A new relatlonshlp between Ion mass and effective cyclotron frequency Is derived for Ions stored In a cublc cell and detected by uslng Fourler transform mass spectrometry. An assessment of colllslonal damping on mass measurement error Is also made. I t Is concluded that frequency perturbation by colllslonal damping, whlle predlcted by the model, Is negllglble at sufflclently low pressure. The mass callbration law is tested at a magnetlc fleld of 1.2 T by uslng major fragment Ions of 1,1,1,2-tetrachloroethane. WHh broad band "chirp" excltatlon of Ions, systematic rather than random errors were dlscovered. The magnitude of these systematic errors increased as the number of Ions Qtored In the cell was Increased. However, H Is predlcted from the calibration law that errors will decrease wHh the square of the magnetic field strength.The Fourier transform mass spectrometer (FTMS) (1) is recognized as a potentially useful analytical instrument because it is capable of high mass range and ultrahigh mass resolution. The demonstration of high resolving power with the FTMS, however, has predated by several years the development of methodology for exact mass measurement and elemental composition assignment.Early attempts to produce a mass calibration scheme sufficiently accurate for elemental composition assignments were not successful. For example, Ledford and McIver (2) reported measurement accuracy ranging from 7 ppm to 151 ppm over the mass range m/z 47 to m/z 264 using an ICR mass spectrometer with electrometer detection. The mass errors were systematic, caused by changes in space charge conditions in the analyzer cell as ions were sequentially observed. Comisarow (3) reported mass measurement accuracy ranging from 0.3 ppm to 80 ppm over the mass range m/z 69 to m / i 1166. The relative measurement errors were found to increase systematically with mass.Mass measurement accuracy of the a few parts per million or less (often sufficient for elemental composition assignment) was achieved in more recent studies. Ledford ( 4 ) et al. investigated mass measurement using a parabolic mass calibration law for cubic analyzer cells. Over a 4 m u mass range, errors of 0.8 ppm were typical, while average errors of 2 ppm over an 18 amu mass range were obtained when a three-parameter fit was used. Wanczek and Allemann (5) reported a novel side-band method for measuring the masses of trapped ions. Errors averaging 1.5 ppm were obtained over the mass range m J z 18 to m/z 170 amu.Although these latter methods (4,5) of calibration represent improvements over earlier work ( 2 , 3 ) , they have not proved to be routinely useful for exact mass determination. One reason is that ion space charge in the analyzer cell affects observed frequencies, and this must be accounted for in accurate measurements. Ledford et al. (4) recognized this in their efforts to develop a mass calibration scheme. They demonstrated that frequency shifts associated with changes in space charge were qualitatively similar to those caused by changes in trap voltage. Whit...
Fast photochemical oxidation of proteins (FPOP) is a chemical footprinting method whereby exposed amino-acid residues are covalently labeled by oxidation with hydroxyl radicals produced by the photolysis of hydrogen peroxide. Modified residues can be detected by standard trypsin proteolysis followed by LC/MS/MS, providing information about solvent accessibility at the peptide and even the amino-acid level. Like other chemical footprinting techniques, FPOP must ensure only the native conformation is labeled. Although oxidation via hydroxyl radical induces unfolding in proteins on a timescale of milliseconds or longer, FPOP is designed to limit •OH exposure to 1 μs or less by employing a pulsed laser for initiation to produce the radicals and a radical-scavenger to limit their lifetimes. We applied FPOP to three oxidation-sensitive proteins and found that the distribution of modification (oxidation) states is Poisson when a scavenger is present, consistent with a single conformation protein modification model. This model breaks down when a scavenger is not used and/or hydrogen peroxide is not removed following photolysis. The outcome verifies that FPOP occurs on a time scale faster than conformational changes in these proteins.
Protein-ligand binding and the concomitant conformational change in the protein are of crucial importance in biophysics and drug design. We report a novel method to quantify protein-ligand interactions in solution by mass spectrometry, titration, and H/D exchange (PLIMSTEX). The approach can determine the conformational change, binding stoichiometry, and affinity in protein-ligand interactions including those that involve small molecules, metal ions, and peptides. Binding constants obtained by PLIMSTEX for four model protein-ligand systems agree with K values measured by conventional methods. At higher protein concentration, the method can be used to determine quickly the binding stoichiometry and possibly the purity of proteins. Taking advantage of concentrating the protein on-column and desalting, we are able to use different concentrations of proteins, buffer systems, salts, and pH in the exchange protocol. High picomole quantities of proteins are sufficient, offering significantly better sensitivity than that of NMR and X-ray crystallography. Automation could make PLIMSTEX a high throughput method for library screening, drug discovery, and proteomics.
