Controlling the structure of sequence‐defined poly(phosphodiester)s for optimal MS/MS reading of digital information

Digital polymers are monodisperse chains with a controlled sequence of co‐monomers, defined as letters of an alphabet, and are used to store information at the molecular level. Reading such messages is hence a sequencing task that can be efficiently achieved by tandem mass spectrometry. To improve their readability, structure of sequence‐controlled synthetic polymers can be optimized, based on considerations regarding their fragmentation behavior. This strategy is described here for poly(phosphodiester)s, which were synthesized as monodisperse chains with more than 100 units but exhibited extremely complex dissociation spectra. In these polymers, two repeating units that differ by a simple H/CH₃ variation were defined as the 0 and 1 bit of the ASCII code and spaced by a phosphate moiety. They were readily ionized in negative ion mode electrospray but dissociated via cleavage at all phosphate bonds upon collisional activation. Although allowing a complete sequence coverage of digital poly(phosphodiester)s, this fragmentation behavior was not efficient for macromolecules with more than 50 co‐monomers, and data interpretation was very tedious. The structure of these polymers was then modified by introducing alkoxyamine linkages at appropriate location throughout the chain. A first design consisted of placing these low dissociation energy bonds between each monomeric bit: while cleavage of this sole bond greatly simplified MS/MS spectra, efficient sequencing was limited to chains with up to about 50 units. In contrast, introduction of alkoxyamine bonds between each byte (i.e. a set of eight co‐monomers) was a more successful strategy. Long messages (so far, up to 8 bytes) could be read in MS³ experiments, where single‐byte containing fragments released during the first activation stage were further dissociated for sequencing. The whole sequence of such byte‐truncated poly(phosphodiester)s could be easily re‐constructed based on a mass tagging system which permits to determine the original location of each byte in the chain. Copyright © 2017 John Wiley & Sons, Ltd.     

Dissociation Channel Dependence on Peptide Size Observed in Electron Capture Dissociation of Tryptic Peptides

Electron capture dissociation (ECD) of a series of five residue peptides led to the observation that these small peptides did not lead to the formation of the usual c/z ECD fragments, but to a, b, y, and w fragments. In order to determine how general this behavior is for small sized peptides, the effect of peptide size on ECD fragments using a complete set of ECD spectra from the SwedECD spectra database was examined. Analysis of the database shows that b and w fragments are favored for small peptide sizes and that average fragment size shows a linear relationship to parent peptide size for most fragment types. From these data, it appears that most of the w fragments are not secondary fragments of the major z ions, in sharp contrast with the proposed mechanism leading to these ions. These data also show that c fragment distributions depend strongly on the nature of C-terminal residue basic site: arginine leads to loss of short neutral fragments, whereas lysine leads to loss of longer neutral fragments. It also appears that b ions might be produced by two different mechanisms depending on the parent peptide size. A model for the fragmentation pathways in competition is proposed. These relationships between average fragment size and parent peptide size could be further exploited also for CID fragment spectra and could be included in fragmentation prediction algorithms.     

Electrochemical investigation of complexation of polymer ligands containing 2,2��-biquinolyl fragments with NiII ions in solution

Polyamic acids containing biquinolyl (biQ) fragments in the polymer backbone are capable of coordinating with NiII ions to form two types of coordination units: with one or two biQ ligands in the Ni coordination environment. The relative number of [Ni(biQ)₂]²⁺ coordination units in the polymer chain increases with an increase in the concentration of the biQ fragments in solution and with a decrease in the conformational rigidity of the polymer. The synthesis of the nickel complexes with the polymer ligands can be carried out using both Ni salts and the electrochemical dissolution of the Ni anode.     

