Local acid environment in poly(ethylene‐ran‐methacrylic acid) ionomers

The local environment of unneutralized carboxylic acid groups in poly(ethylene‐ran‐methacrylic acid) (E/MAA) ionomers neutralized with monovalent (Li and Na) and divalent (Ca and Zn) ions has been investigated with Fourier transform infrared spectroscopy. These unneutralized acid groups interact with one another to form acid dimers, and they associate with existing neutralized complexes. At room temperature, no free acids can be detected for any system, not even for pure E/MAA. With the acid dimer peak (1700 cm^?1) and a known unneutralized acid concentration, the concentration of acids associated with a neutralized complex can be determined. This concentration of associated acids increases with increasing neutralization, reaches a maximum below 50% neutralization, and then decreases toward zero near 80% neutralization. This behavior is perhaps due to the increased driving force for aggregation of the neutralization acids. Although Li, Na, and Ca contain similar concentrations of associated acids over the range of neutralizations, the Zn system contains far fewer associated acids (i.e., more acid dimers) at any particular neutralization level. These results are confirmed by an analysis of the absorbance in the neutralized region (1650–1500 cm^?1). © 2002 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 2833–2841, 2002     

Effects of peptide acetylation and dimethylation on electrospray ionization efficiency

Peptide acetylation and dimethylation have been widely used to derivatize primary amino groups (peptide N‐termini and the ε‐amino group of lysines) for chemical isotope labeling of quantitative proteomics or for affinity tag labeling for selection and enrichment of labeled peptides. However, peptide acetylation results in signal suppression during electrospray ionization (ESI) due to charge neutralization. In contrast, dimethylated peptides show increased ionization efficiency after derivatization, since dimethylation increases hydrophobicity and maintains a positive charge on the peptide under common LC conditions. In this study, we quantitatively compared the ESI efficiencies of acetylated and dimethylated model peptides and tryptic peptides of BSA. Dimethylated peptides showed higher ionization efficiency than acetylated peptides for both model peptides and tryptic BSA peptides. At the proteome level, peptide dimethylation led to better protein identification than peptide acetylation when tryptic peptides of mouse brain lysate were analyzed with LC‐ESI‐MS/MS. These results demonstrate that dimethylation of tryptic peptides enhanced ESI efficiency and provided up to two‐fold improved protein identification sensitivity in comparison with acetylation. Copyright © 2016 John Wiley & Sons, Ltd.     

Global proteomic analysis of lysine acetylation in zebrafish (Danio rerio) embryos

Lysine acetylation is an important post‐translational modification (PTM). Since the development of MS‐based proteomics technology, important roles of lysine acetylation beyond histones have focused on chromatin remodeling during the cell cycle and regulation of nuclear transport, metabolism, and translation. Zebrafish (Danio rerio) is a widely used vertebrate model in genetics and biologic studies. Although studies in several mammalian species have been performed, the mechanism of lysine acetylation in D. rerio embryos is incompletely understood. Here, we investigated the global acetylome in D. rerio embryos by using an MS‐based proteomics approach. We identified 351 acetylated peptides and 377 nonredundant acetylation sites on 189 lysine‐acetylated proteins in 5‐day postfertilization (hpf) embryos of D. rerio. Among lysine‐acetylated peptides, 40.2% indicated three motifs: (ac)KxxxK, (ac)KxxxxK, and Lx(ac)K. Of 190 acetylated proteins, 81 (42.6%) were mainly distributed in the cytoplasm. Gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that lysine acetylation in D. rerio was enriched in metabolic pathways. Additionally, 17 of 30 acetylated ribosomal proteins were evolutionarily conserved between zebrafish and humans. Our results indicate that acetyllysine might have regulatory effects on ribosomal proteins involved in protein biosynthesis.     

Cross‐β Amyloid Nanohybrids Loaded With Cytochrome C Exhibit Superactivity in Organic Solvents

The present study reports the development of a unique class of Cytochrome C (CytC)‐loaded cross‐beta amyloid nanohybrids. The peroxidase activity of the bound CytC increased up to two orders of magnitude in organic solvents compared to the activity of unbound CytC in water. The amyloid sequences used in the study feature the nucleating core ¹⁷LVFF²¹ of the beta amyloid (Aβ), which assembled to form homogenous fibers and nanotubes. The morphology and exposed surface of the amyloid nanohydrids critically modulated the CytC activity. A CytC–Ac‐KLVFFAE‐NH₂ hybrid featuring nanofiber morphology showed 308‐fold higher activity than unbound CytC in water, which increased to 450‐fold with the nanotube morphology of CytC–Ac‐KLVFFA L ‐NH₂. Notably, activity declined substantially when the exposed surface charge was detuned by replacing lysine with histidine, thus underpinning the importance of surface charge. This enzyme–amyloid nanohybrid system could facilitate the technological application of enzymes.     

