Large-scale identification of membrane proteins based on analysis of trypsin-protected transmembrane segments.

Integral membrane proteins are generally under-represented in routine proteomic analyses, mostly because of their relatively low abundance, hydrophobicity and lack of trypsin-cleavage sites. To increase the coverage of membrane proteomes, various strategies have been developed, targeting mostly the extra-membrane segments of membrane proteins. We focused our attention to the rather overlooked hydrophobic transmembrane segments. Such peptides can be isolated after carbonate stripping and protease "shaving" of membranes isolated by simple centrifugation procedure. The treated membranes with embedded hydrophobic peptides can then be solubilized in organic solvents, re-digested with CNBr, delipidated and subjected to LC-MS/MS analysis. We modified the original "hppK" method, and applied it for the analysis of human lymphoma cells. We identified 1224 proteins of which two-thirds were IMPs with 1-16 transmembrane segments. This method allowed us to identify 13 "missing proteins" - proteins with no previous evidence on protein level. BIOLOGICAL SIGNIFICANCE: Integral membrane proteins execute numerous essential functions and represent substantial part of eukaryotic proteomes. Our knowledge of their function and expression is, however, limited. Novel approaches extending our knowledge of membrane proteome are therefore highly desired. As we demonstrate here, a non-conventional method which targets rather overlooked hydrophobic transmembrane segments of integral membrane proteins has wide potential to provide the missing information on the membrane proteome. We show that it can deliver identification and potentially also quantification of hundreds of integral membrane proteins including the so called "missing proteins".

Metabolomics and metabolite profiling: past heroes and future developments.

Following the sequencing of the human and other genomes, much research effort is now invested in post- genomic science, particularly in the related disciplines of proteomics and metabolomics. In this paper, we will attempt to provide an overview of mass spectrometry-based metabolomic strategies, discuss the evolution of metabolomics from its predecessor, Hmetabolite profiling", and provide some pointers to future methodological and technological direction. Current data from the authors' laboratory will also be presented, highlighting our efforts in the field of "targeted metabolomics", namely, "steroidomics in the brain".

Two assays for dihydrocodeine in plasma and in urine and their use to determine the bioavailability of a controlled-release product.

A GC method (20 ng/mL quantitation limit) and an HPLC method (37.5 ng/mL quantitation limit) for the analysis of dihydrocodeine in plasma and an HPLC method for dihydrocodeine in urine (2 micrograms/mL quantitation limit) are described. These methods have been used to compare the bioavailability in four subjects of a controlled-release formulation of dihydrocodeine bitartrate (equivalent to 90 mg of base) with that of a solution (equivalent to 3 x 30 mg of base). With each preparation, the mean urinary recovery of dihydrocodeine was approximately 30 mg, and both had a comparable bioavailability. A further study with 12 subjects, each taking the equivalent of 30 mg of base at 12 hourly intervals for 4 d, indicated that the controlled-release formulation produced effective steady-state concentrations of drug whether administered before or after meals, with no accumulation being observed.