Top-down analysis of 30–80 kDa proteins by electron transfer dissociation time-of-flight mass spectrometry

Electron transfer dissociation (ETD)-based top-down mass spectrometry (MS) is the method of choice for in-depth structure characterization of large peptides, small- and medium-sized proteins, and non-covalent protein complexes. Here, we describe the performance of this approach for structural analysis of intact proteins as large as the 80 kDa serotransferrin. Current time-of-flight (TOF) MS technologies ensure adequate resolution and mass accuracy to simultaneously analyze intact 30–80 kDa protein ions and the complex mixture of their ETD product ions. Here, we show that ETD TOF MS is efficient and may provide extensive sequence information for unfolded and highly charged (around 1 charge/kDa) proteins of ～30 kDa and structural motifs embedded in larger proteins. Sequence regions protected by disulfide bonds within intact non-reduced proteins oftentimes remain uncharacterized due to the low efficiency of their fragmentation by ETD. For serotransferrin, reduction of S–S bonds leads to significantly varied ETD fragmentation pattern with higher sequence coverage of N- and C-terminal regions, providing a complementary structural information to top-down analysis of its oxidized form.

Comprehensive identification of the binding sites of cisplatin in hen egg white lysozyme

Platinum drugs have become one of the most important kinds of chemotherapy agents, and the interactions of these drugs with proteins play very important roles in their side effects and drug resistance. However, it is still a challenge to determine the binding sites of platinum drugs in proteins with multiple disulfide bonds and stable three-dimensional structures using mass spectrometry. Here, the interaction between cisplatin and hen egg white lysozyme (HEWL), a multi-disulfide-bond-containing protein with a stable three-dimensional structure, was investigated using Fourier transform ion cyclotron resonance mass spectrometry. Typical disulfide bond reduction with dithiothreitol/tris(2-carboxyethyl)phosphine before trypsin digestion destroyed the binding of cisplatin to HEWL, and no platination sites were found. Efficient trypsin digestion methods for HEWL–cisplatin adducts were developed to avoid the loss of platinum binding to protein. At 55 °C, platinated HEWL was digested directly by trypsin in 6 h, and multiple platinated peptides were observed. In 60 % acetonitrile, the digestion time of platinated HEWL was shortened to 2 h, and most of the platinated peptides were observed. In addition, the reduction of the disulfide bonds of HEWL greatly accelerated the reaction between HEWL and cisplatin, and the potential binding sites of cisplatin in reduced HEWL could be easily recognized. On the basis of the above-mentioned methods, multiple binding sites of cisplatin in HEWL were first identified by mass spectrometry.

Collision-Induced Dissociation Fragmentation Inside Disulfide C-Terminal Loops of Natural Non-Tryptic Peptides

Collision-induced dissociation (CID) spectra of long non-tryptic peptides are usually quite complicated and rather difficult to interpret. Disulfide bond formed by two cysteine residues at C-terminus of frog skin peptides precludes one to determine sequence inside the forming loop. Thereby, chemical modification of S–S bonds is often used in “bottom up” sequencing approach. However, low-energy CID spectra of natural non-tryptic peptides with C-terminal disulfide cycle demonstrate an unusual fragmentation route, which may be used to elucidate the “hidden” C-terminal sequence. Low charge state protonated molecules experience peptide bond cleavage at the N-terminus of C-terminal cysteine. The forming isomeric acyclic ions serve as precursors for a series of b-type ions revealing sequence inside former disulfide cycle. The reaction is preferable for peptides with basic lysine residues inside the cycle. It may also be activated by acidic protons of Asp and Glu residues neighboring the loop. The observed cleavages may be quite competitive, revealing the sequence inside disulfide cycle, although S–S bond rupture does not occur in this case.

Reactions of Hydroxyalkyl Radicals with Cysteinyl Peptides in a NanoESI Plume

In biological systems, carbon-centered small molecule radicals are primarily formed via external radiation or internal radical reactions. These radical species can react with a variety of biomolecules, most notably nucleic acids, the consequence of which has possible links to gene mutation and cancer. Sulfur-containing peptides and proteins are reactive toward a variety of radical species and many of them behave as radical scavengers. In this study, the reactions between alkyl alcohol carbon-centered radicals (e.g., ?CH₂OH for methanol) and cysteinyl peptides within a nanoelectrospray ionization (nanoESI) plume were explored. The reaction system involved ultraviolet (UV) irradiation of a nanoESI plume using a low pressure mercury lamp consisting of 185 and 254 nm emission bands. The alkyl alcohol was added as solvent into the nanoESI solution and served as the precursor of hydroxyalkyl radicals upon UV irradiation. The hydroxyalkyl radicals subsequently reacted with cysteinyl peptides either containing a disulfide linkage or free thiol, which led to the formation of peptide-S-hydroxyalkyl product. This radical reaction coupled with subsequent MS/MS was shown to have analytical potential by cleaving intrachain disulfide linked peptides prior to CID to enhance sequence information. Tandem mass spectrometry via collision-induced dissociation (CID), stable isotope labeling, and accurate mass measurement were employed to verify the identities of the reaction products.