COMPLEX CHARGE RECOMBINATION KINETICS OF THE PHOTOTRAP IN RHODOSPIRILLUM RUBRUM*

Abstract— The dark decay kinetics of excited phototraps in chromatophores prepared from Rhodospirillum rubrum have been quantitatively measured under two different conditions where a direct return of the electron(s) from ubiquinone to the primary donor bacteriochlorophyll cation radical is expected. One of these experimental conditions was with a frozen sample (whole cells, chromatophores, and extracted chromatophores) at 77 K, while the other was a room temperature measurement of a sample of chromatophores from which the loosely bound ubiquinone was first removed by extraction with organic solvents. Results with the latter system were also compared to those obtained with chromatophores plus 1 mM o-phenanthroline. In all cases the kinetics of charge recombination were not pseudo-first order but showed a biphasic pattern. The data at low temperature could be exactly fit by assuming that two major pseudo-first order processes were occurring with half times of 6.5 and 19 ms, 22% decaying by the faster process and 78% by the slower process. Both types of experiments at room temperature could also be fit by assuming two major pseudo-first order processes were occurring, but with somewhat slower half times, 22 and 59 ms. In this case, 20% decayed by the faster process and 80% by the slower process. An equation was developed to fit the data assuming that one ubiquinone molecule was the electron acceptor for two donor units (the duplex model), the dark decay thus taking place in two sequential steps with 50% of the oxidized donor units becoming reduced in each step. The experimental data could be exactly fit using this equation and the above parameters. The data are viewed as best supporting the duplex model for explaining primary photochemical events in these bacteria.     

Direct determination of the primary binding site of cisplatin on cytochrome c by mass spectrometry

Protein—cisplatin interactions lie at the heart of both the effectiveness of cisplatin as a therapeutic agent and side effects associated with cisplatin treatment. Because a greater understanding of the protein—cisplatin interactions at the molecular level can inform the design of cisplatin-like agents for future use, mass spectrometric determination of the binding site of cisplatin on a model protein, cytochrome c, was undertaken in this paper. The monoadduct cytochrome c—Pt(NH₃)₂(H₂O) is found to be the primary adduct produced by the cytochrome c—cisplatin interactions under native conditions. To locate the primary binding site of cisplatin, both free cytochrome c and the cytochrome c adducts underwent trypsin digestion, followed by Fourier transform mass spectrometry (FT-MS) to identify unique fragments in the adduct digest. Four such fragments were found in the adduct digest. Tandem mass spectrometry (MS/MS and MS³ indicates that two fragments are Pt(NH₃)₂(H₂O) bound peptides (Gly56-Glu104 and Asn54-Glu104) with one water associated at the peptide bond Lys79∼Met80, and the other two fragments are heme containing peptides (acety1-Gly1-Lys53 and acety1-Gly1-Lys55). The product-ion spectra of the four fragments reveal that Met65 is the primary binding site of cisplatin on cytochrome c.     

Solution and latex properties of model alkali‐soluble rheology modifiers,synthesized via the reversible addition–fragmentation chain transfer process,and the effects of the ethylene oxide chain length on the rheological properties

Model alkali‐soluble rheology modifiers were synthesized through the reversible addition–fragmentation chain transfer polymerization of methyl methacrylate, methacrylic acid, and three different associative macromonomers containing 20, 50, and 100 ethylene oxide spacer units, respectively. The synthesized polymers showed well‐controlled molar masses and narrow molar mass distributions. The rheological properties of the model alkali‐soluble rheology modifiers were measured in alkali solutions and in the presence of a well‐characterized core–shell emulsion. The steady‐shear viscosity data for the emulsion solutions, thickened with the associative rheology modifiers, were described by the Carreau model. The rheology modifiers containing the macromonomers with the longest ethylene oxide spacer units produced the highest viscosity in the latex systems but the lowest viscosity in alkali solutions. The highest viscosities in alkali solutions were obtained for the rheology modifiers containing macromonomers with 50 ethylene oxide spacer units. © 2004 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 42: 2502–2512, 2004     

Poly(ethylene‐co‐1,4‐cyclohexylenedimethylene terephthalate) copolyesters obtained by ring opening polymerization

