"Mass defect" tags for biomolecular mass spectrometry

We present a new class of "mass defect" tags with utility in biomolecular mass spectrometry. These tags, incorporating element(s) with atomic numbers between 17 (Cl) and 77 (Ir), have a substantially different nuclear binding energy (mass defect) from the elements common to biomolecules. This mass defect yields a readily resolvable mass difference between tagged and untagged species in high-resolution mass spectrometers. We present the use of a subset of these tags in a new protein sequencing application. This sequencing technique has advantages over existing mass spectral protein identification methodologies: intact proteins are quickly sequenced and unambiguously identified using only an inexpensive, robust mass spectrometer. We discuss the potential broader utility of these tags for the sequencing of other biomolecules, differential display applications and combinatorial methods.     

Ionic-liquid-based catch and release mass spectroscopy tags for enzyme monitoring

A novel, inexpensive and versatile ionic-liquid-based catch and release mass spectrometry tag (I-Tag) that facilitates substrate purification, fast, robust and sensitive enzymatic reaction monitoring and quantitative kinetic analysis has been developed. The applicability of the system has been demonstrated in an enzymatic assay with β-1,4-galactosyltransferase.     

Generation of mass tags by the inherent electrochemistry of electrospray for protein mass spectrometry

We present herein a review of our work on the on-line electrochemical generation of mass tags toward cysteine residues in peptides and proteins. Taking advantage of the inherent electrochemical nature of electrospray generated from a microfabricated microspray emitter, selective probes for cysteine were developed and tested for on-line nonquantitative mass tagging of peptides and proteins. The nonquantitative aspect of the covalent tagging thus allows direct counting of free cysteines in the mass spectrum of a biomolecule through additional adduct peaks. Several substituted hydroquinones were investigated in terms of electrochemical properties, and their usefulness for on-line mass tagging during microspray experiments were assessed with L-cysteine, peptides, and intact proteins. Complementarily, numerical simulations were performed to properly understand the respective roles of mass transport, kinetics of electrochemical-chemical reactions, and design of the microspray emitter in the mass tagging overall efficiency. Finally, the on-line electrochemical tagging of cysteine residues was applied to the analysis of tryptic peptides of purified model proteins for protein identification through peptide mass fingerprinting.     

Clickable report tags for identification of modified peptides by mass spectrometry

The identification and quantification of modified peptides are critical for the functional characterization of post-translational protein modifications (PTMs) to elucidate their biological function. Nowadays, quantitative mass spectrometry coupled with various bioinformatic pipelines has been successfully used for the determination of a wide range of PTMs. However, direct characterization of low abundant protein PTMs in bottom-up proteomic workflow remains challenging. Here, we present the synthesis and evaluation of tandem mass spectrometry tags (TMT) which are introduced via click-chemistry into peptides bearing alkyne handles. The fragmentation properties of the two mass tags were validated and used for screening in a model system and analysis of AMPylated proteins. The presented tags provide a valuable tool for diagnostic peak generation to increase confidence in the identification of modified peptides and potentially for direct peptide-PTM quantification from various experimental conditions.     

Polymeric electrolytes based on vinylpyridines

Homopolymers of 4-vinylpyridine and 2-methyl-5-vinylpyridine and copolymers of 2-methyl-5-vinylpyridine with vinyl chloride and vinyl acetate were prepared by radical copolymerization. Organo-inorganic composites were prepared by hydrolytic polycondensation of the copolymers obtained and tetraethoxysilane. From solutions of the (co)polymers and composites, films were prepared by casting, and their protonic conductivity was determined.     

Proteome analyses using accurate mass and elution time peptide tags with capillary LC time-of-flight mass spectrometry

We describe the application of capillary liquid chromatography (LC) time-of-flight (TOF) mass spectrometric instrumentation for the rapid characterization of microbial proteomes. Previously (Lipton et al., Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11049) the peptides from a series of growth conditions of Deinococcus radiodurans have been characterized using capillary LC MS/MS and accurate mass measurements which are captured as an accurate mass and time (AMT) tag database. Using this AMT tag database, detected peptides can be assigned using measurements obtained on a TOF due to the additional use of elution time data as a constraint. When peptide matches are obtained using AMT tags (i.e., using both constraints) unique matches of a mass spectral peak occurs 88% of the time. Not only are AMT tag matches unique in most cases, the coverage of the proteome is high; approximately 3500 unique peptide AMT tags are found on average per capillary LC run. From the results of the AMT tag database search, approximately 900 ORFs detected using LC-TOFMS, with approximately 500 ORFs covered by at least two AMT tags. These results indicate that AMT database searches with modest mass and elution time criteria can provide proteomic information for approximately one thousand proteins in a single run of <3 h. The advantage of this method over using MS/MS based techniques is the large number of identifications that occur in a single experiment as well as the basis for improved quantitation. For MS/MS experiments, the number of peptide identifications is severely restricted because of the time required to dissociate the peptides individually. These results demonstrate the utility of the AMT tag approach using capillary LC-TOF MS instruments, and also show that AMT tags developed using other instrumentation can be effectively utilized.     

