A Combination of Immobilised pH Gradients Improves Membrane Proteomics

Membrane protein analyses have been notoriously difficult due to hydrophobicity and the general low abundance of these proteins compared to their soluble cytosolic counterparts. Shotgun proteomics has become the preferred method for analyses of membrane proteins, in particular the recent development of peptide immobilized pH gradient isoelectric focusing (IPG-IEF) as the first dimension of two-dimensional shotgun proteomics. Recently, peptide IPG-IEF has been shown to be a valuable shotgun proteomics technique through the use of acidic narrow range IPG strips, which demonstrated that small acidic pI increments are rich in peptides. In this study, we assess the utility of both broad range (BR) (pI 3−10) and narrow range (NR) (pI 3.4−4.9) IPG strips for rat liver membrane protein analyses. Furthermore, the use of these IPG strips was evaluated using label-free quantitation to demonstrate that the identification of a subset of proteins can be improved using NR IPG strips. NR IPG strips provided 2603 protein assignments on average (with 826 integral membrane proteins (IMPs)) compared to BR IPG strips, which provided 2021 protein assignments on average (with 712 IMPs). Nonredundant protein analysis demonstrated that in total from all experiments, 4195 proteins (with 1301 IMPs) could be identified with 1428 of these proteins unique to NR IPG strips with only 636 from BR IPG strips. With the use of label-free quantitation methods, 1659 proteins were used for quantitative comparison of which 319 demonstrated statistically significant increases in normalized spectral abundance factors (NSAF) in NR IPG strips compared to 364 in BR IPG strips. In particular, a selection of six highly hydrophobic transmembrane proteins was observed to increase in NSAF using NR IPG strips. These results provide evidence for the use of alternative pH gradients in combination to improve the shotgun proteomic analysis of the membrane proteome.