| 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. |
- 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.
|
|
