The largest category of proteins are involved in ion transport (30%) and include the likes of excitatory amino acid transporter 1 and Na+/K+ ATPase ( subunit). to the lightest region of the gradient as seen with the fully glycosylated Nervana isoforms (observe Figure ?Physique3A).3A). C – Fractionation by two phase affinity partitioning following an initial density gradient fractionation and probed with anti-HRP. The most prominent band behaves the same way as Nervana (observe Figure ?Physique6B)6B) and probably em is /em Nervana (see [28]). An overexposure of the final ConA eluate (ConA over) is included to show that other anti-HRP detectable proteins are also present in the PM portion. em Labeling /em : same as figure ?physique33 for any and B and physique ?figure44 for C. em Loading /em : Equivalent amounts of all fractions were loaded. 1471-2164-11-302-S1.PDF (4.9M) GUID:?04318DC0-C683-473B-8EDD-5AE5FB3A6A3C Additional file 2 MudPIT identification of proteins purified by combination of density gradient centrifugation and 2PAP from em Drosophila /em head microsomes: This table includes a list of all the proteins purified by our optimized protocol and recognized with 95% confidence. The sub-cellular compartment Anethol in which each protein can be found is indicated, along with the quantity of peptides recognized. For single-peptide identifications, the sequence, precursor m/z and score of the peptide have been provided. Cross-references to Additional Files 3 and 4 are also included. 1471-2164-11-302-S2.XLS (146K) GUID:?A0BCB994-6389-4C17-A9B2-56BACCB2539A Additional file 3 Spectra for proteins recognized by single-peptide hit: This table provides the matched peptide and spectrum for all those single-peptide identifications. 1471-2164-11-302-S3.PDF (14M) GUID:?D0109ABB-4E6E-4FB6-92A2-5E7B3F39E044 Additional file 4 Hydropathy plots Anethol for proteins predicted to have transmembrane domains: This table provides the hydropathy plots of all those proteins predicted to have transmembrane domains by the method of Kyte and Doolittle. 1471-2164-11-302-S4.PDF (1.0M) GUID:?3C09C7E5-5DB0-45E5-AFDD-C2BF7D35F41A Additional file 5 Functional categorisation of proteins identified as residents of the plasma membrane: This table classifies the plasma membrane proteins outlined in Additional File 2 on the basis of their cellular function. 1471-2164-11-302-S5.XLS (47K) GUID:?6FB6B433-AD92-4C06-AAF4-D53E4B4F0B76 Abstract Background The plasma membrane (PM) is a compartment of significant interest because cell surface proteins influence the way in which a cell interacts with its neighbours and its extracellular environment. However, PM is Anethol usually hard to isolate because of its low large quantity. Aqueous two-phase affinity purification (2PAP), based on PEG/Dextran two-phase fractionation and lectin affinity for PM-derived microsomes, is an emerging method for the isolation of high purity plasma membranes from several vertebrate sources. In contrast, PM isolation techniques in important invertebrate genetic model systems, such as Rabbit Polyclonal to CCRL1 em Drosophila melanogaster /em , have relied upon enrichment by density gradient centrifugation. To facilitate genetic investigation of activities contributing to the content of the PM sub-proteome, we sought to adapt 2PAP to this invertebrate model to provide a strong PM isolation technique for em Drosophila /em . Results We show that 2PAP alone does not completely remove contaminating endoplasmic reticulum and mitochondrial membrane. However, a novel combination of density gradient centrifugation plus 2PAP results in a strong PM preparation. To demonstrate the utility of this technique we isolated PM from travel heads and successfully recognized 432 proteins using MudPIT, of which 37% are integral membrane proteins from all compartments. Of the 432 proteins, 22% have been previously assigned to the PM compartment, and a further 34% are currently unassigned to any compartment and represent candidates for assignment to the PM. The remainder have previous assignments to other compartments. Conclusion A combination of density gradient centrifugation and 2PAP results in a strong, high purity PM preparation from em Drosophila /em , something neither technique can achieve on its own. This novel preparation should lay the groundwork for the proteomic investigation of the PM in different genetic backgrounds in em Drosophila /em . Our results also identify two key actions in this procedure: The optimization of membrane partitioning in the PEG/Dextran combination, and careful choice of the correct lectin for the affinity purification step in light of variations in bulk membrane lipid composition and glycosylation patterns respectively. This points the way for further adaptations into other systems. Background The plasma membrane (PM) and its associated proteins play an important role in determining how a cell interacts with its neighbours as well as how it responds to components of, and conditions in its extracellular environment. As a reflection of this, more than 50% of the current drug targets lie at the cell surface [1]. The amount of a protein at the cell surface is determined by its rate of delivery, internalization, recycling and degradation. All these parameters are subject to change during normal physiological adjustments, development, varying environmental influences and pathological conditions [2]. Obviously, to monitor such changes em via /em total protein level, when the surface pool is the active Anethol populace, would mask important regulatory changes that.