Recent developments in ion-trap mass spectrometry and related technologies

Due to their versatility, quadrupole ion traps have become popular mass spectrometers in the growing field of proteomics. High sensitivity, user friendliness and low cost are the key features that have contributed to the success of the technology. However, mass measurement accuracy, resolution and mass range are still not comparable to the analytical performances obtained on other mass spectrometers. In the past 5 years, researchers have tried to overcome these drawbacks, focusing their attention on two different aspects of ion-trap mass spectrometry, development of novel types of ion traps and manipulation of the gas-phase ion chemistry, in order to obtain alternative techniques for tandem mass spectrometry analysis. In the field of trapping devices, improvements in instrumental design have led to the linear ion trap, digital ion trap and orbitrap. Activation methods based on electrons, chemically produced by an anion or from irradiation with an electron beam, have demonstrated their utility in providing complementary sequence information for improving confidence in protein identification.     

Direct sequence analysis of proteins by in-source fragmentation during delayed ion extraction.

Continuous segments of amino acid sequence information as long as 41 residues have been deduced by interpretation of matrix-assisted laser desorption/ionization-generated ion signals dominated by Cn fragmentation within the ion source of a linear time-of-flight mass spectrometer utilizing delayed ion extraction. The technique has been applied successively to five proteins of mass 12.2 kDa to 18.3 kDa, yielding segments of continuous sequence as long as 41 residues without the need for prior proteolytic fragmentation. Intact crosslinks such as disulfides or heme linkages interrupt the generation of these data.     

On optimal size and number of reserves for metapopulation persistence

Habitat fragmentation is generally considered to be detrimental to the persistence of natural populations. In nature management, one therefore tends to prefer few large nature reserves over many small nature reserves having equal total area. This paper examines whether this preference is warranted in a metapopulation framework with circular reserves (patches) by formulating the dependence of metapopulation persistence on the size and number of reserves, both of which depend on reserve radius if the total area is kept constant. Two measures of metapopulation persistence are used: R(0), the number of patches colonized by an occupied patch during its lifetime as an occupied patch, and T(e), the expected time to extinction. These two measures are functions of the extinction and colonization rates of the metapopulation. Several mechanisms for the extinction and colonization processes are formulated from which the dependence of these rates on reserve radius is calculated. It turns out that T(e)generally increases with reserve radius for all mechanisms, which supports the preference of few large reserves. However, R(0)supports this preference only in the case of some special, rather unrealistic, mechanisms. In many other, more realistic, cases an intermediate reserve size exists for which metapopulation persistence measured by R(0)is optimal.     

An improved chemical approach toward the C-terminal sequence analysis of proteins containing all natural amino acids.

An improved chemical method, capable of derivatizing all natural amino acids to their corresponding thiohydantoins, is described. This involves activation by acetyl chloride in TFA followed by derivatization with ammonium thiocyanate. Possible interference of reactive side chains was investigated by reacting N-acetylamino acids as well as several peptides with propionyl chloride instead of acetyl chloride. The products were characterized by PDMS mass spectrometry and 1H-NMR. This chemical method allows, for the first time, complete derivatization of N-acetylproline to proline thiohydantoin. Applying this chemistry to peptides with a C-terminal proline, the yields for formation of proline thiohydantoin were found to be up to 60%, depending on the peptide sequence. The previous inability to derivatize C-terminal proline to thiohydantoin was thought to stem from the fact that proline cannot form the oxazolonium ion required for efficient reaction with the thiocyanate ion. However, we have found mass spectrometric evidence for the existence of a proline oxazolonium ion, under basic as well as under acidic conditions. This improvement in derivatization of C-terminal amino acids including proline is a major step forward in the development of a general chemical C-terminal sequencing method that permits the C-terminal sequence analysis of proteins of any amino acid composition.     

The use of a hybrid linear trap/FT-ICR mass spectrometer for on-line high resolution/high mass accuracy bottom-up sequencing.

