Highly Amino Acid Selective Hydrolysis of Myoglobin at Aspartate Residues as Promoted by Zirconium(IV)‐Substituted Polyoxometalates

SDS‐PAGE/Edman degradation and HPLC MS/MS showed that zirconium(IV)‐substituted Lindqvist‐, Keggin‐, and Wells–Dawson‐type polyoxometalates (POMs) selectively hydrolyze the protein myoglobin at Asp? X peptide bonds under mildly acidic and neutral conditions. This transformation is the first example of highly sequence selective protein hydrolysis by POMs, a novel class of protein‐hydrolyzing agents. The selectivity is directed by Asp residues located on the surface of the protein and is further assisted by electrostatic interactions between the negatively charged POMs and positively charged surface patches in the vicinity of the cleavage site.     

Immunobinding‐induced alteration in the electrophoretic mobility of proteins: An approach to studying the preconcentration of an acidic protein under cationic isotachophoresis

The objective of this study is to explore an approach for analyzing negatively charged proteins using paper‐based cationic ITP. The rationale of electrophoretic focusing the target protein with negative charges under unfavorable cationic ITP condition is to modify the electrophoretic mobility of the target protein through antigen‐antibody immunobinding. Cationic ITP was performed on a paper‐based analytical device that was fabricated using fiberglass paper. The paper matrix was modified with (3‐aminopropyl)trimethoxysilane to minimize sample attraction to the surface for cationic ITP. Negatively charged BSA was used as the model target protein for the cationic ITP experiments. No electrophoretic mobility was observed for BSA‐only samples during cationic ITP experimental condition. However, the presence of a primary antibody to BSA significantly improved the electrokinetic behavior of the target protein. Adding a secondary antibody conjugated with amine‐rich quantum dots to the sample further facilitated the concentrating effect of ITP, reduced experiment time, and elevated the stacking ratio. Under our optimized experimental conditions, the cationic ITP‐based paper device electrophoretically stacked 94% of loaded BSA in less than 7 min. Our results demonstrate that the technique has a broad potential for rapid and cost‐effective isotachphoretic analysis of multiplex protein biomarkers in serum samples at the point of care.     

MS2 and Qβ bacteriophages reveal the contribution of surface hydrophobicity on the mobility of non‐enveloped icosahedral viruses in SDS‐based capillary zone electrophoresis

SDS is commonly employed as BGE additive in CZE analysis of non‐enveloped icosahedral viruses. But the way by which SDS interacts with the surface of such viruses remains to date poorly known, making complicate to understand their behavior during a run. In this article, two related bacteriophages, MS2 and Qβ, are used as model to investigate the migration mechanism of non‐enveloped icosahedral viruses in SDS‐based CZE. Both phages are characterized by similar size and surface charge but significantly different surface hydrophobicity (Qβ > MS2, where ‘>’ means ‘more hydrophobic than’). By comparing their electrophoretic mobility in the presence or not of SDS on both sides of the CMC, we show that surface hydrophobicity of phages is a key factor influencing their mobility and that SDS‐virus association is driven by hydrophobic interactions at the surface of virions. The CZE analyses of heated MS2 particles, which over‐express hydrophobic domains at their surface, confirm this finding. The correlations between the present results and others from the literature suggest that the proposed mechanism might not be exclusive to the bacteriophages examined here.     

Multifunctional Stimuli Responsive ABC Terpolymers: From Three‐Compartment Micelles to Three‐Dimensional Network

Summary: A highly asymmetric P2VP₅₈‐PAA₉₂₄‐PBMA₄₈ double hydrophilic block terpolymer exhibits a rich phase behavior as a function of pH. In acidic media (pH 1) three compartment micelles with a positively charged outer corona are formed. At high pH, the above structure is transformed into a three‐dimensional transient network constituted of hydrophobic domains interconnected with negatively charged bridging chains (PAA chains). At even higher pH (14) the network is disrupted and finally exhibits a closed loop sol–gel–sol behavior.