Cyclic oligomer fractions of ethylene terephthalate c(ET)_n and 1,4‐cyclohexylenedimethylene terephthalate c(CT)_n were obtained by cyclodepolymerization of their respective polyesters, the former containing around 80 mol % of trimer and the latter with around 70 mol % of trimer to pentamer. Mixtures of these fractions at selected compositions were subjected to ring opening copolymerization to give a series of poly(ethylene‐co‐cyclohexylenedimethylene terephthalate) copolyesters with ET/CT comonomer ratios ranging from 90/10 to 10/90. The copolyesters were characterized by GPC and NMR, and their thermal properties were evaluated by DSC and TGA. They had essentially the same composition as the feed from which they were produced and had an average‐weight molecular weights between 30,000 and 40,000 g/mol with polydispersities between 2 and 2.7. The distribution of the monomeric units in these copolyesters was essentially at random although it evolved to be a blocky microstructure as the contents in the two comonomers became more dissimilar. Their thermal behavior was the expected one for these types of copolyesters with crystallinity and heating stability decreasing with the content in CT units. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 5954–5966, 2009     

Use of Enzymatic Digestion and Chemical Methylation Followed by Matrix-assisted Laser Desorption/Ionisation (MALDI) Mass Spectrometry to Sequence a 60kDa Commercial Fraction of Cellulose Acetate

Cellulose acetate was characterized by using enzyme in both digestion and chemical derivation and acetolysis. The fragments were normalised and compared on an anhydroglucose scale, using mass spectrometry to identify the different sized fragments. It was determined that at least two sub-populations for cellulose acetate existed within the parent. The macroscopic effect of this variation in the degree of acetylation will be a modification of the structural properties of the polymer chains. It was found that through comparison with enzyme-based degradation, an estimation of the acetylation topography of the cellulose acetate fraction could be made. Enzyme degradation produced a number of oligosaccharides of more than 10 glucose units, presumably resistant to enzyme degradation because they contained acetate groups. Chemical hydrolysis gave a random ladder of short sequences of mainly 3–4 glucose units some of which had a high methyl ether content, that were analysed by mass and converted to an anhydroglucose mass scale. This approach could be used to demonstrate differences between large biopolymers of cellulose acetate that previously gave an overall average rather than a specific ladder average.     

Design of photoresists with reduced environmental impact. II. Water‐soluble resists based on photocrosslinking of poly(2‐isopropenyl‐2‐oxazoline)

The performance of water‐ and solvent‐cast, two‐component photoresist films containing poly(2‐isopropenyl‐2‐oxazoline) or poly(2‐isopropenyl‐2‐oxazoline‐co‐styrene) with a photoacid generator has been investigated. These materials afford negative‐tone images after deep‐UV exposure and development in a suitable medium (water or toluene). Resist solutions prepared from polymers containing at least 80 mol % 2‐isopropenyl‐2‐oxazoline may be cast from and developed in pure water. Features of higher quality can be obtained when the resist is cast from 2‐methoxyethanol, probably because side reactions such as partial hydrolysis of the pendant oxazoline rings in aqueous environments are avoided. It was possible to resolve micrometer scale patterns using ca. 200 mJ/cm² of irradiation at 254 nm, followed by heating 2 min at 130°C and development in water alone. Image quality and etch resistance were improved using copolymers containing up to 20 mol % of styrene repeat units. © 1999 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 37: 1225–1236, 1999     

(η3-allyl)palladium(II) catalysts for the addition polymerization of norbornene derivatives with functional groups

Cycloaliphatic polyolefins with functional groups were prepared by the Pd(II)-catalyzed addition polymerization of norbornene derivatives. Homo- and copolymers containing repeating units based on bicyclo[2.2.1] hept-5-en-2-ylmethyl decanoate (endo/exo-ratio = 80/20), bicyclo[2.2.1]hept-5-ene-2-carboxylic acid methyl ester (exo/endo = 80/20), bicyclo[2.2.1]hept-5-ene-2-methanol (endo/exo = 80/20), and bicyclo[2.2.1]hept-5-ene-2-carboxylic acid (100% endo) were prepared in 49–99% yields with {(η³-allyl)Pd(BF₄)} and {(η³-allyl)Pd(SbF₆)} as catalysts. The catalyst containing the hexafluoroantimonate ion was slightly more active than the tetrafluoroborate based Pd-complex.     

