Detection of multiple protein conformational ensembles in solution via deconvolution of charge-state distributions in ESI MS.

Monitoring the changes in charge-state distributions of protein ions in electrospray ionization (ESI) mass spectra has become one of the commonly accepted tools to detect large-scale conformational changes of proteins in solution. However, these experiments produce only qualitative, low-resolution information. Our goal is to develop a procedure that would produce quantitative data on protein conformational isomers coexisting in solution at equilibrium. To that end, we have examined the evolution of positive ion charge-state distributions in the     

Gas-phase separations of protein and peptide ion fragments generated by collision-induced dissociation in an ion trap

Ion mobility/time-of-flight mass spectrometry techniques have been used to examine distributions of fragment ions generated by collision-induced dissociation (CID) in a quadrupole ion trap. The mobility-based separation step prior to mass-to-charge (m/z) analysis reduces spectral congestion and provides information that complements m/z-based assignments of peaks. The approach is demonstrated by examining fragmentation patterns of insulin chain B (a 30-residue peptide), and ubiquitin (a protein containing 76 amino acids). Some fragments of ubiquitin show evidence for multiple stable conformations.     

Optical detection and charge-state analysis of MALDI-generated particles with molecular masses larger than 5 MDa

Charged polystyrene nanoparticles are generated by matrix-assisted laser desorption/ionization (MALDI) and detected by laser-induced fluorescence (LIF) in a quadrupole ion trap. Employing the LIF technique, observations of individual fluorescent nanospheres (27 nm in diameter and containing 180 fluorescein dye equivalents) have been achieved with an average signal-to-noise ratio of approximately 10. With the trap operating at a frequency around 5 kHz, charge state analysis of the particles reveals that the number of charges carried by the spheres is between 1 and 10. It suggests a mass-to-charge ratio (m/z) in the range of 10(5)-10(6) for the MALDI-generated particles. To effectively trap such large particles (m > 5 MDa), damping of the particles' motions by using approximately 50 mTorr He buffer gas is absolutely required. Similar findings are obtained for particles with a nominal size of 1 microm in diameter, demonstrating that production of charged particles with a molecular mass as high as 10(12) Da is possible using the MALDI technique.     

Matrix interference-free method for the analysis of small molecules by using negative ion laser desorption/ionization on graphene flakes

This work presents a new approach for the analysis of small molecules with direct negative ion laser desorption/ionization (LDI) on graphene flakes. A series of matrix interference-free mass spectra were obtained for the analysis of a wide range of small molecules including peptides, amino acids, fatty acids, as well as nucleosides and nucleotides. The mixture of analytes and graphene flakes suspension were directly pipetted onto a sample plate for LDI-time-of-flight mass spectrometry (TOFMS) analysis. Deprotonated monomeric species [M-H](-) ions were homogeneously obtained on uniform graphene flakes film when negative ion mode was applied. In positive ion mode, the analytes were detected in form of multiple adduct ions such as sodium adduct [M+Na](+), potassium adduct [M+K](+), double sodium adduct [M+2Na-H](+), double potassium adduct [M+2K-H](+), as well as sodium and potassium mixed adduct [M+Na+K-H](+). Better sensitivity and reproducibility were achieved in negative ion mode compared to positive ion mode. It is believed that the new method of matrix interference-free negative ion LDI on graphene flakes may be expanded for LDI-MS analysis of various small molecules.     

Capillary electrophoresis with lamp-based wavelength-resolved fluorescence detection for the probing of protein conformational changes

Native protein fluorescence spectra encompass information on protein conformation. In this study, capillary electrophoresis (CE) combined with lamp-based wavelength-resolved fluorescence detection (wrFlu) is presented as a novel tool for the analysis of protein mixtures and the monitoring of protein unfolding. The CE-wrFlu system provides three-dimensional data (time, emission wavelength, intensity) from which electropherograms and accurate emission spectra of separated proteins can be extracted. For model proteins, linear detector responses (peak height vs concentration) were obtained (R(2) > 0.96) with detection limits (LODs) in the 6-32 nM range. The minimum protein concentration required for precise determination of the maximum emission wavelength by CE-wrFlu was about 15 times the LOD. Unfolding of various model proteins was induced by protein incubation and analysis in background electrolyte (BGE) containing 7.0 M urea. CE-wrFlu of the unfolded species revealed peaks with clear red-shifted spectra, which adequately corresponded to reference spectra obtained on a standard spectrophotometer. Moreover, unfolded proteins showed a significant decrease in effective electrophoretic mobility (after correction for BGE viscosity) due to the increase of their molecular hydrodynamic radii. It is concluded that the CE-wrFlu system provides two independent indicators for changes in protein folding and will allow the simultaneous assessment of protein purity and conformation.     