Fully automated isotopic dimethyl labeling and phosphopeptide enrichment using a microfluidic HPLC phosphochip

Quantitative detection of phosphorylation levels is challenging and requires an expertise in both stable isotope labeling as well as enrichment of phosphorylated peptides. Recently, a microfluidic device incorporating a nanoliter flow rate reversed phase column as well as a titania (TiO₂) enrichment column was released. This HPLC phosphochip allows excellent recovery and separation of phosphorylated peptides in a robust and reproducible manner with little user intervention. In this work, we have extended the abilities of this chip by defining the conditions required for on-chip stable isotope dimethyl labeling allowing for automated quantitation. The resulting approach will make quantitative phosphoproteomics more accessible.

LC-MS/MS Analysis and Comparison of Oxidative Damages on Peptides Induced by Pathogen Reduction Technologies for Platelets

Pathogen reduction technologies (PRT) are photochemical processes that use a combination of photosensitizers and UV-light to inactivate pathogens in platelet concentrates (PCs), a blood-derived product used to prevent hemorrhage. However, different studies have questioned the impact of PRT on platelet function and transfusion efficacy, and several proteomic analyses revealed possible oxidative damages to proteins. The present work focused on the oxidative damages produced by the two main PRT on peptides. Model peptides containing residues prone to oxidation (tyrosine, histidine, tryptophane, and cysteine) were irradiated with a combination of amotosalen/UVA (Intercept process) or riboflavin/UVB (Mirasol-like process). Modifications were identified and quantified by liquid chromatography coupled to tandem mass spectrometry. Cysteine-containing peptides formed disulfide bridges (R-SS-R, ?2 Da; favored following amotosalen/UVA), sulfenic and sulfonic acids (R-SOH, +16 Da, R-SO₃H, +48 Da, favored following riboflavin/UVB) upon treatment and the other amino acids exhibited different oxidations revealed by mass shifts from +4 to +34 Da involving different mechanisms; no photoadducts were detected. These amino acids were not equally affected by the PRT and the combination riboflavin/UVB generated more oxidation than amotosalen/UVA. This work identifies the different types and sites of peptide oxidations under the photochemical treatments and demonstrates that the two PRT may behave differently. The potential impact on proteins and platelet functions may thus be PRT-dependent.

A lipase-based electrochemical biosensor for target DNA

A lipase-based electrochemical biosensor has been fabricated for the quantitative determination of target DNA. It is based on a stem-loop nucleic acid probe labeled with ferrocene containing a butanoate ester that is hydrolyzed by lipase. The other end of the probe DNA is linked, via carboxy groups, to magnetic nanoparticles. The binding of target DNA transforms the hairpin structure of the probe DNA and causes the exposure of ester bonds. This results in the release of electro-active ferrocene after hydrolysis of the ester bonds, and in an observable electrochemical response. The quantity of target DNA in the concentration range between 1?×?10^?12 mol·L^?1 and 1?×?10^?8 mol·L^?1 can be determined by measuring the electrochemical current. The method can detect target DNA with rapid response (30 min) and low interference.

Electrochemical sensing of hydrogen peroxide using metal nanoparticles: a review

We are reviewing the state of electrochemical sensing of H₂O₂ based on the use of metal nanoparticles. The article is divided into subsections on sensors based on nanoparticles made from Ag, Pt, Pd, Cu, bimetallic nanoparticles and other metals. Some sensors display high sensitivity, fast response, and good stability. The review is subdivided into sections on sensors based on heme proteins and on nonenzymatic sensors. We also discussed the challenges of nanoscaled sensors and their future aspects.

Advantages of Monodisperse and Chemically Robust “SpheriCal” Polyester Dendrimers as a “Universal” MS Calibrant

The utilization of dendrimer calibrants as an alternative to peptides and proteins for high mass calibration is explored. These synthetic macromolecules exhibited a number of attractive advantages, including exceptional shelf-lives, broad compatibility with a wide range of matrices and solvents, and evenly spaced calibration masses across the mass range examined, 700–30,000 u. The exceptional purity of these dendrimers and the technical simplicity of this calibration platform validate their broad relevance for high molecular weight mass spectrometry.

