The Spontaneous Loss of Coherence Catastrophe in Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

The spontaneous loss of coherence catastrophe (SLCC) is a frequently observed, yet poorly studied, space-charge related effect in Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS). This manuscript presents an application of the filter diagonalization method (FDM) in the analysis of this phenomenon. The temporal frequency behavior reproduced by frequency shift analysis using the FDM shows the complex nature of the SLCC, which can be explained by a combination of factors occurring concurrently, governed by electrostatics and ion packet trajectories inside the ICR cell

Electron Transfer Dissociation with Supplemental Activation to Differentiate Aspartic and Isoaspartic Residues in Doubly Charged Peptide Cations

Electron-transfer dissociation (ETD) with supplemental activation of the doubly charged deamidated tryptic digested peptide ions allows differentiation of isoaspartic acid and aspartic acid residues using the c + 57 or z• − 57 peaks. The diagnostic peak clearly localizes and characterizes the isoaspartic acid residue. Supplemental activation in ETD of the doubly charged peptide ions involves resonant excitation of the charge reduced precursor radical cations and leads to further dissociation, including extra backbone cleavages and secondary fragmentation. Supplemental activation is essential to obtain a high quality ETD spectrum (especially for doubly charged peptide ions) with sequence information. Unfortunately, the low-resolution of the ion trap mass spectrometer makes detection of the diagnostic peak, [M-60], for the aspartic acid residue difficult due to interference with side-chain loss from arginine and glutamic acid residues

Use of 18O Labels to Monitor Deamidation during Protein and Peptide Sample Processing

Nonenzymatic deamidation of asparagine residues in proteins generates aspartyl (Asp) and isoaspartyl (isoAsp) residues via a succinimide intermediate in a neutral or basic environment. Electron capture dissociation (ECD) can differentiate and quantify the relative abundance of these isomeric products in the deamidated proteins. This method requires the proteins to be digested, usually by trypsin, into peptides that are amenable to ECD. ECD of these peptides can produce diagnostic ions for each isomer; the c· + 58 and z − 57 fragment ions for the isoAsp residue and the fragment ion ((M + nH)(n−1)+· − 60) corresponding to the side-chain loss from the Asp residue. However, deamidation can also occur as an artifact during sample preparation, particularly when using typical tryptic digestion protocols. With 18O labeling, it is possible to differentiate deamidation occurring during trypsin digestion which causes a +3 Da (18O1 + 1D) mass shift from the pre-existing deamidation, which leads to a +1-Da mass shift. This paper demonstrates the use of 18O labeling to monitor three rapidly deamidating peptides released from proteins (calmodulin, ribonuclease A, and lysozyme) during the time course of trypsin digestion processes, and shows that the fast (̃4 h) trypsin digestion process generates no additional detectable peptide deamidations

Protein flexibility is key to cisplatin crosslinking in calmodulin

Chemical crosslinking in combination with Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) has significant potential for studying protein structures and proteinprotein interactions. Previously, cisplatin has been shown to be a crosslinker and crosslinks multiple methionine (Met) residues in apo-calmodulin (apo-CaM). However, the inter-residue distances obtained from nuclear magnetic resonance structures are inconsistent with the measured distance constraints by crosslinking. Met residues lie too far apart to be crosslinked by cisplatin. Here, by combining FTICR MS with a novel computational flexibility analysis, the flexible nature of the CaM structure is found to be key to cisplatin crosslinking in CaM. It is found that the side chains of Met residues can be brought together by flexible motions in both apo-CaM and calcium-bound CaM (Ca4-CaM). The possibility of cisplatin crosslinking Ca4-CaM is then confirmed by MS data. Therefore, flexibility analysis as a fast and low-cost computational method can be a useful tool for predicting crosslinking pairs in protein crosslinking analysis and facilitating MS data analysis. Finally, flexibility analysis also indicates that the crosslinking of platinum to pairs of Met residues will effectively close the nonpolar groove and thus will likely interfere with the binding of CaM to its protein targets, as was proved by comparing assays for cisplatin-modified/unmodified CaM binding to melittin. Collectively, these results suggest that cisplatin crosslinking of apo-CaM or Ca4-CaM can inhibit the ability of CaM to recognize its target proteins, which may have important implications for understanding the mechanism of tumor resistance to platinum anticancer drugs

Strained alkynes derived from 2,2′-dihydroxy-1,1′-biaryls ; synthesis and copper-free cycloaddition with azides

