Backbone Cleavage Research Articles

ETD-Cleavable Linker for Confident Cross-linked Peptide Identifications.

Peptide cross-links formed using the homobifunctional-linker diethyl suberthioimidate (DEST) are shown to be ETD-cleavable. DEST has a spacer arm consisting of a 6-carbon alkyl chain and it cleaves at the amidino groups created upon reaction with primary amines. In ETD MS2 spectra, DEST cross-links can be recognized based on mass pairs consisting of peptide-NH2• and peptide+linker+NH3 ions, and backbone cleavages are more equally distributed over the two constituent peptides compared with collisional activation. Dead ends that are often challenging to distinguish from cross-links are diagnosed by intense reporter ions. ETD mass pairs can be used in MS3 experiments to confirm cross-link identifications. These features provide a simple but reliable approach to identify cross-links that should facilitate studies of protein complexes.

Synthesis of alpha-methyl selenocysteine and its utilization as a glutathione peroxidase mimic.

Selenocysteine (Sec) is the 21st amino acid in the genetic code where this amino acid is primarily involved in redox reactions in enzymes because of its high reactivity toward oxygen and related reactive oxygen species. Sec has found wide utility in synthetic peptides, especially as a replacement for cysteine. One limitation of using Sec in synthetic peptides is that it can undergo β-syn elimination reactions after oxidation, rendering the peptide inactive due to loss of selenium. This limitation can be overcome by substituting Cα-H with a methyl group. The resulting Sec derivative is α-methylselenocysteine ((αMe)Sec). Here, we present a new strategy for the synthesis of (αMe)Sec by alkylation of an achiral methyl malonate through the use of a selenium-containing alkylating agent synthesized in the presence of dichloromethane. The seleno-malonate was then subjected to an enzymatic hydrolysis utilizing pig liver esterase followed by a Curtius rearrangement producing a protected derivative of (αMe)Sec that could be used in solid-phase peptide synthesis. We then synthesized two peptides: one containing Sec and the other containing (αMe)Sec, based on the sequence of glutathione peroxidase. This is the first reported incorporation of (αMe)Sec into a peptide as well as the first reported biochemical application of this unique amino acid. The (αMe)Sec-containing peptide had superior stability as it could not undergo β-syn elimination and it also avoided cleavage of the peptide backbone, which we surprisingly found to be the case for the Sec-containing peptide when it was incubated for 96hours in oxygenated buffer at pH8.0.

Photodissociative Cross-Linking of Diazirine-Tagged Peptides with DNA Dinucleotides in the Gas Phase.

Non-covalent complexes of DNA dinucleotides dAA, dAT, dGG, dGC, and dCG with diazirine-tagged Cys-Ala-Gln-Lys peptides were generated as singly charged ions in the gas phase. Laser photodissociation at 355nm of the diazirine ring in the gas-phase complexes created carbene intermediates that underwent covalent cross-linking to the dinucleotides. The dinucleotides differed in the cross-linking yields, ranging from 27 to 36% for dAA and dAT up to 90-98% for dGG, dGC, and dCG. Collision-induced dissociation tandem mass spectrometry (CID-MS3) of the cross-linked conjugates revealed that fragmentation occurred chiefly in the dinucleotide moieties, resulting in a loss of a nucleobase and backbone cleavages. The CID-MS3 spectra further revealed that cross-links were primarily formed in the 3'-nucleotides for the dAT, dGC, and dCG combinations. Gas-phase and solution structures of dGG complexes with S-tagged CAQK were investigated by Born-Oppenheimer molecular dynamics (BOMD) and density functional theory calculations. The low free-energy complexes had zwitterionic structures in which the peptide was protonated at the N-terminus and in the Lys residue whereas the carboxyl or dGG phosphate were deprotonated, corresponding to the respective (Cys+, Lys+, COO-)+ and (Cys+, Lys+, phosphate-)+ protomeric types. Both types preferred structures in which the peptide N-terminal cysteine carrying the S-photo-tag was aligned with the 3'-guanine moiety. BOMD trajectories at 310K were analyzed for close contacts of the incipient peptide carbene with the positions in dGG that pointed to frequent contacts with the N-1, NH2, and N-7 atoms of 3'-guanine, in agreement with the cross-linking results. Carbene insertion to the guanine N-1-H and NH2 bonds was calculated by density functional and Møller-Plesset perturbational theory to be 350-380kJmol-1 exothermic. Based on calculations, we proposed a mechanism for the carbene reaction with guanine starting with an exothermic attack at N-7 to form a dipolar intermediate that can close an aziridine ring in another exothermic reaction, forming a stable covalent cross link. Graphical Abstract.

