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LPI™ FlowCell containing immobilized functional membrane proteins can be used for various functional assays. The flow cell format allows for iterate or continuous flow-through of solution containing binders such as proteins, peptides, phage particles, ligands/inhibitors etc. Due to continuous flow, several binders can be studied simultaneously.
In the traditional way of identifying antigen epitopes various softwares predict several epitopes based on hydrophobicity, similarity and statistics. In silico only. These peptides might not be the most accessible for the antibody even though they are located in the extramembrane part of the membrane protein. Using LPI™ FlowCell, peptides exposed in the 3D structure of the protein are digested and the peptide that is the most digestible and detectable is chosen ensuring high specificity and affinity of the antibody.
LPI™ FlowCell is very appropriate for quantification studies of membrane proteins with for example the AQUA method. After membrane protein characterization by LPI™ FlowCell and MS analysis, a signature peptide from the protein of interest is chosen, synthesized and labelled with 13C or 15N to create an AQUA peptide. Using LPI™ FlowCell to create AQUA peptides always ensures a high quality and very detectable peptide, as opposed to in-silico digestion. A known amount of AQUA peptide is added to the biological sample and a trypsination is performed in the LPI™ FlowCell. Both native and labelled tryptic fragments are collected and analysed with LC-MS generating ion chromatograms for both peptides. Using peak area ratios, the quantity of native peptide is calculated.
High protein sequencing can be achieved with LPI™ FlowCell kits due to the benefit of the flow cell format. Immobilized proteins are sequentially digested with several different enzymes without centrifugation steps using a multi-step protocol and a regular pipette. The peptides are at the same time fractionated and a very high sequence coverage is achieved.
LPI™ FlowCells can be used for sample preparation of membrane proteins for dow-stream MS analysis for the profiling studies. Immobilized membrane proteins in a flow cell format makes sample handling easy and efficient and leads to high sensitivity and reproducibility of data. An example is a membrane protein profiling of ANAMMOX bacteria performed on the LPI™ FlowCell. Digestion and LC-MS/MS analysis confirmed the presence of known key proteins and enabled detection of additional proteins previously not described in the literature.
