A three-pronged "Pitchfork" strategy enables an extensive description of the human membrane proteome and the...

Integral membrane proteins are under-represented in standard proteomic analyses, mostly because of their low expression and absence of trypsin-cleavage sites in their hydrophobic transmembrane segments. Novel and effective strategies for membrane proteomic analysis aim at soluble N-glycosylated segments of integral membrane proteins (CSC, SPEG, N-glyco-FASP) or selectively target the hydrophobic transmembrane alpha-helical segments employing chemical peptide cleavage by CNBr (hpTC). We combined a solid phase enrichment of glycopeptides (SPEG) with a transmembrane segment-oriented hpTC method and a standard "detergent and trypsin" approach into a three-pronged "Pitchfork" strategy to maximize the membrane proteome coverage in human lymphoma cells. This strategy enabled the identification of >1200 integral membrane proteins from all cellular compartments, including 105 CD antigens, 24 G protein-coupled receptors, and 141 solute carrier transporters. The advantage of the combination lies in the complementarity of the methods. SPEG and hpTC target different sets of membrane proteins. HpTC provided identifications of proteins and peptides with significantly higher hydrophobicity compared to SPEG and detergent-trypsin approaches. Among all identified proteins, we observed 32 so-called "missing proteins". The Pitchfork strategy presented here is universally applicable and enables deep and fast description of membrane proteomes in only 3 LC-MS/MS runs per replicate. SIGNIFICANCE: Integral membrane proteins (IMPs) are encoded by roughly a quarter of human coding genes. Their functions and their specific localization makes IMPs highly attractive drug targets. In fact, roughly half of the currently approved drugs in medicine target IMPs. Our knowledge of membrane proteomes is, however, limited. We present a new strategy for the membrane proteome analysis that combines three complementary methods targeting different features of IMPs. Using the combined strategy, we identified over 1200 IMPs in human lymphoma tissue from all sub-cellular compartments in only 3 LC-MS/MS runs per replicate. The three-pronged "Pitchfork" strategy is universally applicable, and offers a fast way toward a reasonably concise description of membrane proteomes in multiple samples.