Pyrolysis—field ionization mass spetrometric investigation of polymeric heterocycles: I. polyquinolines and polyquinoxalines

Pyrolysis—field ionization mass spectrometry was used for the investigation of the mechanism of the thermal degradation of polymeric heterocycles. Heat-resistant polymers containing aromatic and heterocyclic units such as polyquinolines and polyquinoxalines have a strong tendency to form large condensed systems during pyrolysis, and finally will carbonize. In the course of this process, side-groups (phenyl) and small fragments (NH₃, HCN, acetonitrile, acrylonitrile, etc.) are split off. In addition, large heteroatom-containing fragments such as nitriles can be identified. These, together with some fragments that contain the complete heterocyclic ring, are characteristic of the chain building units (“key fragments”). Furthermore, compounds generated by recombination reactions and intramolecular cyclization are constituents of the pyrolysate.     

Novel network polymers based on calixresorcinarenes

The novel network polymers 2a-2d based on calix[4]resorcinarene derivatives were obtained with yields 2a: 80%, 2b: 90%, 2c: 70%, 2d: 70%. Polymers 2a and 2b containing fragments of tetramethylcalix[4]resorcinarene or tetraphenylcalix[4]resorcinarene as a repeating units were synthesized by condensation cis-tetramethylcalix[4]resorcinarene or cis-tetraphenylcalix[4]resorcinarene with formaldehyde. Polymers 2c and 2d containing fragments of furylcalix[4]resorcinarene were obtained by condensation of formaldehyde with products of calix[4]resorcinarene and furfural interaction. The IR spectra and sorption properties of the polymers were researched. It is found out that 2a-2d can participate in the cation-exchange reaction.     

Block length determination of the block copolymer mPEG‐b‐PS using MALDI‐TOF MS/MS

The molar mass determination of block copolymers, in particular amphiphilic block copolymers, has been challenging with chromatographic techniques. Therefore, methoxy poly(ethylene glycol)‐b‐poly(styrene) (mPEG‐b‐PS) was synthesized by atom transfer radical polymerization (ATRP) and characterized in detail not only by conventional chromatographic techniques, such as size exclusion chromatography (SEC), but also by matrix‐assisted laser/desorption ionization tandem mass spectrometry (MALDI‐TOF MS/MS). As expected, different molar mass values were obtained in the SEC measurements depending on the calibration standards (either PEG or PS). In contrast, MALDI‐TOF MS/MS analysis allowed the molar mass determination of each block, by the scission of the weakest point between the PEG and PS block. Thus, fragments of the individual blocks could be obtained. The PEG block showed a depolymerization reaction, while for the PS block fragments were obtained in the monomeric, dimeric, and trimeric regions as a result of multiple chain scissions. The block length of PEG and PS could be calculated from the fragments recorded in the MALDI‐TOF MS/MS spectrum. Furthermore, the assignment of the substructures of the individual blocks acquired by MALDI‐TOF MS/MS was accomplished with the help of the fragments that were obtained from the corresponding homopolymers. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