Base Editing: Chemistry on a Target Nucleotide in the Genome of Living Cells

Point mutations represent the majority of known human genetic variants associated with disease but are difficult to correct cleanly and efficiently using standard nuclease‐based genome editing methods. In this lecture I will describe the development, application, and evolution of base editing, a new approach to genome editing that directly converts a target base pair to another base pair in living cells without requiring DNA backbone cleavage or donor DNA templates. Through a combination of protein engineering and protein evolution, we recently developed two classes of base editors (BE4 and ABE) that together enable all four types of transition mutations (C to T, T to C, A to G, and G to A) to be efficiently and cleanly installed or corrected at target positions in genomic DNA. The four transition mutations collectively account for most known human pathogenic point mutations. Base editing has been widely used by many laboratories around the world in a wide range of organisms including bacteria, fungi, plants, fish, frogs, insects, mammals, and even human embryos. We have recently expanded the scope of base editing by enhancing its efficiency, product purity, targeting scope, and DNA specificity. By optimizing base editor expression, we developed “max” versions of cytosine and adenine base editors with greatly increase editing efficiency in mammalian cells. We also show that base editing can function in vivo in post‐mitotic somatic cells that do not support homology‐directed repair. To improve the targeting scope of base editing, we used our phage‐assisted continuous evolution (PACE) system to rapidly evolve Cas9 and base editor variants with both broadened PAM compatibility and higher DNA specificity. Finally, we integrated several of these developments to address cell and animal models of human genetic disease. Base editing can be used to correct pathogenic point mutations, introduce disease‐suppressing mutations, and create new models of genetic diseases.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enhancing the synthesis of latex clearing protein by different cultivation strategies

In this study, we improved the synthesis of the latex clearing protein from Gordonia polyisoprenivorans VH2 (Lcp1VH2), a key enzyme for the initial cleavage of the rubber backbone. Cultivations using a recombinant strain of Escherichia coli were optimized to overcome poor solubility of Lcp1VH2 and improve the production yields. Different cultivation temperatures and agitation rates were evaluated in the process to demonstrate their impact on the solubility of Lcp1VH2. A specific maximum production rate of 28.3 mg Lcp1VH2 g−1 cell dry weight h-1 was obtained at 25 °C and at agitation rates between 200–300 rpm. The activity of Lcp1VH2 was strongly influenced by variations in the cultivation temperature with a specific maximum activity of 0.81 U mg−1 in cultures incubated at 30 °C. Besides cultivation-based optimization, also the strategy of fusion protein expression with NusA was successfully applied. The in vivo solubility of the Lcp1VH2 fusion protein was calculated to be 73.1%, which means an enhancement of 5.7-fold in comparison to the solubility of the native Lcp1VH2. The fusion protein of Lcp1VH2 and NusA still exhibited oxygenase activity with polyisoprene latex as a substrate. In fact, NusA-His-Lcp1VH2 reached a 4-fold higher volumetric activity in comparison to Lcp1VH2. Oligo(cis-1,4-isoprene) molecules were produced as degradation products due to the cleavage of the polymer backbone by NusA-His-Lcp1VH2. The formation of oligo-isoprenoid molecules with molecular weights between 236 and 984 Da were confirmed by electrospray ionization-mass spectrometry analysis.

VOC Emissions and Formation Mechanisms from Carbon Nanotube Composites during 3D Printing.