Probing the conformational changes of amyloid beta (Aβ) peptide aggregation is challenging owing to the vast heterogeneity of the resulting soluble aggregates. To investigate the formation of these aggregates in solution, we designed an MS-based biophysical approach and applied it to the formation of soluble aggregates of the Aβ 42 peptide, the proposed causative agent in Alzheimer's disease. The approach incorporates pulsed hydrogen-deuterium exchange coupled with MS analysis. The combined approach provides evidence for a self-catalyzed aggregation with a lag phase, as observed previously by fluorescence methods. Unlike those approaches, pulsed hydrogen-deuterium exchange does not require modified Aβ 42 (e.g., labeling with a fluorophore). Furthermore, the approach reveals that the center region of Aβ 42 is first to aggregate, followed by the C and N termini. We also found that the lag phase in the aggregation of soluble species is affected by temperature and Cu 2+ ions. This MS approach has sufficient structural resolution to allow interrogation of Aβ aggregation in physiologically relevant environments. This platform should be generally useful for investigating the aggregation of other amyloid-forming proteins and neurotoxic soluble peptide aggregates.soluble Aβ oligomers | amyloid beta peptide | copper | electrospray ionization | Finke-Watsky mode P rotein aggregation is one of the immediate causes of Alzheimer's, Parkinson, and Huntington diseases, motivating biophysical studies of the responsible proteins. More than 20 small proteins undergo amyloidosis in humans. In Alzheimer's disease (AD), the aggregation of the 40-or 42-aa-long amyloid beta (Aβ) peptide, generally called Aβ 40 or Aβ 42 , respectively, is proposed to be involved in the onset of the disease (1, 2). Aβ 42 is more amyloidogenic and more neurotoxic than Aβ 40 . Although the amyloid-cascade hypothesis suggests that the Aβ-containing amyloid plaques are responsible for neurodegeneration (3-7), other studies suggest that soluble aggregates of Aβ 42 are more neurotoxic than the amyloid plaques (8-13).The amyloid plaques in AD-affected brains contain high levels of copper, zinc, and iron (14-20). Among these, Cu has drawn the most attention because the Aβ precursor protein is likely a Cuchaperone protein (21). Several studies of Cu 2+ -Aβ 40 interactions show that Cu 2+ can promote Aβ 40 aggregation (14,18,19).The structure of Aβ 42 and its aggregates, although studied extensively, remains of high interest. Studies of amyloid fibrils invoke X-ray crystallography (22-24), EM (19,25,26), and thioflavin T fluorescence (19, 27), revealing the polypeptide's global behavior, whereas NMR studies provide residue-level information for the fibrils (28-30). Nevertheless, we know little about soluble Aβ aggregates owing to their intrinsically high heterogeneity.MS should offer an opportunity for investigating soluble aggregates of Aβ 42 . Thus far, there are no MS-based, timedependent studies of the formation of soluble aggregates. Moreover, there are no ot...
Protein arginine deiminases (PADs) are calcium-dependent histone-modifying enzymes whose activity is dysregulated in inflammatory diseases and cancer. PAD2 functions as an Estrogen Receptor (ER) coactivator in breast cancer cells via the citrullination of histone tail arginine residues at ER binding sites. Although an attractive therapeutic target, the mechanisms that regulate PAD2 activity are largely unknown, especially the detailed role of how calcium facilitates enzyme activation. To gain insights into these regulatory processes, we determined the first structures of PAD2 (27 in total), and through calcium-titrations by X-ray crystallography, determined the order of binding and affinity for the six calcium ions that bind and activate this enzyme. These structures also identified several PAD2 regulatory elements, including a calcium switch that controls proper positioning of the catalytic cysteine residue, and a novel active site shielding mechanism. Additional biochemical and mass-spectrometry-based hydrogen/deuterium exchange studies support these structural findings. The identification of multiple intermediate calcium-bound structures along the PAD2 activation pathway provides critical insights that will aid the development of allosteric inhibitors targeting the PADs.