AFM images of P2VP₅₈‐PAA₉₂₄‐PBMA₄₈ micellar self assemblies deposited on mica.     

Electric migration of α‐hemolysin in supported n‐bilayers: A model for transmembrane protein microelectrophoresis

Proteome analysis involves separating proteins as a preliminary step toward their characterization. This paper reports on the translational migration of a model transmembrane protein (α‐hemolysin) in supported n‐bilayers (n, the number of bilayers, varies from 1 to around 500 bilayers) when an electric field parallel to the membrane plane is applied. The migration changes in direction as the charge on the protein changes its sign. Its electrophoretic mobility is shown to depend on size and charge. The electrophoretic mobility varies as 1/R², with R the equivalent geometric radius of the embedded part of the protein. Measuring mobilities at differing pH in our system enables us to determine the pI and the charge of the protein. Establishing all these variations points to the feasibility of electrophoretic transport of a charged object in this medium and is a first step toward electrophoretic separation of membrane proteins in n‐bilayer systems.     

Long‐term stability of an ambient self‐curable latex based on colloidal dispersions in water of two reactive polymers

An ambient self‐curable latex (ASCL) was prepared via the blending of colloidal dispersions in water of a chloromethylstyrene‐functionalized copolymer and a tertiary‐amine‐functionalized copolymer. Upon casting and drying under ambient conditions, the ASCL could generate crosslinked continuous polymer films. The crosslinking occurred via the Menschutkin reaction (quaternization) between the two types of functional groups. Because this reaction was reversible at high temperatures, the films could be decrosslinked and hence were self‐curable. The prepared ASCL exhibited excellent colloidal and chemical stability during long‐term storage: no significant changes in the colloidal properties, such as the particle size, electrophoretic mobility, and crosslinking reactivity, were observed after 48 months of storage. The electrophoretic measurements indicated that the electrostatic repulsion between the negatively charged particles of the ASCL was responsible for the excellent stability. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 2598–2605, 2005     

Ionic Self‐Assembled Platform of Perylenediimide–Sodium Dodecylsulfate for Detection of Spermine in Clinical Samples

The detection and quantification of spermine in clinical practice is important for early diagnosis of many diseases. Chromatographic and immunoassay‐based methods are commonly used. However, a fluorescence‐based assay could provide real‐time detection. Herein, the synthesis and aggregation properties of a dicationic perylene probe (N ¹‐dodecyl‐N ³‐(4‐phenyl)benzimidazolium‐functionalized perylenediimide ( DAB‐PDI )) used to develop a fluorescent “turn‐on” ensemble for the detection of spermine are discussed. The fluorescence of DAB‐PDI (10 μm , Φ =0.55) is efficiently quenched by negatively charged sodium dodecylsulfate (SDS) through the formation of ionic self‐assembled aggregates (charge ratio of negative (N) in SDS to positive (P) in DAB‐PDI (N/P)=9). This negatively charged ionic self‐assembly between DAB‐PDI and SDS has been characterized by using photophysical, microscopic, dynamic light scattering, isothermal titration calorimetry, and HRMS techniques. The addition of spermine to this ensemble solution results in the breakdown of the DAB‐PDI –SDS ensemble owing to strong binding of spermine with SDS and, as a result, the fluorescence of DAB‐PDI is recovered. This ensemble exhibits high sensitivity and selectivity for spermine detection in water, urine, and blood serum. The lowest limit of detection is 27.5 nm , which is at least about 36 times lower than that required for early diagnosis of cancer (1 to 10 μm for urinary spermine).     