A commercially available, 3D printer nanocomposite filament of carbon nanotubes (CNTs) and acrylonitrile-butadiene-styrene (ABS) was analyzed with respect to its VOC emissions during simulated fused deposition modeling (FDM) and compared with a regular ABS filament. VOC emissions were quantified and characterized under a variety of conditions to simulate the thermal degradation that takes place during FDM. Increasing the residence time and temperature resulted in significant increases in VOC emissions, and the oxygen content of the reaction gas influenced the VOC profile. In agreement with other studies, the primary emitted VOC was styrene. Multiple compounds are reported in this work for the first time as having formed during FDM, including 4-vinylcyclohexene and 2-phenyl-2-propanol. Our results show that printing 222.0 g of filament is enough to surpass the reference concentration for inhalation exposure of 1 mg/m3 according to the EPA's Integrated Risk Information System (IRIS). The presence of CNTs in the filament influenced VOC yields and product ratios through three types of surface interactions: (1) adsorption of O2 on CNTs lowers the available O2 for oxidation of primary backbone cleavage intermediates, (2) adsorption of styrene and other VOCs to CNTs leads to surface-catalyzed degradation, and (3) CNTs act as a trap for certain VOCs and prevent them from entering vapor emissions. While the presence of CNTs in the filament lowered the total VOC emission under most experimental conditions, they increased the emission of the most hazardous VOCs, such as α-methylstyrene and benzaldehyde. The present study has identified an increased risk associated with the use of CNT nanocomposites in 3D printing.

A Quantum Mechanism Study of the C-C Bond Cleavage to Predict the Bio-Catalytic Polyethylene Degradation.

The growing amount of plastic solid waste (PSW) is a global concern. Despite increasing efforts to reduce the residual amounts of PSW to be disposed off through segregated collection and recycling, a considerable amount of PSW is still landfilled and the extent of PSW ocean pollution has become a worldwide issue. Particularly, polyethylene (PE) and polystyrene (PS) are considered as notably recalcitrant to biodegradation due to the carbon-carbon backbone that is highly resistant to enzymatic degradation via oxidative reactions. The present research investigated the catalytic mechanism of P450 monooxygenases by quantum mechanics to determine the bio-catalytic degradation of PE or PS. The findings indicated that the oxygenase-induced free radical transition caused the carbon-carbon backbone cleavage of aliphatic compounds. This work provides a fundamental knowledge of the biodegradation process of PE or PS at the atomic level and facilitates predicting the pathway of plastics’ biodegradation by microbial enzymes.

Relative Strength of Noncovalent Interactions and Covalent Backbone Bonds in Gaseous RNA-Peptide Complexes.

Interactions of ribonucleic acids (RNA) with basic ligands such as proteins or aminoglycosides play a key role in fundamental biological processes. Native top-down mass spectrometry (MS) has recently been extended to binding site mapping of RNA–ligand interactions by collisionally activated dissociation, without the need for laborious sample preparation procedures. The technique relies on the preservation of noncovalent interactions at energies that are sufficiently high to cause RNA backbone cleavage. In this study, we address the question of how many and what types of noncovalent interactions allow for binding site mapping by top-down MS. We show that proton transfer from protonated ligand to deprotonated RNA within salt bridges initiates loss of the ligand, but that proton transfer becomes energetically unfavorable in the presence of additional hydrogen bonds such that the noncovalent interactions remain stronger than the covalent RNA backbone bonds.

Hot nitric acid diffusion in fluoroelastomer composite and its degradation

Fluoroelastomers (FKM) are vital sealing materials in acidic environment and their failure can cause severe safety problems. Therefore, investigation of the degradation behavior and mechanism of FKM materials is of great significant. Herein, we investigate a diffusion model of an acidic solution into an FKM composite and its degradation behavior upon immersion in hot nitric acid solution. The results indicate that the diffusion process of the HNO3 solution into the FKM composite conforms to the Fick diffusion model at a low concentration of nitric acid solution. Besides, the concentration of HNO3 solution affects the diffusion process of solvent molecules and the dissolution process of the filler particles to some extent. SEM showed that the surface topography of the FKM was significantly altered after it was immersed in HNO3 solution. The structural and chemical changes of the FKM were studied using ATR-FTIR, SEM-EDS and MAS NMR, which demonstrated the occurrence of decrosslinking via hydrolysis of the crosslinks and backbone cleavage by dehydrofluorination. This was also manifested by the decrease in crosslinking degree and mechanical properties. The present study is helpful for revealing the chemical changes in FKM in hot HNO3 solution.

