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Technology

The Lipid-based Protein Immobilization technology (LPI™) enables immobilization of native membrane proteins in the form of proteoliposomes, generated directly from cells or tissue. The membrane proteins can be subjected to a variety of protocols in a highly controlled manner, to provide effective and precise preparation of samples for downstream analysis. This technology is based on the use of proprietary surfaces and sample preparation protocols designed to retain the natural environment around the membrane proteins. The LPI technology utilizes detergent-free sample preparation prior to the characterization of membrane proteins with, e.g., mass spectrometry, liquid chromatography or UV detection. LPI SamplePrep kit is the first product based on the LPI technology and is optimized for mass spectrometry analysis of membrane proteins. The general workflow is shown below in Figure 1. The technology can be used for a wide range of applications relating to membrane proteins, including membrane profiling, target identification, expression profiling, associated protein identification and protein subunit identification.

Figure 1: Workflow for LPI SamplePrep kit. The product is intended for preparation of molecular species derived from native or artificial lipid bilayer membranes. The membranes must be supplied to LPI FlowCell as proteoliposomes, prepared by methods such as extrusion or sonication. LPI FlowCell accepts membrane vesicles derived from synthetic hosts, mammalian cell lines, bacteria and yeast, as well as organelle-derived vesicles. After deposition of the protein-carrying vesicles on the platform, multiple washing steps and reagent-addition rounds can be carried out without the loss or dilution of the sample. Protein digestion products, i.e. peptides, are then recovered, ready for sample processing such as LC/MS.

The LPI technology is based on 15 years world-renowned research by Owe Orwars research group where biophysics, biochemistry and bioanalytical chemistry converge into a common theme: the use of lipid membranes and cell membranes to elucidate the function and properties of boundaries in cells. The research has a strong emphasis on lipids and lipid membranes, surface chemistry, surface interactions, microfluidics, and characterization of membrane proteins.

Key features of the LPI SamplePrep kit:

• The lipid bilayer format ensures that the structure and function of all embedded proteins and other membrane-bound species is maintained.

• Protocols are free of gels and detergents.

• Solid-phase immobilization allows for protocol versatility; multiple rounds of chemical treatments are possible.

• The ability to carry out multiple rounds of enzymatic digestions gives better sequence coverage than conventional methods (Figure 2).

• Sample dilution as a result of the kit is minimal.

• Enzymatic digestions are completed sooner than with standard protocols allowing for rapid sample turnover.

• The method offers high sensitivity and reproducibility; low-abundance membrane proteins including those present at 500 copies per cell can be detected.

• The LPI technology reduces costs by decreasing the number of MS analyses by a factor of 5-10 compared to in-gel digestions.

*Erythrocyte Anion exchanger
** Steps 1 and 2 shows peptides cleaved by pepsin and trypsin and step 3 shows the combined result

Figure 2: Better sequence coverage with the LPI SamplePrep kit. 85% coverage of Red blood cell AE1* was observed with Multi-Step Digestion.

 Examples of samples used on the LPI SamplePrep kit:

• Skeletal muscle tissue

• Red blood cells

• Anammox bacteria

• Stem cells

• Insect cells

• Jurkat cells

• E.Coli bacteria

• Mast cells

• Breast carcinoma cells

LPI FlowCell specifications:

• Flow cell volume: 350 µl

• Minimum starting material: 10x106 cells

• Optimal sample concentration: 0.1-1 mg/ml total lipid/protein

• Flow cell binding capacity: ~100 µg proteoliposomes

• Optimal vesicle size: 50-150 nm in diameter

Application examples:

• Expression profiling of stem cells
  Expression profiling of differentiated versus undifferentiated cells. 120 proteins, of which 112 were classified as membrane-associated, were identified in a membrane fraction. Initial semi-quantitative results indicated differences in the membrane protein profiles and gave leads towards several differentiation markers.
   

• Novel proteins in ANAMMOX bacteria
  Membrane protein profiling of ANAMMOX bacteria, specifically proteins involved in energy conversion situated in an organelle-like structure called the anammoxosome. LPI digestion confirmed the presence of known key proteins and enabled detection of additional proteins previously not described in the literature.
   

• Biomarkers in breast cancer cells
  Membrane protein profiling of breast cancer cells, grown under different conditions - normal (normoxi) and low O₂ levels (hypoxi). Roughly 550 proteins were identified in the preparation from cells grown at low O₂. Interest lies in finding differences in the two profiles, thus giving leads towards biomarkers.
   

• In-depth membrane profiling of RBC
  Membrane profiling of red blood cells (RBC), specifically integral membrane proteins. High salt and high pH washes were used in order to remove associated proteins. Approximately 130 membrane proteins were identified, with abundance levels from millions of copies per cell down to a couple of hundred copies per cell (Figure 3). This is currently one of the most comprehensive analyses made of the RBC membrane proteome.

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