Ellipsometry analysis of conformational change of immobilized protein monolayer on plasma polymer surfaces

The conformational stability of surface immobilized protein monolayers is a key issue in applications requiring preservation of the protein bioactivity such as in biosensors and in vivo implants. Ellipsometry was used to detect conformational changes in a single monolayer of immobilized proteins on plasma polymer surfaces. The areal mass density of immobilized proteins was used to validate the data analysis in the protein denaturation analysis. We observed that the rate of conformation change was strongly dependent on the properties of the immobilized protein. Immobilized catalase showed a significantly slower denaturation rate than the immobilized horseradish peroxidase, indicating that the tetramer catalase is more stable than the immobilized monomer horseradish peroxidase at the surface/air interfaces. The ellipsometry results were in a good agreement with the enzyme activity analysis.     

Cyclophosphoramidate ion as mass defect marker for efficient detection of protein serine phosphorylation

A novel method is reported to modify the phosphate groups on phosphoserine peptides to the corresponding phosphoramidates, using 2-aminobenzylamine. Upon collision-induced dissociation, the modified peptides release the positively charged phosphoramidate that via gas-phase intramolecular elimination forms a cyclophosphoramidate (CyPAA) ion, the protonated form of 1,4-dihydro-2-hydroxy-2-oxobenzo[3,1,2]oxazaphosphorine. The positive nature of the ion eliminates the need for real-time instrument polarity switching and greatly increases the versatility of commonly used mass spectrometers for phosphopeptide analysis. This ion has sufficient mass defect, due to containing a phosphorus atom and a high content of oxygen atoms, which makes mass spectrometers of medium mass resolution and accuracy adequate for separating the ion from isobaric interfering ions. The specificity of the CyPAA ion for detecting phosphoserine peptides in complex peptide mixtures is comparable to the specificity of the phosphotyrosine immonium ion for phosphotyrosine peptides, allowing the efficient data complexity reduction for fast and focused analysis of phosphoserine-containing peptides.     

Investigation of polypeptide conformational transitions with two-dimensional Raman optical activity correlation analysis, applying autocorrelation and moving window approaches

The study of conformational transitions in polypeptides is not only important for the understanding of folding mechanisms responsible for the self-assembly of proteins but also for the investigation of the misfolding of proteins that can result in diseases including cystic fibrosis, Alzheimer's, and Parkinson's diseases. Our recent studies developing two-dimensional Raman optical activity (ROA) correlation analysis have proven to be successful in the investigation of polypeptide conformational transitions. However, the complexity of the ROA spectra, and the 2D correlation synchronous and asynchronous plots, makes data analysis detailed and complex, requiring great care in interpretation of 2D correlation rules. By utilizing the 2D correlation approaches of autocorrelation and moving windows it has been possible to gain further information from the ROA spectral data sets in a simpler and more consistent way. The most significant spectral intensity changes have been easily identified, facilitating appropriate interpretation of synchronous plots, and transition phases have been identified in the moving window plots, directly relating spectral intensity changes to the perturbation.     