An educational module to explore CRISPR technologies with a cell-free transcription-translation system.

Within the last 6 years, CRISPR-Cas systems have transitioned from adaptive defense systems in bacteria and archaea to revolutionary genome-editing tools. The resulting CRISPR technologies have driven innovations for treating genetic diseases and eradicating human pests while raising societal questions about gene editing in human germline cells as well as crop plants. Bringing CRISPR into the classroom therefore offers a means to expose students to cutting edge technologies and to promote discussions about ethical questions at the intersection of science and society. However, working with these technologies in a classroom setting has been difficult because typical experiments rely on cellular systems such as bacteria or mammalian cells. We recently reported the use of an E. coli cell-free transcription-translation (TXTL) system that simplifies the demonstration and testing of CRISPR technologies with shorter experiments and limited equipment. Here, we describe three educational modules intended to expose undergraduate students to CRISPR technologies using TXTL. The three sequential modules comprise (i) designing the RNAs that guide DNA targeting, (ii) measuring DNA cleavage activity in TXTL and (iii) testing how mutations to the targeting sequence or RNA backbone impact DNA binding and cleavage. The modules include detailed protocols, questions for group discussions or individual evaluation, and lecture slides to introduce CRISPR and TXTL. We expect these modules to allow students to experience the power and promise of CRISPR technologies in the classroom and to engage with their instructor and peers about the opportunities and potential risks for society.

Investigation of GTP-dependent dimerization of G12X K-Ras variants using ultraviolet photodissociation mass spectrometry.

Mutations in the GTPase enzyme K-Ras, specifically at codon G12, remain the most common genetic alterations in human cancers. The mechanisms governing activation of downstream signaling pathways and how they relate back to the identity of the mutation have yet to be completely defined. Here we use native mass spectrometry (MS) combined with ultraviolet photodissociation (UVPD) to investigate the impact of three G12X mutations (G12C, G12V, G12S) on the homodimerization of K-Ras as well as heterodimerization with a downstream effector protein, Raf. Electrospray ionization (ESI) was used to transfer complexes of WT or G12X K-Ras bound to guanosine 5'-diphosphate (GDP) or GppNHp (non-hydrolyzable analogue of GTP) into the gas phase. Relative abundances of homo- or hetero-dimer complexes were estimated from ESI-MS spectra. K-Ras + Raf heterocomplexes were activated with UVPD to probe structural changes responsible for observed differences in the amount of heterocomplex formed for each variant. Holo (ligand-bound) fragment ions resulting from photodissociation suggest the G12X mutants bind Raf along the expected effector binding region (β-interface) but may interact with Raf via an alternative α-interface as well. Variations in backbone cleavage efficiencies during UV photoactivation of each variant were used to relate mutation identity to structural changes that might impact downstream signaling. Specifically, oncogenic upregulation for hydrogen-bonding amino acid substitutions (G12C, G12S) is achieved by stabilizing β-interface interactions with Raf, while a bulkier, hydrophobic G12V substitution leads to destabilization of this interface and instead increases the proximity of residues along the α-helical bundles. This study deciphers new pieces of the complex puzzle of how different K-Ras mutations exert influence in downstream signaling.

Software for top-down proteomic identification of a plasmid-borne factor (and other proteins) from genomically sequenced pathogenic bacteria using MALDI-TOF-TOF-MS/MS and post-source decay