Electrochemical sensor for nitrobenzene based on carbon paste electrode modified with a poly(3,4-ethylenedioxythiophene) and carbon nanotube nanocomposite

A sensitive and selective electrochemical sensor for the determination of nitrobenzene (NB) was developed based on a carbon paste electrode (CPE) modified with a nanocomposite prepared from the conducting polymer poly(3,4-ethylenedioxythiophene) and carbon nanotubes. The modified CPE exhibits good conductivity, a large surface area, and excellent catalytic activity towards the electrochemical reduction of NB. Under optimal conditions, the modified CPE is capable of detecting NB in the 0.25 to 43 μM concentration range and with a detection limit at 83 nM. Moreover, the sensor is highly stable and reusable, and free of interferences by other commonly present nitro compounds. It was used to determine NB in wastewater samples.

Ultra-sensitive electrochemical DNA biosensor based on signal amplification using gold nanoparticles modified with molybdenum disulfide,graphene and horseradish peroxidase

We have developed an ultra-sensitive electrochemical DNA biosensor by assembling probe ssDNA on a glassy carbon electrode modified with a composite made from molybdenum disulfide, graphene, chitosan and gold nanoparticles. A thiol-tagged DNA strand coupled to horseradish peroxidase conjugated to AuNP served as a tracer. The nanocomposite on the surface acts as relatively good electrical conductor for accelerating the electron transfer, while the enzyme tagged gold nanoparticles provide signal amplification. Hybridization with the target DNA was studied by measuring the electrochemical signal response of horseradish peroxidase using differential pulse voltammetry. The calibration plot is linear in the 5.0?×?10^?14 and 5.0?×?10^?9 M concentration range, and the limit of detection is 2.2?×?10^?15 M. The biosensor displays high selectivity and can differentiate between single-base mismatched and three-base mismatched sequences of DNA. The approach is deemed to provide a sensitive and reliable tool for highly specific detection of DNA.

Study of an Unusual Advanced Glycation End-Product (AGE) Derived from Glyoxal Using Mass Spectrometry

Glycation is a post-translational modification (PTM) that affects the physiological properties of peptides and proteins. In particular, during hyperglycaemia, glycation by α-dicarbonyl compounds generate α-dicarbonyl-derived glycation products also called α-dicarbonyl-derived advanced glycation end products. Glycation by the α-dicarbonyl compound known as glyoxal was studied in model peptides by MS/MS using a Fourier transform ion cyclotron resonance mass spectrometer. An unusual type of glyoxal-derived AGE with a mass addition of 21.98436 Da is reported in peptides containing combinations of two arginine-two lysine, and one arginine-three lysine amino acid residues. Electron capture dissociation and collisionally activated dissociation results supported that the unusual glyoxal-derived AGE is formed at the guanidino group of arginine, and a possible structure is proposed to illustrate the 21.9843 Da mass addition.

NADH oxidation using modified electrodes based on lactate and glucose dehydrogenase entrapped between an electrocatalyst film and redox catalyst-modified polymers

We have developed a simple and efficient method for the enhanced loading of silver nanoparticles onto carbon nanospheres, and how this method can be used to design an electrochemical sensor for hydrogen peroxide (HP). A glassy carbon electrode was modified with hemoglobin, carbon nanospheres, and by enhanced loading of silver nanoparticles onto the carbon nanospheres via spontaneous polymerization of dopamine. The hemoglobin exhibits a remarkable electrocatalytic activity for the reduction of HP. The electrochemical response to HP is linear range in the 1.0–147.0?μM concentration range, with a detection limit of 0.3?μM at a signal-to-noise ratio of 3.

Electrochemical determination of paraquat using a DNA-modified carbon ionic liquid electrode

Deoxyribonucleic acid (DNA) was electrochemically deposited on a carbon ionic liquid electrode to give a biosensor with excellent redox activity towards paraquat as shown by cyclic voltammetry and differential pulse voltammetry. Experimental conditions were optimized with respect to sensing paraquat by varying the electrochemical parameters, solution pH, and accumulation time of DNA. Under the optimized conditions, a linear relation exists between the reduction peak current and the concentration of paraquat in the range from 5?×?10^?8 mol L^?1 to 7?×?10^?5 mol L^?1, with a detection limit of 3.6?×?10^?9 mol L^?1. The utility of the method is illustrated by successful analysis of paraquat in spiked real water samples.