Preparation, separation, and conformational analysis of differentially sulfated heparin octasaccharide isomers using ion mobility mass spectrometry

Heparin is a linear sulfated polysaccharide widely used in medicine because of its anticoagulant properties. The various sulfation and/or acetylation patterns on heparin impart different degrees of conformational change around the glycosidic bonds and subsequently alter its function as an anticoagulant, anticancer, or antiviral drug. Characterization of these structures is important for eventual elucidation of its function but presents itself as an analytical challenge due to the inherent heterogeneity of the carbohydrates. Heparin octasaccharide structural isomers of various sulfation patterns were investigated using ion mobility mass spectrometry (IMMS). In addition to distinguishing the isomers, we report the preparation and tandem mass spectrometry analysis for multiple sulfated or acetylated oligosaccharides. Herein, our data indicate that heparin octasaccharide isomers were separated on the basis of their structural conformations in the ion mobility cell. Subsequent to this separation, isomers were further distinguished using product ions resulting from tandem mass spectrometry. Overall, IMMS analysis was used to successfully characterize and separate individual isomers and subsequently measure their conformations.     

Gas-phase proton-transfer chemistry coupled with TOF mass spectrometry and ion mobility-MS for the facile analysis of poly(ethylene glycols) and PEGylated polypeptide conjugates

Gas-phase ion/molecule chemistry has been combined with ion mobility separation and time-of-flight mass spectrometry to enable the characterization of large poly(ethylene glycol)s (PEGs) and PEGylated molecules (>40 kDa). A facile method is presented in which gas-phase superbases are reacted in the high-pressure source region of commercial TOF mass spectrometers to manipulate the charge states of large ions generated by electrospray ionization (ESI). Charge stripping decreases the spectral congestion typically observed in ESI mass spectra of high molecular weight polydisperse PEGylated molecules. From these data, accurate average molecular weights and molecular weight distributions for synthetic polymers and PEGylated proteins are determined. The average MW measured for PEGylated Granulocyte colony-stimulating factor (rh-GCSF, 40 726.2 Da) is in good agreement with the theoretical value, and a 16 Da mass shift is easily observed in the spectrum of an oxidized form of the heterogeneous PEGylated protein. Ion mobility separations can fractionate PEGs of different chain length; when coupled with charge stripping ion/molecule reactions, ion mobility mass spectrometry (IMMS) offers several analytical advantages over mass spectrometry alone for the characterization of large PEGylated molecules including enhanced dynamic range, increased sensitivity, and specificity. Low abundance free PEG in a PEGylated peptide preparation, which is not directly detectable by mass spectrometry, can be easily observed and accurately quantified with gas-phase ion/molecule chemistry combined with ion mobility mass spectrometry.     

Reconstruction of cluster distributions at 100% detection efficiency for a track-nanodosimetric counter through a Bayesian analysis

A Bayesian unfolding has been applied to ionisation distributions due to 5.4 MeV alpha particles in a 20-nm site obtained using Monte Carlo simulations, taking into account different detection efficiency conditions. The ideal case of a target volume with uniform efficiency has been investigated to study the dependence of the reconstruction on prior distributions. A simplified approach has been used to treat the case of a target volume with non-uniformly distributed efficiency, like the sensitive volume of the track-nanodosimetric counter. Our results point out that Bayesian analysis provides a effective tool for reconstructing the true ionisation distributions, well beyond the maximum measured cluster size.     

Effect of molecular weight on conformational changes of PEO: an infrared spectroscopic analysis

Effect of molecular weight on conformation, helix structure (H structure) and trans planar structure (T structure), of Poly(ethylene oxide) (PEO) has been investigated in detail by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimeter. Two main diffraction peaks at about 2θ = 19° and 23° are discovered, and XRD patterns reveal that the unit cell of crystalline PEO belongs to the monoclinic lattice. The crystallinity decreases from 93.82 to 59.62 %, and the deviation of crystalline temperature of PEO-0.5 is larger than those of the other three under four reheated cycles. From FTIR results, a red shift about 11 cm^?1 is observed in the stretching vibration of –C–O–C– with increasing molecular weight, suggesting the presence of chain–chain interactions to restrict the stretching vibration of -C–O–C– in main chains. Meanwhile, the bending region of –C–C–O– at about 533 cm^?1 sensitive to tension shifts to lower wavenumber, and a new peak at about 510 cm^?1 emerges with increasing molecular weight, which is the indicator of internal tension/strain and orientation. Furthermore, the peak intensity ratios of H structure decrease with increasing molecular weight. In contrast, T structure increases dramatically. Consequently, with respect to molecular weight, the possible interactions, entanglements and tie molecules, of PEO molecular chains to explain the difference between H and T structure is proposed, which is in agreement with the experimental observations quite well.     