A plasmid-borne factor (hypothetical protein) was identified in three Shiga toxin-producing Escherichia coli O113:H21 strains using antibiotic-induction, MALDI-TOF-TOF tandem mass spectrometry (MS/MS), post-source decay (PSD) and top-down proteomic analysis. The plasmid-encoded factor was identified from three pairs of complementary fragment ions (b/y) which were also used to obtain a more accurate calculation of the mass of the protein than that possible from MS analysis. A software application tool was developed to rapidly search amino acid sequences from whole genome sequencing-derived data to match the observed protein mass and fragment ions on the assumption that the fragment ions are the result of polypeptide backbone cleavage on the C-terminal side of aspartic acid and glutamic acid residues, i.e. aspartic acid effect. Manual inspection and top-down proteomic analysis confirmed the correctness of the identification of this protein using this new application tool. Although the function of the plasmid-borne factor is not known, its antibiotic-induced expression may suggest a role in antimicrobial stress. This approach was also used to identify three highly conserved bacterial cold-shock proteins: CspC, CspE, CsbD from one of the STEC strains. In addition to polypeptide backbone cleavage, metastable CsbD showed multiple small dissociative losses perhaps suggesting ammonia loss from the four arginine residues in its sequence. This phenomenon had been previously observed for metastable ubiquitin which also has four arginine residues.

Photoinduced Tyrosine Side Chain Fragmentation in IgG4-Fc: Mechanisms and Solvent Isotope Effects.

Immunoglobulin gamma (IgG) monoclonal antibodies (mAbs) are glycoproteins that have emerged as powerful and promising protein therapeutics. During the process of production, storage and transportation, exposure to ambient light is inevitable, which can cause protein physical and chemical degradation. For mechanistic studies of photodegradation, we have exposed IgG4-Fc to UV light. The photoirradiation of IgG4-Fc with monochromatic UVC light at λ = 254 nm and UVB light with λmax = 305 nm in air-saturated solutions revealed multiple photoproducts originating from tyrosine side chain fragmentation at Tyr300, Tyr373, and Tyr436. Tyr side chain fragmentation yielded either Gly or various backbone cleavage products, including glyoxal amide derivatives. A mechanism is proposed involving intermediate Tyr radical cation formation, either through direct light absorption of Tyr or through electron transfer to an initial Trp radical cation, followed by elimination of quinone methide. Product formation showed either no (cleavage of Tyr373) or significant (cleavage of Tyr436) inverse product solvent isotope effects (SIEs), indicating a role for proton transfer in the cleavage mechanism of Tyr436. The role of electron transfer in the cleavage of Tyr436 was further investigated through mutation of an adjacent Trp381. This is the first observation of a photoinduced Tyr side chain cleavage reactions in a protein.

Characterization of supramolecular peptide-polymer bioconjugates using multistage tandem mass spectrometry

Electrospray ionization multistage tandem mass spectrometry (ESI-MSn) was employed to examine the non-covalent complexes between poly(styrene sulfonate) (PSS) and poly-l-lysine (PLL). During single-stage ion activation, the PLL peptide chain mainly underwent backbone cleavages without disruption of the non-covalent interaction which could only be broken via sequential application of electron transfer dissociation (ETD) and collisionally activated dissociation (CAD), indicating strong binding interactions between the two polyelectrolyte chains. Such binding properties make PSS a potential “non-covalent (supramolecular) label” for determining the surface accessibility of basic residues on a peptide or protein. To probe this premise, non-covalent complexes of substance P and PSS were characterized by ESI-MSn using different ion activation methods. Both MS2 and MS3 experiments on the substance P + PSS complex resulted in the formation of bn (on CAD) or cn (on ETD) fragments attached non-covalently to the intact PSS chain. All peptide fragments containing the intact PSS chain included Arg1, Lys3, and Gln5, pointing out that these residues, which are located near the N-terminus, are most likely involved in the non-covalent interaction with PSS. In contrast, Gln6 was excluded from this fragment series, attesting a much weaker interaction with PSS due to lesser accessibility. The strong tendency of PSS to bind peptides non-covalently at sites that can be elucidated by MSn demonstrates a proof-of-concept for the capacity of this approach to unveil higher order structure in proteins.

Characterization of Protein Disulfide Linkages by MS In-Source Dissociation Comparing to CID and ETD Tandem MS.