Solution Structure and Model Membrane Interactions of P‐113, a Clinically Active Antimicrobial Peptide Derived from Human Saliva

P‐113, AKRHHGYKRKFH‐NH₂, was derived from human saliva and found to possess clinical activity against fungus infections in HIV patients with oral candidiasis. We have determined the solution structure of P‐113 bound to membrane‐mimetic SDS micelles by two‐dimensional NMR methods. The SDS micelle‐bound structure of P‐113 adopts an α‐helical segment and the positively charged residues are clustered together to form a hydrophilic patch. A variety of biophysical and biochemical experiments, including circular dichroism, fluorescence spectroscopy and microcalorimetry, were used to show that P‐113 interacted with negatively charged phospholipid vesicles and induced dye release from these vesicles. However, its dye leakage efficiency is much less than the results of previously reported antimicrobial peptides. These results suggest that the antimicrobial activity of P‐113, unlike other antimicrobial peptides, may act not only through binding to and destabilization of the microbial membrane but also through a specific protein receptor on the microbial cell surface.     

Electrophoretic mobility of oil droplets in electrolyte and surfactant solutions

Electrophoretic mobility of oil droplets of micron sizes in PBS and ionic surfactant solutions was measured in this paper. The experimental results show that, in addition to the applied electric field, the speed and the direction of electrophoretic motion of oil droplets depend on the surfactant concentration and on if the droplet is in negatively charged SDS solutions or in positively charged hexadecyltrimethylammonium bromide (CTAB) solutions. The absolute value of the electrophoretic mobility increases with increased surfactant concentration before the surfactant concentration reaches to the CMC. It was also found that there are two vortices around the oil droplet under the applied electric field. The size of the vortices changes with the surfactant and with the electric field. The vortices around the droplet directly affect the drag of the flow field to the droplet motion and should be considered in the studies of electrophoretic mobility of oil droplets. The existence of the vortices will also influence the determination and the interpretation of the zeta potential of the oil droplets based on the measured mobility data.     

Novel pH‐responsive mixed‐charge copolymer brushes based on carboxylic acid and quaternary amine monomers

In this study, we report on the fabrication of tunable mixed‐charged copolymer brushes consisting of negatively charged carboxylic acid monomer (4‐vinylbenzoic acid, VBA) and positively charged quaternary amine monomer ((ar‐vinylbenzyl)trimethylammonium chloride) via reversible addition–fragmentation chain transfer‐mediated polymerization. The copolymer brushes have negative charge under neutral and basic conditions, and are positively charged under acidic conditions owing to the protonation of the carboxylate groups. The copolymer brushes revealed a unique reversible wetting behavior with pH. The reversible properties of the copolymer brushes can be employed to regulate the adsorption of charged biomacromolecules such as DNA and proteins. © 2013 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013     

Quantitative proteomics by fluorescent labeling of cysteine residues using a set of two cyanine‐based or three rhodamine‐based dyes

Despite all remarkable progress in gel‐based proteomics in recent years, there is still need to further improve quantification by decreasing the detection limits and increasing the dynamic range. These criteria are achieved best by fluorescent dyes that specifically stain the proteins either by adsorption after gel electrophoresis (in‐gel staining) or covalent coupling prior to gel electrophoresis (in‐solution staining). Here we report a multiplex analysis of protein samples using maleimide‐activated cyanine‐based (Cy3 and Cy5) and rhodamine‐based dyes (Dy505, Dy535, and Dy635) to permanently label all thiol‐groups of cysteine‐containing proteins. The detection limits in SDS‐PAGE were about 10 ng per band and even 2 ng for BSA due to its high content of cysteine residues. Thus only 5 μg protein of a mouse brain homogenate were analyzed by 2‐DE. Both cyanine‐ and rhodamine‐based dyes also stained proteins that did not contain cysteines, probably by reaction with amino groups. This side reactivity did not limit the method and might even extend its general use to proteins missing cysteine residues, but at a lower sensitivity. The dynamic range was more than two orders of magnitude in SDS‐PAGE and the Dy‐fluorophores did not alter the mobility of the tested proteins. Thus, a mixture of Dy505‐, Dy555‐, and Dy635‐labeled Escherichia coli lysates were separated by 2‐DE in a single gel and the three spot patterns relatively quantified.     

