The proteome of normal male urine from a commercial pooled source has been examined using direct liquid chromatography-tandem mass spectrometry (LC-MS/MS). The entire urinary protein mixture was denatured, reduced and enzymatically digested prior to LC-MS/MS analysis using a hybrid-quadrupole time-of-flight mass spectrometer (Q-TOF) to perform data-dependent ion selection and fragmentation. To fragment as many peptides as possible, the mixture was analyzed four separate times, with the mass spectrometer selecting ions for fragmentation from a subset of the entire mass range for each run. This approach requires only an autosampler on the HPLC for automation (i.e, unattended operation). Across these four analyses, 1.450 peptide MS/MS spectra were matched to 751 sequences to identify 124 gene products (proteins and translations of expressed sequence tags). Interestingly, the experimental time for these analyses was less than that required to run a single two-dimensional gel.

No abstract

With an emphasis on obtaining a multitude of high quality tandem mass spectrometry spectra for protein identification, instrumental parameters are described for the liquid chromatography-tandem mass spectrometry analysis of trypsin digested unfractionated urine using a hybrid quadrupole-time-of-flight (Q-TOF) mass spectrometer. Precursor acquisition rates of up to 20 distinct precursors/minute in a single analysis were obtained through the use of parallel precursor selection (four precursors/survey period) and variable collision induced dissociation integration time (1 to 6 periods summed). Maximal exploitation of the gas phase fractionated ions was obtained through the use of narrow survey scans and iterative data-dependent analyses incorporating dynamic exclusion. The impact on data fidelity as a product of data-dependent selection of precursor ions from a dynamically excluded field is discussed with regards to sample complexity, precursor selection rates, survey scan range and facile chemical modifications. Operational and post-analysis strategies are presented to restore data confidence and reconcile the greatest number of matched spectra.

Soluble CD14 (sCD14) is a 55-kDa serum protein that binds lipopolysaccharide (LPS) and mediates LPS-dependent responses in a variety of cells. Using recombinant sCD14 expressed in Chinese hamster ovary (CHO) cells, we examined the structural characteristics of sCD14 and sCD14.LPS complexes. The circular dichroism and fluorescence spectra of the sCD14 indicate that it contains substantial beta-sheet (40%) and a well-defined tertiary structure with the tryptophan residues located in environments with different degrees of hydrophobicity and solvent exposure. The spectra of the sCD14.LPS complex are identical within experimental error to the uncomplexed sCD14. Changes in surface accessibility upon LPS binding were examined using limited proteolysis with endoproteinase Asp-N. This analysis revealed that aspartic acid residues at amino acids 57, 59, and 65 are susceptible to cleavage by Asp-N, while the same residues are protected from proteolytic cleavage in the sCD14.LPS complex. These results suggest that a region including amino acids 57 to 64 is involved in LPS binding by sCD14.

and the ‡ ‡Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada N-Linked glycosylation is a post-translational modification occurring in many eukaryotic secreted and surface-bound proteins and has impact on diverse physiological and pathological processes. Similarly important is the generation of glycosylphosphatidylinositol linkers, which anchor membrane proteins to the cell. Both protein modifications depend on the central nucleotide sugar UDP-N-acetylglucosamine (UDP-GlcNAc). The enzymatic reactions leading to generation of nucleotide sugars are established, yet most of the respective genes still await cloning. We describe the characterization of such a gene, EMeg32, which we identified based on its differential expression in murine hematopoietic precursor cells. We further demonstrate regulated expression during embryogenesis. EMeg32 codes for a 184-amino acid protein exhibiting glucosamine-6-phosphate acetyltransferase activity. It thereby holds a key position in the pathway toward de novo UDP-GlcNAc synthesis. Surprisingly, the protein associates with the cytoplasmic side of various intracellular membranes, accumulates prior to mitosis, and copurifies with the cdc48 homolog p97/valosin-containing protein.Asparagine (N)-linked oligosaccharides are components of most secreted and surface-bound proteins of mammalian cells. Carbohydrate structures on cell surface glycoproteins are instrumental for cell-cell or cell-matrix interactions, immune reactions and tumor development, whereas sugar modifications on secreted proteins are important for their transport, their biological activity and their clearance from the circulation (1, 2).The diversity of sugar units within complex or hybrid Nlinked oligosaccharides is generated by the controlled action of specific glycosyltransferases. The process starts in the endoplasmic reticulum (ER) 1 with the transfer of an oligomannose precursor from the lipid carrier dolichol phosphate to asparagines within the nascent protein chain (3). Maturation of the oligomannose precursor occurs during passage of the attached protein through cis-, medial, and trans-Golgi and is mediated by several trimming enzymes (e.g. mannosidases) and glycosyltransferases (for a review, see Ref. 2).At the basis of each N-linked carbohydrate is N-acetylglucosamine (GlcNAc), whose initial transfer to dolichol phosphate is achieved in the form of its nucleotide sugar donor UDPGlcNAc. GlcNAc not only constitutes the first aminosugar residue linked to asparagine in all N-glycosylated proteins, but also serves as an important module in generating complex or hybrid N-linked oligosaccharide structures as well as O-linked oligosaccharides (3). In addition, a number of proteoglycans contain GlcNAc linked to the respective core protein or other aminosugar residues. It also serves in generating glycosylphosphatidylinositol (GPI) linkers, which are responsible for anchoring a variety of cell surface molecules to the plasma membrane (4 -6). The initial reaction of GPI assembly (the transfer of GlcNAc fro...