteomic analysis of breast tumors confirms the mRNA intrinsic molecular subtypes using different classifiers : A large-scale analysis of fresh frozen tissue samples
- 2016
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Ingår i: Breast Cancer Research. - : Springer Science and Business Media LLC. - 1465-5411 .- 1465-542X. ; 18:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Breast cancer is a complex and heterogeneous disease that is usually characterized by histological parameters such as tumor size, cellular arrangements/rearrangments, necrosis, nuclear grade and the mitotic index, leading to a set of around twenty subtypes. Together with clinical markers such as hormone receptor status, this classification has considerable prognostic value but there is a large variation in patient response to therapy. Gene expression profiling has provided molecular profiles characteristic of distinct subtypes of breast cancer that reflect the divergent cellular origins and degree of progression. Methods: Here we present a large-scale proteomic and transcriptomic profiling study of 477 sporadic and hereditary breast cancer tumors with matching mRNA expression analysis. Unsupervised hierarchal clustering was performed and selected proteins from large-scale tandem mass spectrometry (MS/MS) analysis were transferred into a highly multiplexed targeted selected reaction monitoring assay to classify tumors using a hierarchal cluster and support vector machine with leave one out cross-validation. Results: The subgroups formed upon unsupervised clustering agree very well with groups found at transcriptional level; however, the classifiers (genes or their respective protein products) differ almost entirely between the two datasets. In-depth analysis shows clear differences in pathways unique to each type, which may lie behind their different clinical outcomes. Targeted mass spectrometry analysis and supervised clustering correlate very well with subgroups determined by RNA classification and show convincing agreement with clinical parameters. Conclusions: This work demonstrates the merits of protein expression profiling for breast cancer stratification. These findings have important implications for the use of genomics and expression analysis for the prediction of protein expression, such as receptor status and drug target expression. The highly multiplexed MS assay is easily implemented in standard clinical chemistry practice, allowing rapid and cheap characterization of tumor tissue suitable for directing the choice of treatment.
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