Sequential Dy(OTf)3‐Catalyzed Solvent‐Free Per‐O‐Acetylation and Regioselective Anomeric De‐O‐Acetylation of Carbohydrates

Dysprosium(III) trifluoromethanesulfonate‐catalyzed per‐O ‐acetylation and regioselective anomeric de‐O ‐acetylation of carbohydrates can be tuned by adjusting the reaction medium. In this study, the per‐O ‐acetylation of unprotected sugars by using a near‐stoichiometric amount of acetic anhydride under solvent‐free conditions resulted in the exclusive formation of acetylated saccharides as anomeric mixtures, whereas anomeric de‐O ‐acetylation in methanol resulted in a moderate‐to‐excellent yield. Reactions with various unprotected monosaccharides or disaccharides followed by a semi‐one‐pot sequential conversion into the corresponding acetylated glycosyl hemiacetal also resulted in high yields. Furthermore, the obtained hemiacetals could be successfully transformed into trichloroimidates after Dy(OTf)₃‐catalyzed glycosylation.     

Mass spectrometry‐based proteomics of oxidative stress: Identification of 4‐hydroxy‐2‐nonenal (HNE) adducts of amino acids using lysozyme and bovine serum albumin as model proteins

Modification of proteins by 4‐hydroxy‐2‐nonenal (HNE), a reactive by‐product of ω6 polyunsaturated fatty acid oxidation, on specific amino acid residues is considered a biomarker for oxidative stress, as occurs in many metabolic, hereditary, and age‐related diseases. HNE modification of amino acids can occur either via Michael addition or by formation of Schiff‐base adducts. These modifications typically occur on cysteine (Cys), histidine (His), and/or lysine (Lys) residues, resulting in an increase of 156 Da (Michael addition) or 138 Da (Schiff‐base adducts), respectively, in the mass of the residue. Here, we employed biochemical and mass spectrometry (MS) approaches to determine the MS “signatures” of HNE‐modified amino acids, using lysozyme and BSA as model proteins. Using direct infusion of unmodified and HNE‐modified lysozyme into an electrospray quadrupole time‐of‐flight mass spectrometer, we were able to detect up to seven HNE modifications per molecule of lysozyme. Using nanoLC‐MS/MS, we found that, in addition to N‐terminal amino acids, Cys, His, and Lys residues, HNE modification of arginine (Arg), threonine (Thr), tryptophan (Trp), and histidine (His) residues can also occur. These sensitive and specific methods can be applied to the study of oxidative stress to evaluate HNE modification of proteins in complex mixtures from cells and tissues under diseased versus normal conditions.     

Charge transfer dissociation of phosphocholines: gas‐phase ion/ion reactions between helium cations and phospholipid cations

Phospholipid cations formed by electrospray ionization were subjected to excitation and fragmentation by a beam of 6 keV helium cations in a process termed charge transfer dissociation (CTD). The resulting fragmentation pattern in CTD is different from that of conventional collision‐induced dissociation, but analogous to that of metastable atom‐activated dissociation and electron‐induced dissociation. Like collision‐induced dissociation, CTD yields product ions indicative of acyl chain lengths and degrees of unsaturation in the fatty acyl moieties but also provides additional structural diagnostic information, such as double bond position. Although CTD has not been tested on a larger lipid sample pool, the extent of structural information obtained demonstrates that CTD is a useful tool for lipid structure characterization, and a potentially useful tool in future lipidomics workflows. CTD is relatively unique in that it can produce a relatively strong series of 2+ product ions with enhanced abundance at the double bond position. The generally low signal‐to‐noise ratios and spectral complexity of CTD make it less appealing than OzID or other radical‐induced methods for the lipids studies here, but improvements in CTD efficiency could make CTD more appealing in the future. Copyright © 2017 John Wiley & Sons, Ltd.     