The capability for accurate mass measurements is an important attribute of Fourier transform mass spectrometry (FTMS). Unlike other instrumental methods in mass spectrometry, FTMS still offers significant opportunities to improve mass measurement accuracy (MMA), making it an area of research. This review covers the published literature in FTMS from the late 1970s to the present. We discuss the development and evolution of mass calibration that give accuracies in the low ppm range. We sketch the derivation and show the common foundation of these mass calibration procedures. We also describe the relation of mass calibration and the fundamentals of ion motion and space-charge effects, and we review efforts to improve the basic calibration procedure particularly those that correct for effects of space charge. The advent of matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) have opened the door for FTMS to be used for analyzing at high performance biopolymers, including proteins, oligodeoxynucleotides (ODNs), oligosaccharides, and synthetic polymers. We also discuss the utility of FTMS for accurate mass measurement in these areas and some practical ways to improve MMA. # 2004 Wiley Periodicals, Inc., Mass Spec Rev 24:286-309, 2005
We report a study of sub-millisecond protein folding with amino-acid residue resolution achieved with a two-laser pump/probe experiment with analysis by mass spectrometry. The folding of a test protein, barstar, can be triggered by a laserinduced temperature jump (T jump) from ~0 °C to ~ room temperature. Subsequent reactions via FPOP (fast photochemical oxidation of proteins) at various fractional millisecond points after the T jump leads to oxidative modification of solvent-accessible side chains whose “protection” changes with time and extent of folding. The modifications are identified and quantified by LC-MS/MS following proteolysis. Among all the segments that form secondary structure in the native state, helix1 shows a decreasing trend of oxidative modification during the first 0.1-1 ms of folding while others do not change in this time range. Residues I5, H17, L20, L24 and F74 are modified less in the intermediate state than the denatured state, likely due to full or partial protection of these residues as folding occurs. We propose that in the early folding stage, barstar forms a partially solvent-accessible hydrophobic core consisting of several residues that have long-range interaction with other, more remote residues in the protein sequence. Our data not only are consistent with the previous conclusion that barstar fast folding follows the nucleation-condensation mechanism with the nucleus centered on helix1 formed in a folding intermediate but also show the efficacy of this new approach to following protein folding on the sub millisecond time range.
We report a new mass spectrometry-based approach to study protein-folding dynamics at the sub millisecond time. The strategy couples temperature jump with fast photochemical oxidation of proteins (FPOP) whereby folding/unfolding is followed by changes in oxidative modifications by OH radical reactions. Using a flow system containing the protein barstar as a model, we altered the protein's equilibrium conformation by temperature jump and demonstrated that its reactivity with OH free radicals serves as a reporter of the conformational change. Furthermore, we found that the time-dependent increase in mass, owing to free-radical oxidation, is a measure of the rate constant for the transition from the unfolded to the first intermediate state. This advance offers the promise that, when extended with mass-spectrometry based proteomic analysis, the sites and kinetics of folding/unfolding can also be followed at sub millisecond times.Deciphering protein folding is essential for understanding biological processes and developing therapeutic approaches to misfolding-related diseases 1-3 . Protein folding can be followed in equilibrium experiments, which monitor protein states as a function of temperature or denaturant concentration, and in kinetics, to give a time-dependent conformational change.Starting in the early 1990s, mass spectrometry (MS) emerged as an effective tool for supporting both thermodynamic and kinetic protein-folding studies. The advantage of modern MS is its ability to measure extents of protein modification and pinpoint their locations. For pulsed H/D amide exchange and other pulsed covalent labeling kinetics 4, 5 , MS detection can now track folding events down to the millisecond timescale. Protein folding, however, often occurs more rapidly (in microseconds or even less 6 ), which has been a difficult range to access in current MS-based studies.Here we describe a new approach to investigate protein folding. We use two lasers, one to provide a temperature-jump (T-jump) and a second to generate reagents to footprint the consequences of the T-jump. This is an example of "pump/probe" 7, 8 , but it is distinguished by the use of chemical reactions as the structural probe rather than the usual spectroscopic approaches. To our knowledge, the first irreversible labeling accompanying protein conformational change was demonstrated by Jha and Udgaonkar 9 , who explored protein folding on the msec timescale by combining mutagenesis, pulsed thio-labeling and global protein analysis by MS. Recently, Konermann et al. combined rapid mixing and fast photochemical oxidation of proteins (FPOP) to observe protein unfolding 10 /folding 11 intermediates at times in the 10 ms range. Our approach takes advantage of FPOP, developed in our laboratory 12 , whereby pulsed laser-photolysis yields hydroxyl radicals that mgross@wustl.edu. NIH Public AccessAuthor Manuscript J Am Chem Soc. Author manuscript; available in PMC 2011 November 10. We chose barstar (10.3 kDa) as a test protein because it is denatured at 0 °C and fo...
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