Ion detection by Fourier transform ion cyclotron resonance: the effect of initial radial velocity on the coherent ion packet

Ion detection by Fourier transform ion cyclotron resonance (FT-ICR) is accomplished by observing a coherent ion packet produced from an initially random ensemble of ions. The coherent packet is formed by excitation with a resonant oscillating electric field. Ions that are out of phase with the applied radio frequency (rf) electric field experience a continuous misalignment of the electric field vector. The misalignment creates a net force of the electric field perpendicular to ion motion. The perpendicular component of the rf electric field creates a frequency shift resulting in phase synchronization of the ion ensemble. The phase coherence of the ion packet affects both the sensitivity and the resolution of FT-ICR.     

Determination of electrophoretic mobility distributions through the analysis of individual mitochondrial events by capillary electrophoresis with laser-induced fluorescence detection.

Here we report on the analysis of mitochondrial preparations by capillary electrophoresis with postcolumn laser-induced fluorescence detection. Individual mitochondria are detected by fluorescent labeling with the mitochondrion-selective probe, 10-nonyl acridine orange. Interactions between the organelles and the capillary walls are controlled by derivatization of the capillaries with poly(acryloylaminopropanol). As expected from the presence of charged groups in their outer membranes, isolated mitochondria have intrinsic electrophoretic mobilities. This property may be influenced by variations in size, morphology, membrane composition, and damage caused during the isolation procedure. The mobility distributions of mitochondria isolated from NS1 and CHO cells ranged from -1.2 x 10(-4) to -4.3 x 10(-4) cm2 V(-1) s(-1) and -0.8 x 10(-4) to -4.2 x 10(-4) cm2 V(-1) s(-1), respectively. Furthermore, there seems to be no correlation between the density of the mitochondrial fraction and the resultant electrophoretic mobility distribution. These results suggest a new method for characterization of organelle fractions and for counting individual organelles.     

Whole protein dissociation in a quadrupole ion trap: identification of an a priori unknown modified protein

A protein mixture derived from a whole cell lysate fraction of Saccharomyces cerevisiae, which contains roughly 19 proteins, has been analyzed to identify an a priori unknown modified protein using a quadrupole ion trap tandem mass spectrometer. Collection of the experimental data was facilitated by collision-induced dissociation and ion/ion proton-transfer reactions in multistage mass spectrometry procedures. Ion/ion reactions were used to manipulate charge states of both parent ions and product ions for the purpose of concentrating charge into the parent ion of interest and to reduce the product ion charge states for determination of product ion mass and abundance. The identification of the protein was achieved by matching the uninterpreted product ion spectrum against protein sequence databases with varying degrees of annotation, coupled with a scoring scheme weighted for the relative abundances of the experimentally observed product ions and the frequency of fragmentations occurring at preferential sites. The protein was identified to be an acetylated yeast heat shock protein, HS12_Yeast (11.6 kDa), with the initiating methionine residue removed. This constitutes the first example of the identification of an a priori unknown protein that is not present in an annotated protein database using a "top-down" approach with a quadrupole ion trap. This example illustrates the utility of relatively low cost instrumentation with modest mass analysis characteristics for the identification of modified proteins without recourse to enzymatic digestion. It also illustrates how experimental data can be used interactively with protein databases when the modified protein of interest is not initially present in the database.     

Label-acquired magnetorotation as a signal transduction method for protein detection: aptamer-based detection of thrombin

This paper presents a new signal transduction method, called label-acquired magnetorotation (LAM), for the measurement of the concentration of proteins in solution. We demonstrate the use of LAM to detect the protein thrombin using aptamers, with a limit of detection of 300 pM. LAM is modeled after a sandwich assay, with a 10 μm nonmagnetic "mother" sphere as the capture component and with 1 μm magnetic "daughter" beads as the labels. The protein-mediated attachment of daughter beads to the mother sphere forms a rotating sandwich complex. In a rotating magnetic field, the rotational frequency of a sandwich complex scales with the number of attached magnetic beads, which scales with the concentration of the protein present in solution. This paper represents the first instance of the detection of a protein using LAM.     