pH‐Sensitive Mechanical Properties of Elastin‐Based Hydrogels

Ionizable amino acids in protein‐based hydrogels can confer pH‐responsive behavior. Because elastin‐like polypeptides (ELPs) have an established sequence and can crosslink to form hydrogels, they are an ideal system for creating pH‐sensitive materials. This study examines different parameters that might affect pH‐sensitive behavior and characterizes the mechanical and physical properties between pH 3 and 11 of three ELP‐based crosslinked hydrogels. The first finding is that varying the amount of crosslinker affects the overall stiffness and resilience of the hydrogels but does not strongly affect water content, swelling ratio, or pH sensitivity. Second, the choice of two popular tag sequences, which vary in histidine and aspartic acid content, does not have a strong effect on pH‐sensitive properties. Last, selectively blocking lysine and tyrosine residues through acetylation significantly decreases the pH‐sensitive zeta potential. Acetylated hydrogels also demonstrate different behavior at low pH values with reduced swelling, reduced water content, and higher stiffness. Overall, this work demonstrates that ELP hydrogels with ionizable groups are promising materials for environmentally‐responsive applications such as drug delivery, tissue engineering, and microfluidics.     

Acetylation of the HIV-1 Tat protein: an in vitro study

In the last few years, the understanding of lysine acetylation as a regulatory post-translational modification of proteins in cell signalling cascades has increased. It is now known that not only histones but also non-histone factors can serve as substrates of different acetyltransferase enzymes. Acetylated lysine residues in non-histone factors are often identified using radioactive labelling experiments and immunochemical analysis of synthetic peptides. In this study of the human immunodeficiency virus 1 (HIV-1) Tat protein, we demonstrate the benefits of matrix-assisted laser desorption/ionisation mass spectrometry, proteolytic digestion and Edman sequencing for the mapping of acetylation sites. We confirmed that the HIV-1 Tat protein is acetylated in vitro by the acetyltransferase p300 at a specific lysine residue at position 50 in its RNA binding region. Furthermore, we showed that the Tat cysteine-rich region is acetylated at multiple cysteine residues in the absence of enzyme. Since this non-enzymatic cysteine acetylation occurs independently from the surrounding peptide sequence, we consider the presence of cysteine residues in acetylated peptides an important factor for the interpretation of in vitro acetylation assays in general.Abbreviations aa Amino acid - AcCoA Acetyl coenzyme A - acm Acetamidomethyl - ARM Arginine-rich motif - CRR Cysteine-rich region - HAT Histone acetyltransferaseThis article is dedicated to Harald zur Hausen on the occasion of his retirement as head of the German Cancer Research Center (Deutsches Krebsforschungszentrum) with gratitude and appreciation for 20 years of leadership     

Construction of L‐Lysine Sensor by Layer‐by‐Layer Adsorption of L‐Lysine 6‐Dehydrogenase and Ferrocene‐Labeled High Molecular Weight Coenzyme Derivative on Gold Electrode

A ferrocene‐labeled high molecular weight coenzyme derivative (PEI‐Fc‐NAD) and a thermostable NAD‐dependent L ‐lysine 6‐dehydrogenase (LysDH) from thermophile Geobacillus stearothermophilus were used to fabricate a reagentless L ‐lysine sensor. Both LysDH and PEI‐Fc‐NAD were immobilized on the surface of a gold electrode by consecutive layer‐by‐layer adsorption (LBL) technique. By the simple LBL method, the reagentless L ‐lysine sensor, with co‐immobilization of the mediator, coenzyme, and enzyme was obtained, which exhibited current response to L ‐lysine without the addition of native coenzyme to the analysis system. The amperometric response of the sensor was dependent on the applied potential, bilayer number of PEI‐Fc‐NAD/LysDH, and substrate concentration. A linear current response, proportional to L ‐lysine concentration in the range of 1–120 mM was observed. The response of the sensor to L ‐lysine was decreased by 30% from the original activity after one month storage.     

Determination of the Degree of Charge‐Transfer Contributions to Surface‐Enhanced Raman Spectroscopy

We explore the application of a previously suggested formula for determining the degree of charge transfer in surface‐enhanced Raman scattering (SERS). SERS is often described as a phenomenon which obtains its enhancement from three major sources, namely the surface plasmon resonance, charge‐transfer resonances as well as possible molecular resonances. At any chosen excitation wavelength, it is possible to obtain contributions from several sources and this has led to considerable confusion. The formula for the degree of charge transfer enables one to separate these effects, but it requires that spectra be obtained either at two or more different excitation wavelengths or as a function of applied potential. We apply this formula to several examples, which display rather large charge‐transfer contributions to the spectrum. These are p‐aminothiophenol (PATP), tetracyano‐ ethylene (TCNE) and piperidine. In PATP we can show that several lines of the same symmetry give the same degree of charge transfer. In TCNE we are able to identify the charge‐transfer transition, which contributes to the effect, and are able to independently determine the degree of charge transfer by wavenumber shifts. This enables a comparison of the two techniques of measurement. In piperidine, we present an example of molecule to metal charge transfer and show that our definition of charge transfer is independent of direction.     