In this work we present a hybrid linear trap/Fourier transform ion cyclotron resonance (ICR) mass spectrometer to perform protein sequencing using the bottom-up approach. We demonstrate that incorporation of the linear trap greatly enhances the overall performance of the hybrid system for the study of complex peptide mixtures separated by fast high-performance liquid chromatography gradients. The ability to detect in the linear trap enables employment of automatic gain control to greatly reduce space charging in the ICR cell irregardless of ion flux. Resulting accurate mass measurements of 2 ppm or better using external calibration are achieved for the base peak as well as ions at 2% relative abundance. The linear trap is used to perform ion accumulation and activation prior to detection in the ICR cell which increases the scan rate. The increased duty cycle allows for data-dependent mass analysis of coeluting peptides to be acquired increasing protein sequence coverage without increasing the gradient length. In addition, the linear trap could be used as an ion detection device to perform simultaneous detection of tandem mass spectra with full scan mass spectral detection in the ICR cell resulting in the fastest scan cycles for performing bottom-up sequencing of protein digests. Comparisons of protein sequence coverage are presented for product ion detection in the linear trap and ICR cell.     

Intact protein analysis for site-directed mutagenesis overexpression products: plasmid-encoded R67 dihydrofolate reductase

Mass spectrometry is currently the method of choice for the analysis of recombinant protein expression products. By combining proteolytic digestion with peptide mapping and tandem mass spectrometry techniques, verification of site-directed mutagenesis products can be obtained. The proteolytic digestion step converts a purified recombinant protein into a mixture that must be reseparated, thus greatly increasing the analysis time associated with the confirmation of site-directed mutagenesis products. Ion/ion reaction chemistry combined with quadrupole ion trap mass spectrometry provides a fast and efficient way to analyze intact proteins for the correct site-directed mutagenesis products, without heavy reliance on the proteolytic digestion step. Analysis of a series of protein variants (I68M, I68Q, Y69F, and Q67Y) from plasmid-encoded R67 dihydrofolate reductase using ion/ion reaction chemistry confirmed the presence of the correct site-directed mutagenesis products. For the I68M mutant, ion/ion separations detected the presence of extensive degradation from the N-terminal end of the protein. In the case of the Q67Y mutant, a mixture of Q67Y and Q67C species was detected by employing tandem mass spectrometry combined with ion/ion reactions. The ion/ion reaction technique was also performed on a partially purified lysate of the Q67Y/C mixture and successfully screened for the presence of both components in a complex mixture. The ion/ion reaction approach achieved the same results as the proteolytic-digestion-based methodology in a much shorter analysis time.     

Locating and identifying posttranslational modifications by in-source decay during MALDI-TOF mass spectrometry

A technique is described for identifying and locating posttranslational modifications (PTMs) in peptides and proteins of known sequence by interpretation of c(n) ion signals generated by in-source decay during delayed ion extraction in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Sites of phosphorylation in seven synthetic peptides were determined, as was the location of both the heme group and N,N,N-trimethyllysine in yeast cytochrome c. A semi-automated data analysis process facilitates the identification of segments of the sequence on each side of the PTM, permitting its placement at the junction of the segments and definition of the added mass. A graphical display facilitates illustration of both the location and mass of the PTM.     

A hydrodynamic theory of ion conductance through Ohmic pores

A method of calculating the size of membrane pores lacking strong ionic selectivity is presented. By treating the flow of ions through a small channel as a hydrodynamic phenomenon, the electrical conductance becomes a function of the ratio of ion radius to channel radius. Thus when both the channel conductance and the ion size are known, the radius of the channel may be estimated. The method gives good agreement among radii predicted from conductances of four different alkali cations in alamethicin pores.     