Polymeric materials based on oligodieneurethane-epoxy oligomers

Differential-scanning calorimetry in the dynamic mode was used to study the curing kinetics of a PDI-3AK macromolecular oligodieneurethane-epoxy oligomer with isomethyltetrahydrophthalic anhydride in the presence of a number of catalysts, modifiers, and fillers. The effect of their chemical nature on physicomechanical properties of cured polymers was examined.     

Facile, divergent route to bis-Zn(II)dipicolylamine type chemosensors for pyrophosphate

A new, two-step synthesis has been developed for a series of bis-DPA-type ligands whose dinuclear Zn(II) complexes function as fluorescent anion sensors. The Zn(II) complexes exhibit good selectivity for PP_i over other anions in aqueous medium (pH 7.5) and may be used to monitor the extent of enzyme-catalysed reactions, in which PP_i is produced or consumed.     

Polymeric waveguides based on photopolymerizing methacrylate compositions

Contact photolithography was used to fabricate three-layer polymeric waveguide structures with transverse dimensions of 50 × 90 μm on the basis of two photopolymerizing methacrylate compositions with different refractive indices. The optical loss on the wavelength of 632.8 nm was 0.37 ± 0.14 dB cm^?1. This loss is mostly due to the light scattering by the material of the waveguide layer produced by bulk polymerization.     

Confident assignment of intact mass tags to human salivary cystatins using top-down Fourier-transform ion cyclotron resonance mass spectrometry

A hybrid linear ion-trap Fourier-transform ion cyclotron resonance mass spectrometer was used for top-down characterization of the abundant human salivary cystatins, including S, S1, S2, SA, SN, C, and D, using collisionally activated dissociation (CAD) after chromatographic purification of the native, disulfide intact proteins. Post-translational modifications and protein sequence polymorphisms arising from single nucleotide polymorphisms (SNPs) were assigned from precursor and product ion masses at a tolerance of 10 ppm, allowing confident identification of individual intact mass tags. Cystatins S, S1, S2, SA, and SN were cleaved of a N-terminal 20 amino acid signal peptide and cystatin C a 26-residue peptide, to yield a generally conserved N-terminus. In contrast, cystatin D isoforms with 24 and 28 amino acid residue N-terminal truncations were found such that their N-termini were not conserved. Cystatin S1 was phosphorylated at Ser3, while S2 was phosphorylated at Ser1 and Ser3, in agreement with previous work. Both cystatin D isoforms carried the polymorphism C46R (SNP: rs1799841). The 14,328 Da isoform of cystatin SN previously assigned with polymorphism P31L due to a SNP (rs2070856) was found only in whole saliva. Parotid secretions contained no detectable cystatins while whole saliva largely mirrored the contents of submandibular/sublingual (SMSL) secretions. With fully characterized cystatin intact mass tags it will now be possible to examine the correlation between the abundance of these molecules and human health and disease.     

Impedance‐based viscoelastic flow cytometry

Elastic nature of the viscoelastic fluids induces lateral migration of particles into a single streamline and can be used by microfluidic based flow cytometry devices. In this study, we investigated focusing efficiency of polyethylene oxide based viscoelastic solutions at varying ionic concentration to demonstrate their use in impedimetric particle characterization systems. Rheological properties of the viscoelastic fluid and particle focusing performance are not affected by ionic concentration. We investigated the viscoelastic focusing dynamics using polystyrene (PS) beads and human red blood cells (RBCs) suspended in the viscoelastic fluid. Elasto‐inertial focusing of PS beads was achieved with the combination of inertial and viscoelastic effects. RBCs were aligned along the channel centerline in parachute shape which yielded consistent impedimetric signals. We compared our impedance‐based microfluidic flow cytometry results for RBCs and PS beads by analyzing particle transit time and peak amplitude at varying viscoelastic focusing conditions obtained at different flow rates. We showed that single orientation, single train focusing of nonspherical RBCs can be achieved with polyethylene oxide based viscoelastic solution that has been shown to be a good candidate as a carrier fluid for impedance cytometry.     

