We present a novel microfluidic solid-phase extraction (μSPE) device for the affinity enrichment of biotinylated membrane proteins from whole cell lysates. and was comprised of 3 600 micropillars. Activation of the PMMA micropillars by UV/O3 treatment permitted generation of surface-confined carboxylic acid groups and the covalent attachment of NeutrAvidin onto the μSPE device surfaces which was used to affinity select biotinylated MCF-7 Tyrphostin AG 879 membrane proteins directly from whole cell lysates. The inclusion of a disulfide linker within the biotin Tyrphostin AG 879 moiety permitted release of the isolated membrane proteins via DTT incubation. Very low levels (~20 fmol) of membrane proteins could be isolated and recovered with ~89% effectiveness having a bed capacity of 1 1.7 pmol. Western blotting indicated no traces of cytosolic proteins in the membrane protein fraction as compared to significant contamination using a commercial detergent-based method. We highlight long term avenues for enhanced extraction effectiveness and increased dynamic range of the μSPE device using computational simulations of different micropillar geometries to guide future device designs. Intro Membrane proteins play important tasks in the pathology and physiology of biological cells including regulating the trafficking of ions and solutes in/out of the cell cell-to-cell relationships and reactions to stimuli through surface receptors.1 Specific modifications to membrane proteins have been linked to different pathologic claims such as tumor neurological disorders and diabetes.2 Because of the interest in discovering and validating disease-specific protein signatures with diagnostic value or discovering fresh drug focuses on for personalized therapeutics studies aimed at the identification characterization and quantification of membrane proteins has increased over the past few years. Most notably several biopharmaceuticals that target membrane proteins are already becoming utilized for the treatment of tumors lymphomas and autoimmune diseases.3 Membrane proteins symbolize approximately one-third of all proteins encoded from the human being genome.4 5 Yet only a small fraction of the cell surface proteome has been characterized due to analytical difficulties including: (i) Low abundance especially compared to the cytosolic proteins;1 6 (ii) low frequency of tryptic cleavage sites in transmembrane domains;7 (iii) the heterogeneity of membrane proteins; and (iv) their hydrophobicity making them prone to precipitation and aggregation and thus sensitive to solubilization.7 8 A number of analytical approaches have been developed to aid in the analysis of membrane proteins for example ultracentrifugation 9 10 affinity selection of revised or non-modified membrane proteins (antibody- or lectin-based approaches) 11 12 two-phase partitioning13 14 and extraction.15 16 For example detergent-based membrane protein recovery has been demonstrated to be as efficient as >90%; however this effectiveness was demonstrated for any mitochondrial membrane protein and recovery of a plasma membrane protein was only 50%.17 Two important issues are apparent: (i) It is imperative to specifically isolate plasma membrane proteins as transmission pathways must be stimulated by external connection;18 and (ii) the effectiveness of Tyrphostin AG 879 detergent isolation intimately depends on the membrane protein’s difficulty and hydrophobicity thereby imparting Rabbit Polyclonal to Histone H2B. variability in extraction efficiency.17 In general the majority of detergent methods fail to produce highly pure isolates of membrane proteins due in large part to contamination from cytosolic proteins.18 Alternatively affinity-based isolation of membrane proteins avoids such variability and has the potential to target plasma membrane proteins specifically. Approaches utilizing antibodies for affinity isolation are challenged by the fact that the appropriate antibodies must be available for the necessary targets; one runs the risk of neglecting portions of the membrane proteome.19 Tyrphostin AG 879 Recently improved techniques for the enrichment of membrane proteins both employed streptavidin-coated magnetic beads to enrich plasma membrane proteins that were acquired by lysing biotinylated cells from a human lung carcinoma cell line. The method resulted in a 400-fold enrichment of plasma membrane proteins relative to the Tyrphostin AG 879 endoplasmic reticulum which was a major contaminant in the membrane portion dramatically reduced.