Preparation of a nanocomposite film from poly(diallydimethyl ammonium chloride) and gold nanoparticles by in-situ electrochemical reduction, and its application to SERS spectroscopy and sensing of ascorbic acid

A nanocomposite film is described that is composed of alternating layers of poly(diallydimethyl ammonium chloride) and gold nanoparticles that interact through electrostatic forces. The films of varying thickness were prepared by the layer-by-layer technique, and Au-NPs were generated by electrochemical reduction of hexachloroauric acid. The composite films were characterized by UV?Cvis spectroscopy, X-ray photoelectron spectroscopy, and cyclic voltammetry. Most nanocomposite films exhibit linear, uniform, and regular layer-by-layer growth during the process of formation. The films exhibit unique performance in terms of surface enhanced Raman scattering and electrocatalytic activitiy towards the oxidation of ascorbic acid.

Novel approach for electrochemical preparation of sulfur nanoparticles

An electrochemical method is presented for the preparation of sulfur nanoparticles (S-NPs) from thiosulfate ion. The particle size of the S-NPs can be adjusted between 35 and 65 nm by varying parameters such as the initial concentration of thiosulfate. The solvent/non-solvent precipitation method was also applied to the preparation of S-NPs for comparison. In this case, the use of hot alcohol and cold water as solvent/non-solvent system along with 100 ml·min^?1 flow rate for co-mixing of non-solvent resulted in the formation of S-NPs in a typical size of 250 nm that are fairly homogeneous in shape and have a narrow particle size distribution. The results revealed that, in comparison to the precipitation process, the electro-synthetic method offers simplicity, higher efficiency, improved size control, and less environmental contamination.

Biosensor based on a glassy carbon electrode modified with tyrosinase immmobilized on multiwalled carbon nanotubes

We describe a biosensor for phenolic compounds that is based on a glassy carbon electrode modified with tyrosinase immobilized on multiwalled carbon nanotubes (MWNTs). The MWNTs possess excellent inherent electrical conductivity which enhances the electron transfer rate and results in good electrochemical catalytic activity towards the reduction of benzoquinone produced by enzymatic reaction. The biosensor was characterized by cyclic voltammetry, and the experimental conditions were optimized. The cathodíc current is linearly related to the concentration of the phenols between 0.4???M and 10???M, and the detection limit is 0.2???M. The method was applied to the determination of phenol in water samples.

Use of quantum dots in the development of assays for cancer biomarkers

Biomarker assays may be useful for screening and diagnosis of cancer if a set of molecular markers can be quantified and statistically differentiated between cancerous cells and healthy cells. Markers of disease are often present at very low concentrations, so methods capable of low detection limits are required. Quantum dots (QDs) are nanoparticles that are emerging as promising probes for ultrasensitive detection of cancer biomarkers. QDs attached to antibodies, aptamers, oligonucleotides, or peptides can be used to target cancer markers. Their fluorescent properties have enabled QDs to be used as labels for in-vitro assays to quantify biomarkers, and they have been investigated as in-vivo imaging agents. QDs can be used as donors in assays involving fluorescence resonance energy transfer (FRET), or as acceptors in bioluminescence resonance energy transfer (BRET). The nanoparticles are also capable of electrochemical detection and are potentially useful for “lab-on-a-chip” applications. Recent developments in silicon QDs, non-blinking QDs, and QDs with reduced-size and controlled-valence further make these QDs bioanalytically attractive because of their low toxicity, biocompatibility, high quantum yields, and diverse surface modification flexibility. The potential of multiplexed sensing using QDs with different wavelengths of emission is promising for simultaneous detection of multiple biomarkers of disease.

Rapid colorimetric determination of reduced and oxidized glutathione using an end point coupled enzymatic assay

A simple and rapid colorimetric coupled enzymatic assay for the determination of glutathione is described. The proposed method is based on the specific reaction catalyzed by γ-glutamyltransferase, which transfers the γ-glutamyl moiety from glutahione to an acceptor, with the formation of the γ-glutamyl derivative of the acceptor and cysteinylglycine. The latter dipeptide is a substrate of leucyl aminopeptidase, which hydrolyzes cysteinylglycine to glycine and cysteine that can be easily measured spectrophotometrically. The proposed method was used to measure the content of glutathione in acid extracts of bovine lens, to follow the NADPH-dependent reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) catalyzed by the enzyme glutathione reductase and to determine the glutathione content in human astrocytoma ADF cells subjected to oxidative stress. The results obtained showed that the method can be suitably used for the determination of GSH and GSSG in different biological samples and to monitor tissue or cell redox status under different conditions. It is also applicable for following reactions involving GSH and/or GSSG.