Fluorophore-linked zinc(II)dipicolylamine coordination complexes as sensors for phosphatidylserine-containing membranes

A series of Zn²⁺-2,2′-dipicolylamine (Zn²⁺-DPA) coordination complexes with an attached NBD fluorophore are synthesized and evaluated as fluorescent sensors. The sensors do not respond to vesicles composed of zwitterionic phosphatidylcholine, but the NBD fluorescence emission is enhanced in the presence of anionic vesicles. A sensor with two Zn²⁺-DPA units and a hydrophilic tris(ethyleneoxy) linker produced a larger emission enhancement than an analogue with a butyl linker, and titration with 1:1 POPC:POPS vesicles lead to an apparent phospholipid association constant of 5.3×10⁴ M⁻¹. The sensor can detect the presence of vesicles containing as little as 5% phosphatidylserine. The sensing effect apparently requires a membrane surface because the sensors do not respond to a phosphatidylserine derivative that is monodispersed in aqueous solution.     

Immunosensor based on carbon nanotube/manganese dioxide electrochemical tags

This article reports on carbon nanotube/manganese dioxide (CNT–MnO₂) composites as electrochemical tags for non-enzymatic signal amplification in immunosensing. The synthesized CNT–MnO₂ composites showed good electrochemical activity, electrical conductivity and stability. The electrochemical signal of CNT–MnO₂ composites coated glassy carbon electrode (GCE) increased by nearly two orders of magnitude compared to bare GCE in hydrogen peroxide (H₂O₂) environment. CNT–MnO₂ composite was subsequently validated as electrochemical tags for sensitive detection of α-fetoprotein (AFP), a tumor marker for diagnosing hepatocellular carcinoma. The electrochemical immunosensor demonstrated a linear response on a log-scale for AFP concentrations ranging from 0.2 to 100 ng mL⁻¹. The limit of detection (LOD) was estimated to be 40 pg mL⁻¹ (S/N = 3) in PBS buffer. Further measurements using AFP spiked plasma samples revealed the applicability of fabricated CNT–MnO₂ composites for clinical and diagnostic applications.     

Multipurpose peptide tags for protein isolation

A multifunctional peptide tag (HYDHYD) consisting of histidine, tyrosine and aspartate residues was fused to the N-terminal ends of green fluorescent protein (GFP), lactate dehydrogenase (LDH) and human hemoglobin (Hb), proteins which were subjected to ion-exchange chromatography (IEC), aqueous two-phase system partition, immobilized metal-ion affinity chromatography (IMAC), and hydrophobic interaction chromatography (HIC). Tagged GFP was retained significantly longer (>1 column volume) in both HIC and IEC. It exhibited 3x greater partition in favor of the hydrophobic phase in a two-phase system and 96% could be bound to an IMAC column which did not bind native GFP.     

A novel method for identification and relative quantification of N-terminal peptides using metal-element-chelated tags coupled with mass spectrometry

By means of the reaction between a DOTA-NHS-ester bifunctional reagent and N-terminal peptides of proteins,and then chelation of lanthanide metal ions as tags,we established a novel method for the identification of N-terminal peptides of proteins and their relative quantification using metal-element-chelated tags coupled with mass spectrometry.The experimental results indicate that metal elements are able to completely label N-terminal peptides at the protein level.The N-terminal peptides are enriched as the peptides digested with trypsin are selectively eliminated by isothiocyanate-coupled silica beads.We successfully identified the N-terminal peptides of 158 proteins of Thermoanaerobacter tengcongensis incubated at 55 and 75°C,among which N-terminal peptides of 24 proteins are partially acetylated.Moreover,metal-element tags with high molecule weights make it convenient for N-terminal peptides consisting of less than 6 amino acids to be identified;these make up 55percent of the identified proteins.Finally,we developed a general approach for the relative quantification of proteins based on N-terminal peptides.We adopted lysozyme and ribonuclease B as model proteins;the correlation coefficients(R2)of the standard curves for the quantitative method were 0.9994 and 0.9997,respectively,with each concentration ratio ranging from0.1 to 10 and both relative standard derivations(RSD)measured at less than 5%.In T.tengcongensis at two incubation temperatures,80 proteins possess quantitative information.In addition,compared with the proteins of T.tengcongensis incubated at 55°C,in T.tengcongensis incubated at 75°C,7 proteins upregulate whereas 16 proteins downregulate,and most differential proteins are related to protein synthesis.     

Simple synthesis of deuterium and 13C labeled trifluoromethyl phenyldiazirine derivatives as stable isotope tags for mass spectrometry

The synthesis of trifluoromethyl diazirine with a stable isotope tag is reported. We found that both Friedel-Crafts acylation and reduction of aryl carbonyl to methylene, using commercially available stable-isotope reagents, were utilized for the synthesis of diazirinyl fatty acid derivatives. The stable isotope labeled diazirine may be valuable for identifying binding sites by mass spectrometry.     

Polymeric composites based on alicyclic polyimide and polyaniline

Specific features of modification of an alicyclic polyimide with polyaniline were examined. The thus obtained new polymeric composites exhibit better capability for silver plating.