Hydroxamic Acid‐Piperidine Conjugate is an Activated Catalyst for Lysine Acetylation under Physiological Conditions

Lysine acylation of proteins is an essential chemical reaction for posttranslational modification and as a means of protein modification in various applications. N,N‐Dimethyl‐4‐aminopyridine (DMAP) derivatives are widely‐used catalysts for lysine acylation of proteins; however, the DMAP moiety mostly exists in a protonated, and thus deactivated, form under physiological conditions due to its basicity. An alternative catalytic motif furnishing higher acylation activity would further broaden the possible applications of chemical lysine acylation. We herein report that the hydroxamic acid‐piperidine conjugate Ph‐HXA is a more active catalytic motif for lysine acetylation than DMAP under physiological conditions. In contrast to DMAP, the hydroxamic acid moiety is mostly deprotonated under aqueous neutral pH, resulting in a higher concentration of the activated form. The Ph‐HXA catalyst is also more tolerant of deactivation by a high concentration of glutathione than DMAP. Therefore, Ph‐HXA might be a suitable catalytic motif for target protein‐selective and site‐selective acetylation in cells.     

A quantitative analysis of histone methylation and acetylation isoforms from their deuteroacetylated derivatives: application to a series of knockout mutants

The core histones, H2A, H2B, H3 and H4, undergo post‐translational modifications (PTMs) including lysine acetylation, methylation and ubiquitylation, arginine methylation and serine phosphorylation. Lysine residues may be mono‐, di‐ and trimethylated, the latter resulting in an addition of mass to the protein that differs from acetylation by only 0.03639 Da, but that can be distinguished either on high‐performance mass spectrometers with sufficient mass accuracy and mass resolution or via retention times. Here we describe the use of chemical derivatization to quantify methylated and acetylated histone isoforms by forming deuteroacetylated histone derivatives prior to tryptic digestion and bottom‐up liquid chromatography‐mass spectrometric analysis. The deuteroacetylation of unmodified or mono‐methylated lysine residues produces a chemically identical set of tryptic peptides when comparing the unmodified and modified versions of a protein, making it possible to directly quantify lysine acetylation. In this work, the deuteroacetylation technique is used to examine a single histone H3 peptide with methyl and acetyl modifications at different lysine residues and to quantify the relative abundance of each modification in different deacetylase and methylase knockout yeast strains. This application demonstrates the use of the deuteroacetylation technique to characterize modification ‘cross‐talk’ by correlating different PTMs on the same histone tail. Copyright © 2013 John Wiley & Sons, Ltd.     

Proton‐Coupled Self‐Assembly of a Porphyrin‐Naphthalenediimide Dyad

The construction of an n–p heterojunction through the self‐assembly of a dyad based on tetraphenylporphyrin (TPP) and 1,4,5,8‐naphthalenedimide (NDI) ( 1 ) is described. Proton transfer from the lysine head group of 1 to the porphyrin ring occurs concomitantly with self‐assembly into 1D nanorods in CHCl₃. TEM and AFM studies showed that the nanorods are formed by the lateral and vertical fusion of multilameller vesicles into networks and hollow ribbons, respectively. These intermediate structures transitioned to nanorods over the course of 4–6 days. Time‐resolved spectroscopy revealed that photoinduced charge separation occurs with rate constants that depend on the nature of the aggregation.     

A Highly Selective Amperometric Hydrogen Peroxide Sensor Based on Silicomolybdate‐Doped‐Glutaraldehyde‐Cross‐Linked Poly‐L‐Lysine Film Modified Glassy Carbon Electrode

Silicomolybdate‐doped‐glutaraldehyde‐cross‐linked poly‐L ‐lysine (PLL‐GA‐SiMo) film modified glassy carbon electrode was prepared by means of electrostatically trapping the silicomolybdate anion in the cationic film. The PLL‐GA‐SiMo film was stable and the charge transport through the film was fast. The modified electrode shows excellent electrocatalytic activity towards hydrogen peroxide reduction with significant reduction of overpotential, however, not responded to potential interferrents such as dopamine, ascorbic acid and uric acid. This unique feature of PLL‐GA‐SiMo modified electrode allowed for the development of a highly selective method for the determination of H₂O₂ in the presence of interferents.