Bengt Bjellqvist

Quantitative membrane proteomics applying narrow range peptide isoelectric focusing for studies of small cell lung cancer resistance mechanisms

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.

Elimination of basic gaps at high pH values in 2‐DE

The appearance of gaps, vertical lanes lacking protein spots at the cathodic end of 2‐D maps generated with wide range IPG‐strips exceeding a pH value of 9, is shown to depend on the electro‐osmotic transport of water into the IPG‐strip. Substitution of urea solution with water is demonstrated to increase the hydrolysis rate of polyacrylamide in IPG‐strips explaining the gap formation. The use of 8 M urea or thiourea/urea solutions in the electrode wick enables overnight focusing without the appearance of gaps.