Sequential abundant ion fragmentation analysis (SAIFA): an alternative approach for phosphopeptide identification using an ion trap mass spectrometer

Phosphorylation has been the most studied of all the posttranslational modifications of proteins. Mass spectrometry has emerged as a powerful tool for phosphomapping on proteins/peptides. Collision-induced dissociation (CID) of phosphopeptides leads to the loss of phosphoric or metaphosphoric acid as a neutral molecule, giving an intense neutral loss product ion in the mass spectrum. Dissociation of the neutral loss product ion identifies peptide sequence. This method of data-dependent constant neutral loss (DDNL) scanning analysis has been commonly used for mapping phosphopeptides. However, preferential losses of groups other than phosphate are frequently observed during CID of phosphopeptides. Ions that result from such losses are not identified during DDNL analysis due to predetermined scanning for phosphate loss. In this study, we describe an alternative approach for improved identification of phosphopeptides by sequential abundant ion fragmentation analysis (SAIFA). In this approach, there is no predetermined neutral loss molecule, thereby undergoing sequential fragmentation of abundant peak, irrespective of the moiety lost during CID. In addition to improved phosphomapping, the method increases the sequence coverage of the proteins identified, thereby increasing the confidence of protein identification. To the best of our knowledge, this is the first report to use SAIFA for phosphopeptide identification.     

Top-down MS, a powerful complement to the high capabilities of proteolysis proteomics

For the characterization of protein sequences and post-translational modifications by MS, the 'top-down' proteomics approach utilizes molecular and fragment ion mass data obtained by ionizing and dissociating a protein in the mass spectrometer. This requires more complex instrumentation and methodology than the far more widely used 'bottom-up' approach, which instead uses such data of peptides from the protein's digestion, but the top-down data are far more specific. The ESI MS spectrum of a 14 protein mixture provides full separation of its molecular ions for MS/MS dissociation of the individual components. False-positive rates for the identification of proteins are far lower with the top-down approach, and quantitation of multiply modified isomers is more efficient. Bottom-up proteolysis destroys the information on the size of the protein and the connectivities of the peptide fragments, but it has no size limit for protein digestion. In contrast, the top-down approach has a approximately 500 residue, approximately 50 kDa limitation for the extensive molecular ion dissociation required. Basic studies indicate that this molecular ion intractability arises from greatly strengthened electrostatic interactions, such as hydrogen bonding, in the gas-phase molecular ions. This limit is now greatly extended by variable thermal and collisional activation just after electrospray ('prefolding dissociation'). This process can cleave 287 inter-residue bonds in the termini of a 1314 residue (144 kDa) protein, specify previously unidentified disulfide bonds between eight of 27 cysteines in a 1714 residue (200 kDa) protein, and correct sequence predictions in two proteins, one of 2153 residues (229 kDa).     

Microscopic model for selective permeation in ion channels.

Ionic permeation in the selectivity filter of ion channels is analyzed by a microscopic model based on molecular kinetic theory. The energy and flux equations are derived by assuming that: (a) the selectivity filter is formed by a symmetrical array of carbonyl groups; (b) ion movement is near the axis of the channel; (c) a fraction of water molecules is separated from the ion while it moves across the selectivity filter; (d) the applied voltage drops linearly across the selectivity filter; (e) ions move independently. Energy profiles, single channel conductances, and the degree of hydration of K+ in a hypothetical K+ channel are examined by varying the following microscopic parameters: ion radius and mass, channel radius, number of effective water dipoles, and number of carbonyl groups. The i-V curve is linear up to +/- 170 mV. If the positions of energy maxima and minima are fixed, this linear range is reduced to +/- 50 mV. Channel radius and ion-water interactions are found to be two major channel structural determinants for selectivity sequences. Both radius and mass of an ion are important in selectivity mediated by these interactions. The theory predicts a total of 15 possible kinetic selectivity sequences for alkali cations in ion channels with a single selectivity filter.     

Osmotic lysis of corpora cardiaca using distilled water reveals the presence of partially processed peptides and peptide fragments