Aggregation‐Induced Emission Active Probe for Light‐Up Detection of Anionic Surfactants and Wash‐Free Bacterial Imaging

Anionic surfactants are widely used in daily life and industries, but their residues can cause serious damage to the environment. The current detection methods for anionic surfactants suffer from various limitations and a new detection strategy is highly desirable. Based on 2‐(2‐hydroxyphenyl)benzothiazole fluorogen with aggregation‐induced emission characteristics, we have developed a fluorescent probe HBT‐C₁₈ for selective and sensitive detection of anionic surfactants. By in situ formation of catanionic aggregates or micelles with anionic surfactants, the emission intensity of the HBT‐C₁₈ probe can increase with increasing keto/enol emission ratio through restriction of intramolecular motion and excited‐state intramolecular proton‐transfer mechanisms. The probe can also be used for wash‐free imaging of bacteria enveloped by a negatively charged outer membrane. The results of this study provide a new strategy for sensitive detection of anionic surfactants and wash‐free bacterial imaging.     

Intramolecular Charge Interactions as a Tool to Control the Coiled‐Coil‐to‐Amyloid Transformation

Under the influence of a changed environment, amyloid‐forming proteins partially unfold and assemble into insoluble β‐sheet rich fibrils. Molecular‐level characterization of these assembly processes has been proven to be very challenging, and for this reason several simplified model systems have been developed over recent years. Herein, we present a series of three de novo designed model peptides that adopt different conformations and aggregate morphologies depending on concentration, pH value, and ionic strength. The design strictly follows the characteristic heptad repeat of the α‐helical coiled‐coil structural motif. In all peptides, three valine residues, known to prefer the β‐sheet conformation, have been incorporated at the solvent‐exposed b, c, and f positions to make the system prone to amyloid formation. Additionally, pH‐controllable intramolecular electrostatic repulsions between equally charged lysine (peptide A) or glutamate (peptide B) residues were introduced along one side of the helical cylinder. The conformational behavior was monitored by circular dichroism spectroscopic analysis and thioflavin T fluorescence, and the resulting aggregates were further characterized by transmission electron microscopy. Whereas uninterrupted α‐helical aggregates are found at neutral pH, Coulomb repulsions between lysine residues in peptide A destabilize the helical conformation at acidic pH values and trigger an assembly into amyloid‐like fibrils. Peptide B features a glutamate‐based switch functionality and exhibits opposite pH‐dependent folding behavior. In this case, α‐helical aggregates are found under acidic conditions, whereas amyloids are formed at neutral pH. To further validate the pH switch concept, peptide C was designed by including serine residues, thus resulting in an equal distribution of charged residues. Surprisingly, amyloid formation is observed at all pH values investigated for peptide C. The results of further investigations into the effect of different salts, however, strongly support the crucial role of intramolecular charge repulsions in the model system presented herein.     

Poly(L‐lysine)‐Induced Aggregation of Single‐Strand Oligo‐DNA‐Modified Gold Nanoparticles

Single‐strand oligo‐DNA‐modified Au nanoparticles (AuNPs) undergo aggregation in the presence of poly(L ‐lysine) (PLL), which is attributed to the interactions between the oligo‐DNA and PLL. These interactions between the oligo‐DNA and PLL were identified to be electrostatic when the lysine residues of PLL were positively charged and to be hydrogen bonding when the residues were deprotonated. The aggregation was promoted with an increase in the pH value at a pH level lower than the pK_a value of PLL (pK_a≈10.0) due to the gradual deprotonation of the lysine residues and thus suppressed electrostatic interactions between the positively charged lysine residues of PLL and the negatively charged backbone phosphate groups of the oligo‐DNA. At pH levels higher than the pK_a value of PLL, the aggregation was identified to be dominated by the hydrogen bonds between the bases of the oligo‐DNA and the deprotonated lysine residues of PLL. This study prompts the possibility that the spectral, and thus color, change of AuNPs upon aggregation can be used as a probe to follow the interactions between oligo‐DNA and polypeptides.     