Direct characterization of disulfide linkages in proteins by mass spectrometry has been challenging. Here, we report analysis of disulfide linkages in insulin variant, endothelin 3, and relaxin 2 by in-source dissociation (ISD) during LC-MS. A duplet insulin peptide from Glu-C digestion that contains peptides p1 and p2 (from chains A and B, respectively) was selected as a model peptide. This duplet peptide has an inter-chain disulfide bond between p1 and p2, and an intra-chain disulfide bond in p1. To compare the gas-phase fragmentation, it was subjected to ISD MS and MS/MS methods, including collision-induced dissociation (CID) and electron transfer dissociation (ETD). The pattern and efficiency of peptide backbone and disulfide cleavage varied with these dissociation methods. ETD, CID, and ISD were able to generate single backbone, double backbone, and triple (double backbone and single disulfide bond) cleavages in this model peptide, respectively. Specifically, CID did not cleave disulfide bonds and ETD was able to only cleave the inter-chain disulfide bond at low efficiency, limiting their usage in this disulfide analysis. In contrast, ISD was able to cleave the intra-chain disulfide bond in addition to peptide backbone, creating multiple fragment ions that allow accurate assignment of both intra- and inter-chain disulfide linkages. ISD was also successfully applied to determine double disulfide linkages in endothelin 3 and relaxin 2 peptides. This study contributes to the fundamental understanding of disulfide bond cleavages in different gas-phase fragmentations and provides an efficient cleavage strategy for identification of disulfide bonds in proteins by ISD ESI-MS. Graphical Abstract.

W-Type ions formed by electron transfer dissociation of Cys-containing peptides investigated by infrared ion spectroscopy.

In mass spectrometry‐based peptide sequencing, electron transfer dissociation (ETD) and electron capture dissociation (ECD) have become well‐established fragmentation methods complementary to collision‐induced dissociation. The dominant fragmentation pathways during ETD and ECD primarily involve the formation of c‐ and z •‐type ions by cleavage of the peptide backbone at the N─Cα bond, although neutral losses from amino acid side chains have also been observed. Residue‐specific neutral side chain losses provide useful information when conducting database searching and de novo sequencing. Here, we use a combination of infrared ion spectroscopy and quantum‐chemical calculations to assign the structures of two ETD‐generated w‐type fragment ions. These ions are spontaneously formed from ETD‐generated z •‐type fragments by neutral loss of 33 Da in peptides containing a cysteine residue. Analysis of the infrared ion spectra confirms that these z •‐ions expel a thiol radical (SH•) and that a vinyl C═C group is formed at the cleavage site. z •‐type fragments containing a Cys residue but not at the cleavage site do not spontaneously expel a thiol radical, but only upon additional collisional activation after ETD.

Electron transfer dissociation mass spectrometry of acidic phosphorylated peptides cationized with trivalent praseodymium.

The lanthanide ion praseodymium, Pr(III), was employed to study metallated ion formation and electron transfer dissociation (ETD) of 27 biological and model highly acidic phosphopeptides. All phosphopeptides investigated form metallated ions by electrospray ionization (ESI) that can be studied by ETD to yield abundant sequence information. The ions formed are [M+Pr-H]2+ , [M+Pr]3+ , and [M+Pr+H]4+ . All biological phosphopeptides with a chain length of seven or more residues generate [M+Pr]3+ . For biological phosphopeptides, [M+Pr]3+ undergoes more backbone cleavage by ETD than [M+Pr - H]2+ and, in some cases, full sequence coverage occurs. Acidic model phosphorylated hexa-peptides and octa-peptides, composed of alanine residues and one phosphorylated residue, form exclusively [M+Pr - H]2+ by ESI. Limited sequence information is obtained by ETD of [M+Pr - H]2+ with only metallated product ions being generated. For two biological phosphopeptides, [M+Pr+H]4+ is observed and may be due to the presence of at least one residue with a highly basic side chain that facilitates the addition of an extra proton. For the model phosphopeptides, more sequence coverage occurs when the phosphorylated residue is in the middle of the sequence than at either the N- or C-terminus. ETD of the metallated precursor ions formed by ESI generates exclusively metallated and nonmetallated c- and z-ions for the biological phosphopeptides, while metallated c-ions, z-ions, and a few y-ions form for the model phosphopeptides. Most of the product ions contain the phosphorylated residue indicating that the metal ion binds predominantly at the deprotonated phosphate group. The results of this study indicate that ETD is a promising tool for sequencing highly acidic phosphorylated peptides by metal adduction with Pr (III) and, by extension, all nonradioactive lanthanide metal ions.