Abstract. A simple, single-step aqueous extraction method has been developed to study the neuropeptide content of small neuroendocrine organs. Perifusion of these tissues with deionized water causes osmotic bursting of the cells and release of their content into the surrounding fluid. The neuropeptides are immediately retained from the perifusion fluid using disposable C18 cartridges. After one separation step and mass spectrometry, it was possible to identify a large number of known neuropeptides from the corpora cardiaca of Locusta migratoria (L). Also present in the extract were a number of neuropeptide fragments and two incompletely processed peptides. Using this method, a 959Da peptide present in the corpora cardiaca was sequenced de novo . The full sequence, deduced using Collision Induced Dissociation Tandem Mass Spectrometry (CID MS/MS), is Ser-Pro-Leu-Asp-Ala-His-His-Leu-Ala. This nonapeptide is predicted from the gene encoding the ion transport peptide precursor and from the gene encoding the ion transport-like peptide precursor. In both cases, this nonapeptide, which was named ion transport peptide-copeptide, is flanked by the signal sequence at the N -terminus and a dibasic cleavage site (Lys-Arg) at the C -terminus. This structural feature is common to many physiologically important locust preproneuropeptides and indicates that this copeptide might have a physiological function, but this is not yet known.     

Allometry and curvature in the long bones of quadrupedal mammals

The allometric relationships between basic structural proportions in long bones are examined in the humerus, radius, femur and tibia for a diverse group of 42 terrestrial quadrupedal mammals that span a size range from 0.02–6000 kg. Non-linear scaling is found for length vs. diameter in the tibia and radius, suggesting that the mechanical constraints on the skeleton differ within large and small body-size mammals. Curvature normalized to mid-shaft radius scales differently in the different long bones. Curvature is poorly related to size in the proximal limb bones (humerus and femur) while it decreases systematically with size in the tibia (mass exponent −0.13). The scaling of normalized curvature in the radius is unique among long bones. Variability of curvature in the radius is reduced at any size in comparison to that found in the other long bones. Normalized curvature is constant within the small body size group (0.02 to approximately 100 kg) while it decreases sharply with size within animals over 100 kg body mass. The unusual scaling found in the radius is probably the result of this bone's close alignment with the extrinsic forces which act on it during locomotion. The change in scaling within the radius for animals of different size may be indicative of more general size-dependent mechanical trade-offs which are masked by the complex loading circumstances of the other long bones.     

东亚钳蝎蝎毒素BmKBT基因组序列的克隆及其分析

东亚钳蝎 (ButhusmartensiiKarsch ,BmK)蝎毒素BmKBT(又名BmKabT)是一个在初级结构上相似于β类哺乳动物毒素和功能接近于α类哺乳动物毒素的Na+ 通道毒素 .基于从毒腺cDNA文库中筛选得到的全长BmKBT前体核苷酸序列设计引物 ,以蝎基因组总DNA为模板进行聚合酶链式反应 (PCR) ,将PCR产物克隆至T载体、测序 .序列分析表明 :在BmKBT信号肽编码区的 3′端的- 4位Gly密码子的第 1位与第 2位碱基中有 1个长 2 2 5nt的内含子 ,插入位点距离该基因的起始密码子 4 6nt ,AT含量为 78 7% ,其内含子可能的剪接分枝位点距离 3′剪接受体位点 4 7nt.内含子的大小及其基因组织结构分析表明 :BmKBT具有与α类哺乳动物毒素类似的基因组织结构 ,进一步说明BmKBT是一个介于α类和β类Na+ 通道毒素之间的中间型蝎毒素 ,可以作为研究蝎毒素分子进化的合适材料     

Advanced Organic Electrode Materials for Rechargeable Sodium‐Ion Batteries

Benefiting from the high abundance and low cost of sodium resource, rechargeable sodium‐ion batteries (SIBs) are regarded as promising candidates for large‐scale electrochemical energy storage and conversion. Due to the heavier mass and larger radius of Na⁺ than that of Li⁺, SIBs with inorganic electrode materials are currently plagued with low capacity and insufficient cycling life. In comparison, organic electrode materials display the advantages of structure designability, high capacity and low limitation of cationic radius. However, organic electrode materials also encounter issues such as high‐solubility in electrolyte and low conductivity. Here, recently reported organic electrode materials, which mainly include the reactions based on either carbon‐oxygen double bond or carbon‐nitrogen double bond, and doping reactions, are systematically reviewed. Furthermore, the design strategies of organic electrodes are comprehensively summarized. The working voltage is regulated through controlling the lowest unoccupied molecular orbital energies. The theoretical capacity can be enhanced by increasing the active groups. The dissolution is inhibited with elevating the intermolecular forces with proper molecular weight. The conductivity can be improved with extending conjugated structures. Future research into organic electrodes should focus on the development of full SIBs with aqueous/aprotic electrolytes and long cycling stability.     