BOP: A basic phenylalanine‐rich oligo‐peptide located on the surface of glycolate oxidase influences its pI values

Glycolate oxidase (GO) consists of identical subunits and therefore should show one definite pI value, but the isolated GO exhibited a range of pIs. This study investigated the underlying cause of this phenomenon. GO was purified and showed a molecular weight of 40 kDa by SDS‐PAGE. Elution behavior on DEAE‐cellulose chromatography and cellulose acetate membrane electrophoresis indicated that the purified GO was highly basic (pI>10.0). Repeated IEF and cIEF analysis showed that the pI of the purified GO was in the range of 10.0–3.25, either in a smear form or as distinct bands. 2‐DE analysis showed that the 40 kDa subunit of GO displayed variable pIs from 9.6 to 3.65. It was likely that the purified GO was actually a complex consisted of GO and an unknown protein. CE‐SDS, SDS‐cellulose acetate membrane electrophoresis and amino acid compositions indicated that the unknown protein was a highly basic polymer (BP) consisting of basic and phenylalanine‐rich oligo‐peptide (BOP). Many BOPs are located on the surface of the acidic GO via ionic and hydrophobic interactions and formed GO‐BOP complex (GC), resulting in a highly basic GC although GO itself was acidic. This hypothesis was further supported by the facts that anti‐GC serum failed to recognize GO, and GC showed a peak at 257 nm although GO has few phenylalanine residues. Irregular and incomplete disassociation between GO and BOP was observed in IEF and cIEF, resulting in various intermediates with different ratios of GO/BOP, which could be the reason for the range of pIs observed for GO.     

Quaternization of Vinyl/Alkynyl Pyridine Enables Ultrafast Cysteine‐Selective Protein Modification and Charge Modulation

Quaternized vinyl‐ and alkynyl‐pyridine reagents were shown to react in an ultrafast and selective manner with several cysteine‐tagged proteins at near‐stoichiometric quantities. We have demonstrated that this method can effectively create a homogenous antibody–drug conjugate that features a precise drug‐to‐antibody ratio of 2, which was stable in human plasma and retained its specificity towards Her2+ cells. Finally, the developed warhead introduces a +1 charge to the overall net charge of the protein, which enabled us to show that the electrophoretic mobility of the protein may be tuned through the simple attachment of a quaternized vinyl pyridinium reagent at the cysteine residues. We anticipate the generalized use of quaternized vinyl‐ and alkynyl‐pyridine reagents not only for bioconjugation, but also as warheads for covalent inhibition and as tools to profile cysteine reactivity.     

Specific glutamic acid residues in targeted proteins induce exaggerated retardations in Phos‐tag SDS‐PAGE migration

We describe two unique proteins, Escherichia coli ClpX and human histone H2A, that show extremely retarded migrations relative to their molecular weights in Phos‐tag SDS‐PAGE, despite being nonphosphorylated. Although ClpX separated into multiple migration bands in Phos‐tag gels, the separation was not due to phosphorylation. The N‐terminal 47–61 region of ClpX was responsible for producing multiple phosphorylation‐independent structural variants, even under denaturing conditions, and some of these variants were detected as highly up‐shifted bands. By systematic Ala‐scanning mutation analysis in the N‐47–61 region, we concluded that the Glu‐51 or Glu‐54 residue was responsible for the appearance of exaggerated mobility‐shifting bands. Histone H2A showed a much slower migration in Phos‐tag gels in comparison with other major histones having similar molecular weights, and we found that the Glu‐62 or Glu‐65 residue caused the retarded migration. In addition, Phos‐tag SDS‐PAGE permitted us to detect a shift in the mobility of the phosphorylated form of histone H2A from that of the nonphosphorylated one. This is the first report showing that exaggerated retardation in the migration of a certain protein in Phos‐tag SDS‐PAGE is induced by interactions between the Phos‐tag molecule and the carboxylate group of a specific Glu residue on the target.     