A series of strained alkynes were prepared from 2,2′-dihydroxy-biaryls. Several were characterised by X-ray crystallography, revealing strained C(sp)–C(sp)–C(sp3) bond angles in the range of 163–167°. Their cycloadditions with azides proceed without a catalyst. Functionalised versions of these reagents have potential applications to materials synthesis and bioconjugations

Generation of maghemite nanocrystals from iron–sulfur centres

Iron oxide nano-crystals 0.1–1.1 μm in diameter were generated on sulfur-doped amorphous carbon surfaces by electron beam irradiation of the novel 13e− high-spin complex [Fe(4-methyl-1,2-benzenedithiolate)2][NHEt3] encapsulated in a triblock copolymer. Possible relevance to iron nano-mineralization from Fe–S ferredoxin proteins and iron dysregulation in neurological disorders is discussed. Graphical abstract: Generation of maghemite nanocrystals from iron–sulfur centres Iron is an essential element for mammals and, amongst many other functions, plays an important role in the human brain.1 Recent research has indicated a strong association between iron dysregulation and Alzheimer's disease (AD), although it is unknown how the chemical and magnetic state of iron is linked to AD pathogenesis.2–4 Reports from Collingwood et al., and Dobson et al., for example, have shown the presence of iron oxide as the mixed oxidation state mineral, magnetite (Fe3O4) in AD tissue, a possible source of redox-active iron, but it remains unclear how this kind of iron mineral forms in the tissue.5,6 These unsolved and important questions have led us to consider how atomic resolution microscopy might provide new insight into nanoscale iron mineralization. Recently we reported methodology for studies of the nano-mineralisation of osmium, gold, ruthenium and iridium from their respective 1,2-dicarba-closo-dodecarborane-1,2-dithiolate complexes encapsulated in polymer micelles upon electron beam irradiation.7–9 Here we report the synthesis and characterization of the novel 13e− iron(iii) complex [Fe(4-methyl-1,2-benzenedithiolate)2][NHEt3] (1), containing Fe–S bonds analogous to those in the ubiquitous iron–sulfur ferredoxin proteins. Importantly, recent research has indicated a strong relationship between neurodegenerative disorders and defective Fe–S clusters.10,11 We have characterized complex 1 using Mössbauer, Raman and far-infra red spectroscopy, and investigated the generation of iron nanocrystals from 1 encapsulated in a poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) polymer (Scheme 1) by electron beam irradiation, and used electron energy-loss spectra (EELS) to identify the oxidation state of iron and its coordination environment in the nanocrystals

Coupling electron capture dissociation and the modified Kendrick mass defect for sequencing of a poly(2-ethyl-2-oxazoline) polymer

With increasing focus on the structural elucidation of polymers, advanced tandem mass spectrometry techniques will play a crucial role in the characterization of these compounds. In this contribution, synthesis and analysis of methyl initiated and xan-thate terminated poly (2-ethyl-2-oxazoline) using Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry was achieved. Electron capture dissociation (ECD) produced full end group characterization as well as back bone fragmentation including complete sequence coverage of the polymer. A method of fragment ion characterization is also presented with the use of the high resolution modified Kendrick mass defect plots as a means of grouping fragments from the same fragmentation pathways together. This type of data processing is applicable to all tandem mass spectrometry techniques for polymer analysis but is made more effective with high mass accuracy methods. ECD FT-ICR MS demonstrates its promising role as a structural characterization technique for polyoxazoline species

MIND: A Double-Linear Model To Accurately Determine Monoisotopic Precursor Mass in High-Resolution Top-Down Proteomics

Top-down proteomics approaches are becoming ever more popular, due to the advantages offered by knowledge of the intact protein mass in correctly identifying the various proteoforms that potentially arise due to point mutation, alternative splicing, post-translational modifications, etc. Usually, the average mass is used in this context; however, it is known that this can fluctuate significantly due to both natural and technical causes. Ideally, one would prefer to use the monoisotopic precursor mass, but this falls below the detection limit for all but the smallest proteins. Methods that predict the monoisotopic mass based on the average mass are potentially affected by imprecisions associated with the average mass. To address this issue, we have developed a framework based on simple, linear models that allows prediction of the monoisotopic mass based on the exact mass of the most-abundant (aggregated) isotope peak, which is a robust measure of mass, insensitive to the aforementioned natural and technical causes. This linear model was tested experimentally, as well as in silico, and typically predicts monoisotopic masses with an accuracy of only a few parts per million. A confidence measure is associated with the predicted monoisotopic mass to handle the off-by-one-Da prediction error. Furthermore, we introduce a correction function to extract the “true” (i.e., theoretically) most-abundant isotope peak from a spectrum, even if the observed isotope distribution is distorted by noise or poor ion statistics. The method is available online as an R shiny app: https://valkenborg-lab.shinyapps.io/mind

