The structure of N-linked glycans is determined by a complex, anabolic, intracellular pathway but the exact role of individual glycans is not always clear. Characterization of carbohydrates attached to glycoproteins is essential to aid understanding of this complex area of biology. Specific mass spectral detection of glycopeptides from protein digests may be achieved by on-line HPLC-MS, with selected ion monitoring (SIM) for diagnostic product ions generated by cone voltage fragmentation, or by precursor ion scanning for terminal saccharide product ions, which can yield the same information more rapidly. When glycosylation is heterogeneous, however, these approaches can result in spectra that are complex and poorly resolved. We have developed methodology, based around precursor ion scanning for ions of high m/z, that allows site specific detection and structural characterization of glycans at high sensitivity and resolution. These methods have been developed using the standard glycoprotein, fetuin, and subsequently applied to the analysis of the N-linked glycans attached to the scrapieassociated prion protein, PrP Sc . These glycans are highly heterogeneous and over 30 structures have been identified and characterized site specifically. Product ion spectra have been obtained on many glycopeptides confirming structure assignments. The glycans are highly fucosylated and carry Lewis X or sialyl Lewis X epitopes and the structures are in-line with previous results. [Abbreviations: Hex-Hexose, C 6 H 12 O 6 carbohydrates, including mannnose and galactose; HexNAc-N-acetylhexosamine, C 8 H 15 NO 6 carbohydrates, including N-acetylglucosamine and N-acetylgalactosamine; GlcNAc-N-acetylglucosamine; GalNAc-N-acetylgalactosamine; Fuc-Fucose; NeuAC-N-acetylneuraminic acid or sialic acid; TSE-Transmissible Spongiform Encephalopathy.] (J Am Soc Mass Spectrom 2002Spectrom , 13, 1065Spectrom -1077
A method is developed for measuring collision cross sections of gas-phase biomolecules using a slightly modified commercial triple quadrupele instrument. The modifications allow accurate stopping potentials to be measured for ions exiting the collision region of the instrument. A simple model allows these curves to be converted to cross sections. In order to account for certain poorly defined experimental parameters (exact ion energy, absolute pressure in the collision cell, etc.) variable parameters are included in the model. These parameters are determined on a case by case basis by normalizing the results to the well known cross section of singly charged bradykinin. Two relatively large systems were studied (cytochrome c and myoglobin) so comparisons could be made to literature values. A number of new peptide systems were then studied in the 9 -14 residue range. These included singly and doubly charged ions of luteinizing hormone releasing hormone (LHRH) substance P, and bombesin in addition to bradykinin. The experimental cross sections were in very good agreement with predictions from extensive molecular dynamics modeling. One interesting result was the experimental observation that the cross section of the doubly charged ions of LHRH, substance P, and bombesin were all smaller than those of the corresponding singly charged ions. Molecular dynamics did not reproduce this result, predicting doubly charged cross sections of the same magnitude or slightly larger than for the singly charged species. The experimental results appear to be correct, however. Possible shortcomings in the modeling procedure for multiply charged ions were suggested that might account for the discrepancy. (Int J Mass Spectrom 195/196 (2000) 685-697)
Certain polysulphated polyanions have been shown to have prophylactic effects on the progression of transmissible spongiform encephalopathy disease, presumably because they bind to prion protein (PrP). Until now, the difficulty of obtaining large quantities of native PrP has precluded detailed studies of these interactions. We have over-expressed murine recombinant PrP (recPrP), lacking its glycophosphoinositol membrane anchor, in modified mammalian cells. Milligram quantities of secreted, soluble and partially glycosylated protein were purified under non-denaturing conditions and the identities of mature-length aglycosyl recPrP and two cleavage fragments were determined by electrospray MS. Binding was assessed by surface plasmon resonance techniques using both direct and competitive ligand-binding approaches. recPrP binding to immobilized polyanions was enhanced by divalent metal ions. Polyanion binding was strong and showed complex association and dissociation kinetics that were consistent with ligand-directed recPrP aggregation. The differences in the binding strengths of recPrP to pentosan polysulphate and to other sulphated polyanions were found to parallel their in vivo anti-scrapie and in vitro anti-scrapie-specific PrP formation potencies. When recPrP was immobilized by capture on metal-ion chelates it was found, contrary to expectation, that the addition of polyanions promoted the dissociation of the protein.
The transmissible spongiform encephalopathies are characterized by conversion of a host protein, PrP C (cellular prion protein), to a protease-resistant isoform, PrP Sc (prion protein scrapie isoform). The importance of the highly¯exible, N-terminal region of PrP has recently become more widely appreciated, particularly the biological activities associated with its metal ion-binding domain and its potential to form a poly(L-proline) II (PPII) helix. Circular dichroism spectroscopy of an N-terminal peptide, PrP 37±53 , showed that the PPII helix is formed in aqueous buffer; as it also contains an Xaa±Pro±Gly consensus sequence, it may act as a substrate for the collagenmodifying enzyme prolyl 4-hydroxylase. Direct evidence for this modi®cation was obtained by mass spectrometry and Edman sequencing in recombinant mouse PrP secreted from stably transfected Chinese hamster ovary cells. Almost complete conversion of proline to 4-hydroxyproline occurs speci®cally at residue Pro44 of this murine protein; the same hydroxylated residue was detected, at lower levels, in PrP Sc from the brains of scrapie-infected mice. Cation binding and/or post-translational hydroxylation of this region of PrP may regulate its role in the physiology and pathobiology of the cell.
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