Electron capture dissociation at 86 K of the linear peptide Substance P produced just two backbone fragments, whereas at room temperature eight backbone fragments were formed. Similarly, with the cyclic peptide gramicidin S, just one backbone fragment was formed at 86 K but five at room temperature. The observation that some backbone scissions are active and others inactive, when all involve N™C ␣ cleavages and have a high rate constant, indicates that the more specific fragments at low temperatures reflects the reduced conformation heterogeneity at low temperatures. This is supported by reduced or inactive hydrogen loss, a channel that has previously been shown to be affected by conformation. The conclusion that the ECD fragments are a snapshot of the conformational (intramolecular solvation shell) heterogeneity helps explain how the relative intensities of ECD fragments can be different on different instrument and highlights the common theme in methodologies used to increase sequence coverage, namely an increase in the conformational heterogeneity of the precursor ion population. ( ploys reactions of multiprotonated peptides/ proteins with low energy electrons to generate peptide fragments. The high sequence coverage and the ability to retain labile groups after ECD have allowed posttranslational modifications (PTMs) and point mutations (PMs) in peptides and proteins to be both identified and localized. Such performance is of great value for the analysis of the proteome, particularly that of diseased organisms. PTMs and PMs are widespread and are frequently associated with disease [5][6][7], for example over 80 different point mutations have been found in transthyretin (most of which lead to autosomal disorders) [8].To be able to fully characterize such modified proteins, 100% sequence coverage is required (so-called top-down proteomics). Several methods have been developed to increase the sequence coverage of ECD, which can be grouped into either infrared illumination [9 -11] or collisional activation [12]. These methods increase the average internal energy of the ions, which also affects the conformation of these gas-phase ions [13].There is experimental and theoretical evidence that ECD is directed by the internal solvation of the (neutralized) proton [9, 14 -18]. If the reaction progresses faster than the electron-proton recombination energy is randomized and thermal fluctuations are small, specific fragments would be expected from a single (frozen) conformer, reflecting the solvation shell, specific for that conformation, surrounding the neutralized proton. Thermal fluctuations that result in a dynamic solvation shell, but not conformational change (local fluctuations rather than a global change), would produce a greater number of fragments. Finally, multiple conformations as well as thermal fluctuations would produce yet more fragments. Because the methods used to increase the sequence coverage of ECD all increase the internal energy of the ions, and so affect the conformation of the gas-phase ions, the...
Phosphorylation of proteins is essential in intracellular signal transduction pathways in eukaryotic and prokaryotic cells. Histidine phosphorylation plays an important role in two-component signal transduction in bacteria. In this study, we describe the characterization of a synthetic histidine-phosphorylated peptide with four different mass spectrometric (MS) fragmentation techniques: Collision-induced dissociation (CID), electron capture dissociation, electron-transfer dissociation, and electron detachment dissociation. Furthermore, LC-MS methods were developed to detect histidine-phosphorylated peptides, which are acid-labile, in more complex samples. From these results, we concluded that nonacidic solvent systems or fast LC methods provide the best conditions for separation of histidine-phosphorylated peptides prior to electrospray ionization mass spectrometry analysis. Electron-based fragmentation methods should be used for determination of histidine phosphorylation sites, since CID results in very facile phosphate-related neutral losses. The developed LC-MS/MS methods were successfully applied to a tryptic digest of the cytoplasmic part of the histidine kinase EnvZ, which was in vitro autophosphorylated. Finally, a new method is described for nonretentive solid-phase extraction of histidine-phosphorylated peptides using polymeric Strata-X microcolumns.
The phosphatidylcholine transfer protein (PC-TP) is a specific transporter of phosphatidylcholine (PC) between membranes. To get more insight into its physiological function, we have studied the localization of PC-TP by microinjection of fluorescently labeled PC-TP in foetal bovine heart endothelial (FBHE) cells and by expression of an enhanced yellow fluorescent protein-PC-TP fusion protein in FBHE cells, human umbilical vein endothelial cells, and HepG2 cells. Analysis by confocal laser scanning microscopy showed that PC-TP was evenly distributed throughout the cytosol with an apparently elevated level in nuclei. By measuring the fluorescence recovery after bleaching it was established that PC-TP is highly mobile throughout the cell, with its transport into the nucleus being hindered by the nuclear envelope. Given the proposed function of PC-TP in lipid metabolism, we have tested a number of compounds (phorbol ester, bombesin, A23187, thrombin, dibutyryl cyclic AMP, oleate, clofibrate, platelet-derived growth factor, epidermal growth factor, and hydrogen peroxide) for their ability to affect intracellular PC-TP distribution. Only clofibrate (100 M) was found to have an effect, with PC-TP moving to mitochondria within 5 min of stimulation. This relocation did not occur with PC-TP(S110A), lacking the putative protein kinase C (PKC)-dependent phosphorylation site, and was restricted to the primary endothelial cells. Relocation did not occur in HepG2 cells, possibly due to the fact that clofibrate does not induce PKC activation in these cells.
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