This application note clearly demonstrates the benefits of using the LPI™ FlowCell in a two-step digestion protocol using a shave-and-conquer approach. New peptides are identified in the second digestion step leading to improved sequence coverage, an increased number of confident protein identifications and a completely new set of proteins not detected in the first digestion step. Membrane protein vesicles are immobilized on the surfaces of LPI™ Maxi FlowCell, where the flow cell design allows for easy exchange of solutions containing e.g. proteases. This feature is highly suitable for obtaining multiple complementary peptide fractions from a single sample, greatly enhancing the protein sequence information obtainable from a membrane preparation analysis.
This study demonstrates the use of Nanoxis LPI™ FlowCell in conjunction with LC-MS/MS to profile the protein content of a plasma membrane preparation derived from Arabidopsis thaliana, with respect to identity, membrane association types and subcellular locations. A total of 249 proteins were identified, there of 138 proteins (55%) were classed as membrane associated. Subcellular localization analysis of the membrane proteins revealed that the dominant membrane compartment represented was the plasma membrane (88).