Ion hydration in nanopores and the molecular basis of selectivity

Using a simple model, it is shown that the cost of constraining a hydrated potassium ion inside a narrow pore is smaller than the cost of constraining hydrated sodium or lithium ions in pores of radius around 1.5 A. The opposite is true for pores of radius around 2.5 A. The reason for the selectivity in the first region is that the potassium ion allows for a greater distortion of its hydration shell and can therefore maintain a better coordination, and the reason for the reverse selectivity in the second region is that the smaller ions retain their hydration shells in these pores. This is relevant to the molecular basis of ion selective channels, and since this mechanism does not depend on the molecular details of the pore, it could also operate in all sorts of nanotubes.     

Allometric relationship of postmolt net ion uptake, ventilation, and circulation in the freshwater crayfish Procambarus clarkii: intraspecific scaling

There are few intraspecific studies relating physiological parameters to body mass. This study relates scaling of ionic regulation and respiratory parameters with body mass in crayfish (Procambarus clarkii). These animals were chosen because of their direct development, spanning four orders of magnitude in body mass. Usually, these animals are hyperregulators and must maintain hemolymph electrolyte levels above those in the ambient freshwater. This is especially important in the postmolt, when ion imbalance can occur. Maintaining hemolymph ion levels above ambient involves active processes that are independently related to metabolic rate, ventilation, and circulation. Therefore, this study investigates relationships among size and ionic regulation, heart rate, and ventilation in crayfish, spanning a size range of 0.003-24 g. Postmolt net ion uptake of Ca, titratable base, Na, Cl, and NH4 increase with body mass (positive allometry) with slopes of 0.92, 0.79, 0.90, 0.84, and 0.87, respectively. Between 72% and 97% of variation in ionic regulation was related to body mass. The slopes differed from each other for Ca and titratable base but not for Na, Cl, and NH4. For heart rate and ventilation rate, different relationships were derived for animals smaller and larger than 0.01 g (between first and third instar). Animals larger than 0.01 g show a negative allometric relationship between heart rate and body size ([body mass](0.15)), while smaller animals show positive allometry with body size, but only 29% of variation in heart rate is explained by body size alone. For ventilation rates, the negative allometry with body size for animals larger than 0.01 g is present, but less than 15% of variation in ventilation rate is explained by size, while for smaller animals the size dependency disappears. Based on these results, predictions of physiological parameters such as ionic regulation based on body size are useful in crayfish, but estimates of respiratory parameters and body size should be used with caution.     

Precursor ion discovery on a hybrid quadrupole-time-of-flight mass spectrometer for gonadotropin-releasing hormone detection in complex biological mixtures

The gonadotropin-releasing hormone (GnRH) family includes several hypophysiotropic peptides occupying a central position in the regulatory loop controlling reproduction. Studies are still under way to clarify its biological role and evolutionary implication. Although sequencing of multiple genomes is bringing further advances in the understanding of the evolution of GnRH, there is still a need for biochemical studies aiming to identify GnRH from different species. Using a hybrid quadrupole-time-of-flight (Q-TOF) instrument, a new method for selective and sensitive GnRH detection and characterization from tissue extracts has been developed. The method uses the “precursor ion discovery” mode based on the capability of the Q-TOF analyzer to quickly record alternate mass spectra at low and high collision energy of precursor and product ion spectra, respectively, following liquid chromatographic separation of complex biological mixtures. The method exploits the selective detection of a specific b₂ product ion at m/z 249.1, corresponding to the N-terminus dipeptide pyroglutamic acid-histidine, highly conserved among nearly all species (22 of 24), and deriving from the preferential fragmentation of GnRHs carrying the dipeptide. Importantly, the method also includes acquisition of the product ion spectra from any candidate precursor ion, thereby allowing the determination of sequence information to confirm the GnRH identity or to isolate new ones.