Induction of a chiral nematic phase in smectic polymers

A number of new photosensitive copolyacrylates of different composition were obtained by the copolymerization of chiral photochromic benzilidene-p-menthane- 3-one acrylic monomers with a smectogenic monomer containing a hexyloxyphenylbenzoate mesogenic group. The chiral, photochromic monomers differ by the length of the aliphatic spacer and the aromatic fragment. It was found that the introduction of a small number of chiral units into the copolymers (5 mol%) leads to the “degeneration” of the smectic C phase, which characterizes the hexyloxyphenylbenzoate homopolymer, and to the formation of the smectic A phase. An unusual effect of chiral nematic phase induction was observed for copolymers containing chiral side groups with two ring aromatic fragments. It should be pointed out that the chiral nematic phase does not occur in the case of the homopolymers of both initial comonomers. An explanation of this effect, based on the consideration of the chemical structure of the chiral and hexyloxyphenylbenzoate units, was suggested. The optical properties of cholesteric copolymers were investigated; the helical twisting power of the chiral groups of different structures was calculated. The possibility of using such copolymers as new photosensitive materials was demonstrated. Received: 16 December 1999/Accepted: 1 February 2000     

Poly[1‐(trimethylgermyl)‐1‐propyne] and poly[1‐(trimethylsilyl)‐1‐propyne] with various geometries: Their synthesis and properties

The polymerization of 1,2‐disubstituted acetylenes [1‐(trimethylgermyl)‐1‐propyne and 1‐(trimethylsilyl)‐1‐propyne] initiated by Nb‐ and Ta‐based catalytic systems was studied within a wide temperature range (?10 to +80 °C) with solvents (cyclohexane, CCl₄, toluene, anisol, and n‐chlorobutane) with variable dielectric constants (2.023–7.390). Conditions ensuring the synthesis of poly[1‐(trimethylsilyl)‐1‐propyne] (PTMSP) containing 20–80% cis units and poly[1‐(trimethylgermyl)‐1‐propyne] (PTMGP) containing 3–65% cis units were determined. The PTMSP and PTMGP samples were amorphous, exhibited a two‐phase structure characterized by the presence of less ordered regions and regions with an enhanced level of ordering, and differed in solubility. A correlation was found between the cis/trans ratio and the morphology, the geometrical density of PTMSP and PTMGP films, and the gas permeability of the polymers. The gas permeability and solubility behavior of PTMSP and PTMGP were examined in terms of the molecular characteristics of the polymer samples (the thermodynamic Kuhn segment and the Kerr electrooptic effect). It was demonstrated that the gas permeability, as well as the solubility of the polymers, was defined by their supramolecular ordering, which depended on the lengths of continuous sequences composed of units of analogous microstructures and on the flexibility of macrochains. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 2133–2155, 2003     

Travelling‐wave ion mobility mass spectrometry and negative ion fragmentation of hybrid and complex N‐glycans

Nitrogen collisional cross sections (CCSs) of hybrid and complex glycans released from the glycoproteins IgG, gp120 (from human immunodeficiency virus), ovalbumin, α1‐acid glycoprotein and thyroglobulin were measured with a travelling‐wave ion mobility mass spectrometer using dextran as the calibrant. The utility of this instrument for isomer separation was also investigated. Some isomers, such as Man₃GlcNAc₃ from chicken ovalbumin and Man₃GlcNAc₃Fuc₁ from thyroglobulin could be partially resolved and identified by their negative ion fragmentation spectra obtained by collision‐induced decomposition (CID). Several other larger glycans, however, although existing as isomers, produced only asymmetric rather than separated arrival time distributions (ATDs). Nevertheless, in these cases, isomers could often be detected by plotting extracted fragment ATDs of diagnostic fragment ions from the negative ion CID spectra obtained in the transfer cell of the Waters Synapt mass spectrometer. Coincidence in the drift times of all fragment ions with an asymmetric ATD profile in this work, and in the related earlier paper on high‐mannose glycans, usually suggested that separations were because of conformers or anomers, whereas symmetrical ATDs of fragments showing differences in drift times indicated isomer separation. Although some significant differences in CCSs were found for the smaller isomeric glycans, the differences found for the larger compounds were usually too small to be analytically useful. Possible correlations between CCSs and structural types were also investigated, and it was found that complex glycans tended to have slightly smaller CCSs than high‐mannose glycans of comparable molecular weight. In addition, biantennary glycans containing a core fucose and/or a bisecting GlcNAc residue fell on different mobility‐m/z trend lines to those glycans not so substituted with both of these substituents contributing to larger CCSs. Copyright © 2016 John Wiley & Sons, Ltd.     

