| 1. |
- Eriksson, Hanna, et al.
(författare)
-
Quantitative membrane proteomics applying narrow range peptide isoelectric focusing for studies of small cell lung cancer resistance mechanisms
- 2008
-
Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 28:5C, s. 3275-3276
-
Tidskriftsartikel (refereegranskat)abstract
- Drug resistance is often associated with upregulation of membrane-associated drug-efflux systems, and thus global membrane proteomics methods are valuable tools in the search for novel components of drug resistance phenotypes. Herein we have compared the microsomal proteome from the lung cancer cell line H69 and its isogenic Doxorubicin-resistant subcell line H69AR. The method used includes microsome preparation, iTRAQ labeling followed by narrow range peptide IEF in an immobilized pH-gradient (IPG-IEF) and LC-MS/MS analysis. We demonstrate that the microsomal preparation and iTRAQ labeling is reproducible regarding protein content and composition. The rationale using narrow range peptide IPG-IEF separation is demonstrated by its ability to: (i) lowering the complexity of the sample by two-thirds while keeping high proteome coverage (96%), (ii) providing high separation efficiency, and (iii) allowing for peptide validation and possibly identifications of post-transcriptional modifications. After analyzing one-fifth of the IEF fractions (effective pH range of 4.0-4.5), a total of 3704 proteins were identified, among which 527 were predicted to be membrane proteins. One of the proteins found to be differentially expressed was Serca 2, a calcium pump located in the ER membrane that potentially could result in changes of apoptotic response toward Doxorubicin.
|
|
| 2. |
- Orre, Lukas M., et al.
(författare)
-
FUNCTIONAL STUDIES OF S100A6 USING PROTEOMICS
- 2008
-
Ingår i: Anticancer Research. - : INT INST ANTICANCER RESEARCH. - 0250-7005 .- 1791-7530. ; 28:5C, s. 3430-3431
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
|
|
| 3. |
- Orre, Lukas M., et al.
(författare)
-
p53 is involved in clearance of ionizing radiation-induced RAD51 foci in a human colon cancer cell line
- 2006
-
Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 342:4, s. 1211-7
-
Tidskriftsartikel (refereegranskat)abstract
- We have investigated p53-related differences in cellular response to DNA damaging agents, focusing on p53s effects on RAD51 protein level and sub-cellular localization post exposure to ionizing radiation. In a human colon cancer cell line, HCT116 and its isogenic p53-/- subcell line we show here p53-independent RAD51 foci formation but interestingly the resolution of RAD51 foci showed clear p53 dependence. In p53 wt cells, but not in p53-/- cells, RAD51 protein level decreased 48 h post irradiation and fluorescence immunostaining showed resolution of RAD51 foci and relocalization of RAD51 to nucleoli at time points corresponding to the decrease in RAD51 protein level. Both cell lines rejoined DNA double strand breaks efficiently with similar kinetics and p53 status did not influence sensitivity to DNA damaging agents. We suggest that p53 has a role in RAD51 clearance post DSB repair and that nucleoli might be sites of RAD51 protein degradation.
|
|
| 4. |
- Pernemalm, Maria, et al.
(författare)
-
Evaluation of three principally different intact protein prefractionation methods for plasma biomarker discovery.
- 2008
-
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 7:7, s. 2712-2722
-
Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to evaluate three principally different top-down protein prefractionation methods for plasma: high-abundance protein depletion, size fractionation and peptide ligand affinity beads, focusing in particular on compatibility with downstream analysis, reproducibility and analytical depth. Our data clearly demonstrates the benefit of high-abundance protein depletion. However, MS/MS analysis of the proteins eluted from the high-abundance protein depletion column show that more proteins than aimed for are removed and, in addition, that the depletion efficacy varies between the different high-abundance proteins. Although a smaller number of proteins were identified per fraction using the peptide ligand affinity beads, this technique showed to be both robust and versatile. Size fractionation, as performed in this study, focusing on the low molecular weight proteome using a combination of gel filtration chromatography and molecular weight cutoff filters, showed limitations in the molecular weight cutoff precision leading detection of high molecular weight proteins and, in the case of the cutoff filters, high variability. GeLC-MS/MS analysis of the fractionation methods in combination with pathway analysis demonstrates that increased fractionation primarily leads to high proteome coverage of pathways related to biological functions of plasma, such as acute phase reaction, complement cascade and coagulation. Further, the prefractionation methods in this study induces limited effect on the proportion of tissue proteins detected, thereby highlighting the importance of extensive or targeted downstream fractionation.
|
|
| 5. |
- Stranneheim, Henrik, et al.
(författare)
-
A comparison between protein profiles of B cell subpopulations and mantle cell lymphoma cells
- 2009
-
Ingår i: Proteome Science. - : Springer Science and Business Media LLC. - 1477-5956. ; 7, s. 43-
-
Tidskriftsartikel (refereegranskat)abstract
- Background: B-cell lymphomas are thought to reflect different stages of B-cell maturation. Based on cytogenetics and molecular markers, mantle cell lymphoma (MCL) is presumed to derive predominantly from naive, pre-germinal centre (pre-GC) B lymphocytes. The aim of this study was to develop a method to investigate the similarity between MCL cells and different B-cell compartments on a protein expression level. Methods: Subpopulations of B cells representing the germinal centre (GC), the pre-GC mantle zone and the post-GC marginal zone were isolated from tonsils using automated magnetic cell sorting (AutoMACS) of cells based on their expression of CD27 and IgD. Protein profiling of the B cell subsets, of cell lines representing different lymphomas and of primary MCL samples was performed using top-down proteomics profiling by surface-enhanced laser detection/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Results: Quantitative MS data of significant protein peaks (p-value < 0.05) separating the three B-cell subpopulations were generated. Together, hierarchical clustering and principal component analysis (PCA) showed that the primary MCL samples clustered together with the pre- and post-GC subpopulations. Both primary MCL cells and MCL cell lines were clearly separated from the B cells representing the GC compartment. Conclusion: AutoMACS sorting generates sufficient purity to enable a comparison between protein profiles of B cell subpopulations and malignant B lymphocytes applying SELDI-TOF-MS. Further validation with an increased number of patient samples and identification of differentially expressed proteins would enable a search for possible treatment targets that are expressed during the early development of MCL.
|
|