Degradation of high molecular weight polyacrylamide by alkali-activated persulfate: Reactivity and potential application in filter cake removal before cementing

The interface between annular cement to wellbore rock is crucial for ensuring the oil and gas well integrity and the environmental protection. Poor bonding quality may lead to gas influx into the annulus or even ground and cause health, safety and environment (HSE) hazards including contamination of freshwater-bearing zones, corruption of the cement and casing, sustained casing pressure (SCP) and formation fluid spill to the ground surface. This bonding interface between annular cement and wellbore is given by a filter cake, which is commonly consisted of a bentonite, barite and high molecular weight polymer such as polyacrylamide (PAM) from the drilling process. Therefore, finding an efficient and environment-friendly method to degrade PAM to remove the filter cake before the cementing is highly desired. In this study, a fast and effective method for high molecular weight PAM (Mw=3×107 Da) degradation by an oxidizing OH-/persulfate (PS) system is presented. By optimizing the PS concentration, alkali-to-PS ratio and temperature of the oxidizing solution, it is demonstrated that a high PAM degradation efficiency (nearly 90%) can be obtained within 20 minutes and maintain a pH range suitable for cement casting. The degradation reactions conformed to pseudo-first-order kinetics with an activation energy of 59.87 kJ mol-1. Dominant radicals were analyzed by EPR that both SO4•- and HO• were found in the reaction system, but the HO• played the dominant role in the degradation process. Structure transformation of PAM was studied via GPC, SEM and FTIR. The backbone cleavage and detachment of side groups caused structural failure of polymer-bentonite and make possible removing filter cake. Filter cake removal test demonstrated at least 81% filter cake could be removed under the optimized condition. It’s more effective than ordinary preflush with only 24wt% on average. The results obtained in this work exhibit that polymer degradation by OH-/PS oxidizing system highly helps to filter cake removal and the OH-/PS system is a promising method for filter cake removal before the cementing process.

Effects of intramolecular hydrogen bonds on the collision-induced dissociation of tryptic peptide ions

The protonated basic side chains in tryptic peptide ions can form intramolecular hydrogen bonds with backbone amide carbonyls, which inhibit the backbone cleavage and hinder the peptide sequencing using mass spectrometry. To study the strength of intramolecular hydrogen bonds and its effect on the backbone cleavage, we carried out the energy-dependent collision-induced dissociation (CID) of tryptic peptides and their modified ones. The N-acylation of lysine-terminated peptides was used to convert the primary amines at both lysine and the N-terminus into amides in order to block the primary protonation sites. The guanidination was employed to transform lysine into homoarginine in order to imitate arginine-terminated peptides. Then, N-acylation was applied to the guanidinated peptides to prepare N-acylated, homoarginine-terminated peptides. Singly charged peptide ions were generated by electrospray ionization in a Q-TOF instrument and their CID spectra were obtained as a function of collision energy under nearly single-collision conditions with Xe as the target gas. The energy-dependent survival yield of the precursor ion provided information on the change in dissociation energy of peptides after chemical modifications. We also identified possible sites and numbers of hydrogen bonds that stabilized a charge-solvated structure by comparing the variation in product yields with the location of backbone cleavage. By taking these data together, we estimated the relative strength of intramolecular hydrogen bonds formed in lysine- and homoarginine-terminated peptides before and after N-acylation. Our results demonstrate that N-acylation lowers the dissociation energy of tryptic peptides and facilitates the backbone cleavage by preventing the formation of intramolecular hydrogen bonds, enabling accurate identification of peptides.

Radical processes initiated by ionizing radiation in PEEK and their macroscopic consequences

Poly (ether ether ketone) was irradiated with gamma rays or electron beam to investigate the radical process. The generated paramagnetic species were observed by electron spin resonance spectroscopy at ambient temperature and in liquid nitrogen. The effect of microwave power on saturation of the particular spectra and thermal annealing effects were determined. The following radicals were identified: radical anion, phenoxyl radical, and phenylperoxy radical. Despite the fact that the intermediates were formed as a result of backbone cleavage causing degradation, the macroscopic features were almost unaffected by irradiation up to dose of 1500 kGy.