Metal ion binding to the amyloid beta monomer studied by native top-down FTICR mass spectrometry

Native top-down mass spectrometry is a fast, robust biophysical technique that can provide molecular-scale information on the interaction between proteins or peptides and ligands, including metal cations. Here we have analyzed complexes of the full-length amyloid β (1-42) monomer with a range of (patho)physiologically relevant metal cations using native Fourier transform ion cyclotron resonance mass spectrometry and three different fragmentation methods—collision-induced dissociation, electron capture dissociation, and infrared multiphoton dissociation—all yielding consistent results. Amyloid β is of particular interest as its oligomerization and aggregation are major events in the etiology of Alzheimer’s disease, and it is known that interactions between the peptide and bioavailable metal cations have the potential to significantly damage neurons. Those metals which exhibited the strongest binding to the peptide (Cu2+, Co2+, Ni2+) all shared a very similar binding region containing two of the histidine residues near the N-terminus (His6, His13). Notably, Fe3+ bound to the peptide only when stabilized toward hydrolysis, aggregation, and precipitation by a chelating ligand, binding in the region between Ser8 and Gly25. We also identified two additional binding regions near the flexible, hydrophobic C-terminus, where other metals (Mg2+, Ca2+, Mn2+, Na+, and K+) bound more weakly—one centered on Leu34, and one on Gly38. Unexpectedly, collisional activation of the complex formed between the peptide and [CoIII(NH3)6]3+ induced gas-phase reduction of the metal to CoII, allowing the peptide to fragment via radical-based dissociation pathways. This work demonstrates how native mass spectrometry can provide new insights into the interactions between amyloid β and metal cations

Commissioning and quality assurance for VMAT delivery systems: An efficient time-resolved system using real-time EPID imaging.

PURPOSE: An ideal commissioning and quality assurance (QA) program for Volumetric Modulated Arc Therapy (VMAT) delivery systems should assess the performance of each individual dynamic component as a function of gantry angle. Procedures within such a program should also be time-efficient, independent of the delivery system and be sensitive to all types of errors. The purpose of this work is to develop a system for automated time-resolved commissioning and QA of VMAT control systems which meets these criteria. METHODS: The procedures developed within this work rely solely on images obtained, using an electronic portal imaging device (EPID) without the presence of a phantom. During the delivery of specially designed VMAT test plans, EPID frames were acquired at 9.5 Hz, using a frame grabber. The set of test plans was developed to individually assess the performance of the dose delivery and multileaf collimator (MLC) control systems under varying levels of delivery complexities. An in-house software tool was developed to automatically extract features from the EPID images and evaluate the following characteristics as a function of gantry angle: dose delivery accuracy, dose rate constancy, beam profile constancy, gantry speed constancy, dynamic MLC positioning accuracy, MLC speed and acceleration constancy, and synchronization between gantry angle, MLC positioning and dose rate. Machine log files were also acquired during each delivery and subsequently compared to information extracted from EPID image frames. RESULTS: The largest difference between measured and planned dose at any gantry angle was 0.8% which correlated with rapid changes in dose rate and gantry speed. For all other test plans, the dose delivered was within 0.25% of the planned dose for all gantry angles. Profile constancy was not found to vary with gantry angle for tests where gantry speed and dose rate were constant, however, for tests with varying dose rate and gantry speed, segments with lower dose rate and higher gantry speed exhibited less profile stability. MLC positional accuracy was not observed to be dependent on the degree of interdigitation. MLC speed was measured for each individual leaf and slower leaf speeds were shown to be compensated for by lower dose rates. The test procedures were found to be sensitive to 1 mm systematic MLC errors, 1 mm random MLC errors, 0.4 mm MLC gap errors and synchronization errors between the MLC, dose rate and gantry angle controls systems of 1°. In general, parameters measured by both EPID and log files agreed with the plan, however, a greater average departure from the plan was evidenced by the EPID measurements. CONCLUSION: QA test plans and analysis methods have been developed to assess the performance of each dynamic component of VMAT deliveries individually and as a function of gantry angle. This methodology relies solely on time-resolved EPID imaging without the presence of a phantom and has been shown to be sensitive to a range of delivery errors. The procedures developed in this work are both comprehensive and time-efficient and can be used for streamlined commissioning and QA of VMAT delivery systems