Differences in electrophoretic behavior between linear and branched PEG‐conjugated proteins

The objective of this study was to characterize the differences in electrophoretic behavior between linear and branched PEG‐conjugated proteins. Human growth hormone and alpha‐lactalbumin modified by linear or branched PEGs with molecular weight of 10 kDa were analyzed by SEC, MALDI‐TOF MS, SDS‐PAGE, and microchip CGE (MCGE). Chromatographic and mass spectrometric differences between the linear and branched PEG‐proteins on SEC and MALDI‐TOF MS were small, but their electrophoretic behaviors on SDS‐PAGE and MCGE were significantly different. In particular, MCGE showed significant differences in the peak width and the migration times of linear and branched PEG‐proteins, in which the branched PEG‐proteins exhibited a narrower peak and longer migration time than the linear PEG‐proteins. This phenomenon may explain the longer circulation half‐life for the branched PEG‐proteins observed in previously reported in vivo studies. Consequently, this study indicates that MCGE may be a valuable tool for differentiating linear and branched PEG‐proteins.     

Energetic switch of the proline effect in collision‐induced dissociation of singly and doubly protonated peptide Ala‐Ala‐Arg‐Pro‐Ala‐Ala

Suppression of the selective cleavage at N‐terminal of proline is observed in the peptide cleavage by proteolytic enzyme trypsin and in the fragment ion mass spectra of peptides containing Arg‐Pro sequence. An insight into the fragmentation mechanism of the influence of arginine residue on the proline effect can help in prediction of mass spectra and in protein structure analysis. In this work, collision‐induced dissociation spectra of singly and doubly charged peptide AARPAA were studied by ESI MS/MS and theoretical calculation methods. The proline effect was evaluated by comparing the experimental ratio of fragments originated from cleavage of different amide bonds. The results revealed that the backbone amide bond cleavage was selected by the energy barrier height of the fragmentation pathway although the strong proton affinity of the Arg side chain affected the stereostructure of the peptide and the dissociation mechanism. The thermodynamic stability of the fragment ions played a secondary role in the abundance ratio of fragments generated via different pathways. Fragmentation studies of protonated peptide AACitPAA supported the energy‐dependent hypothesis. The results provide an explanation to the long‐term arguments between the steric conflict and the proton mobility mechanisms of proline effect.     

Anomalous electrophoretic migration of short oligodeoxynucleotides labelled with 5′‐terminal Cy5 dyes

By using a fluorescent exonuclease assay, we reported unusual electrophoretic mobility of 5′‐indocarbo‐cyanine 5 (5′‐Cy5) labelled DNA fragments in denaturing polyacrylamide gels. Incubation time and enzyme concentration were two parameters involved in the formation of 5′‐Cy5‐labelled degradation products, while the structure of the substrate was slightly interfering. Replacement of positively charged 5′‐Cy5‐labelled DNA oligonucleotides (DNA oligos) by electrically neutral 5′‐carboxyfluorescein (5′‐FAM) labelled DNA oligos abolished the anomalous migration pattern of degradation products. MS analysis demonstrated that anomalously migrating products were in fact 5′‐labelled DNA fragments ranging from 1 to 8 nucleotides. Longer 5′‐Cy5‐labelled DNA fragments migrated at the expected position. Altogether, these data highlighted, for the first time, the influence of the mass/charge ratio of 5′‐Cy5‐labelled DNA oligos on their electrophoretic mobility. Although obtained by performing 3′ to 5′ exonuclease assays with the family B DNA polymerase from Pyrococcus abyssi, these observations represent a major concern in DNA technology involving most DNA degrading enzymes.