Trypsin and MALDI matrix pre‐coated targets simplify sample preparation for mapping proteomic distributions within biological tissues by imaging mass spectrometry

Prefabricated surfaces containing α‐cyano‐4‐hydroxycinnamic acid and trypsin have been developed to facilitate enzymatic digestion of endogenous tissue proteins prior to matrix‐assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). Tissue sections are placed onto slides that were previously coated with α‐cyano‐4‐hydroxycinnamic acid and trypsin. After incubation to promote enzymatic digestion, the tissue is analyzed by MALDI IMS to determine the spatial distribution of the tryptic fragments. The peptides detected in the MALDI IMS dataset were identified by Liquid chromatography‐tandem mass spectrometry/mass spectrometry. Protein identification was further confirmed by correlating the localization of unique tryptic fragments originating from common parent proteins. Using this procedure, proteins with molecular weights as large as 300 kDa were identified and their distributions were imaged in sections of rat brain. In particular, large proteins such as myristoylated alanine‐rich C‐kinase substrate (29.8 kDa) and spectrin alpha chain, non‐erythrocytic 1 (284 kDa) were detected that are not observed without trypsin. The pre‐coated targets simplify workflow and increase sample throughput by decreasing the sample preparation time. Further, the approach allows imaging at higher spatial resolution compared with robotic spotters that apply one drop at a time. Copyright © 2016 John Wiley & Sons, Ltd.     

Coupling needle‐trap devices with comprehensive two‐dimensional gas chromatography with high‐resolution time‐of‐flight mass spectrometry to rapidly reveal the chemical transformation of volatile components from sulfur‐fumigated ginseng

Sulfur‐fumigation could alter the quality of white ginseng by damaging the bioactive compounds and generating sulfur‐containing materials. In the present study, coupling needle‐trap devices with comprehensive two‐dimensional gas chromatography and high‐resolution time‐of‐flight mass spectrometry was applied to rapidly reveal chemical transformation of volatile components from sulfur‐fumigated ginseng. Thirty‐two volatile compounds were not in white ginseng samples after sulfur‐fumigation. Furthermore, 20 sulfur‐containing compounds were identified for the first time in volatile oil of sulfur‐fumigated white ginseng. The established approach could be applied to discriminate sulfur‐fumigated white ginseng among commercial samples and to control the quality of white ginseng.     

Synthesis and characterization of new copolyacrylates containing porphyrin units as pendant groups and their use as sensors

Novel copolyacrylates containing various molar ratios of 5,10,15‐tri[p‐(9‐methoxy‐triethylenoxy)phenyl]‐20‐(p‐acryloxyphenyl)porphyrin units in the chains have been synthesized, and their chemical structure was determined by NMR and MALDI‐TOF mass spectrometry. Sensing response of the synthesized copolymers has been tested for trifluoroacetic and hydrochloric acids and nitrogen dioxide analytes. The reported sensing data indicate that porphyrin units are interacting with analytes in a reversible fashion. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Crystallinity in poly-N-methyllaurolactam and copolymers

Fully methylated nylon 12 was prepared by the hydrolytic polymerization of N-methyllaurolactam. The polymer, with about 20% crystallinity, has a crystal structure different from that of nylon 12. The X-ray diffraction patterns of copolymers of N-methyllaurolactam and laurolactam were shown to be composition-dependent. Copolymers containing up to 15% laurolactam units exhibited a crystal structure essentially the same as poly-N-methyllaurolactam. Copolymers containing from 20 to 50% laurolactam are transition structures, whereas copolymers containing more than 50% laurolactam units exhibit a crystal structure characteristic of polylaurolactam. DSC data on the homopolymers and copolymers are also reported.