Multiples,poisson distributions,and chance: An analysis of the Brannigan-Wanner model

Brannigan andWanner argue that the empirical distribution of multiple grades can be more adequately explained in terms of a negative contagious poisson model. This alternative is based on a Zeitgeist theory which places emphasis on the role of communication in scientific discovery. Nonetheless, a detailed analysis indicates the following: (a) mathematically, the simple Poisson is the limiting case of the contagious Poisson when the contagion parameter approaches zero; (b) empirically, the mean and variance are so nearly equal (i. e., the contagion effect is very small) that predictions from the contagious Poisson are virtually equivalent to those of the simple Poisson; (c) in particular, both distributions predict that multiples are less common than singletons and even nulltons, the latter occurring with a probability of over one third (thereby implying that chance plays a much bigger part than Zeitgeist or maturational theories would suggest); (d) estimates from theSimonton, Merton, andOgburn-Thomas data sets all concur that the contagion effect is not only small, but positive besides, yielding a modest positive contagious Poisson that contradicts the principal tenet of the communication interpretation.     

Copper quantum clusters in protein matrix: potential sensor of Pb2+ ion

A one-pot synthesis of extremely stable, water-soluble Cu quantum clusters (QCs) capped with a model protein, bovine serum albumin (BSA), is reported. From matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, we assign the clusters to be composed of Cu(5) and Cu(13) cores. The QCs also show luminescence properties having excitation and emission maxima at 325 and 410 nm, respectively, with a quantum yield of 0.15, which are found to be different from that of protein alone in similar experimental conditions. The quenching of luminescence of the protein-capped Cu QCs in the presence of very low hydrogen peroxide concentration (approximately nanomolar, or less than part-per-billion) reflects the efficacy of the QCs as a potential sensing material in biological environments. Moreover, as-prepared Cu QCs can detect highly toxic Pb(2+) ions in water, even at the part-per-million level, without suffering any interference from other metal ions.     

Development of high-sensitivity ion trap ion mobility spectrometry time-of-flight techniques: a high-throughput nano-LC-IMS-TOF separation of peptides arising from a Drosophila protein extract

A linear octopole trap interface for an ion mobility time-of-flight mass spectrometer has been developed for focusing and accumulating continuous beams of ions produced by electrospray ionization. The interface improves experimental efficiencies by factors of approximately 50-200 compared with an analogous configuration that utilizes a three-dimensional Paul geometry trap (Hoaglund-Hyzer, C. S.; Lee, Y. J.; Counterman, A. E.; Clemmer, D. E. Anal. Chem. 2002, 74, 992-1006). With these improvements, it is possible to record nested drift (flight) time distributions for complex mixtures in fractions of a second. We demonstrate the approach for several well-defined peptide mixtures and an assessment of the detection limits is given. Additionally, we demonstrate the utility of the approach in the field of proteomics by an on-line, three-dimensional nano-LC-ion mobility-TOF separation of tryptic peptides from the Drosophila proteome.     

Aptamer-based rolling circle amplification: a platform for electrochemical detection of protein

Aptamer-based rolling circle amplification (aptamer-RCA) was developed as a novel versatile electrochemical platform for ultrasensitive detection of protein. This method utilized antibodies immobilized on the electrode surface to capture the protein target, and the surface-captured protein was then sandwiched by an aptamer-primer complex. The aptamer-primer sequence mediated an in situ RCA reaction that generated hundreds of copies of a circular DNA template. Detection of the amplified copies via enzymatic silver deposition then allowed enormous sensitivity enhancement in the assay of target protein. This novel aptamer-primer design circumvented time-consuming preparation of the antibody-DNA conjugate for the common immuno-RCA assay. Moreover, the detection strategy based on enzymatic silver deposition enabled a highly efficient readout of the RCA product as compared to a redox-labeled probe based procedure that might exhibit low detection efficiency due to RCA product distance from the electrode. With the platelet-derived growth factor B-chain (PDGF-BB) as a model target, it was demonstrated that the presented method was highly sensitive and specific with a wide detection range of 4 orders of magnitude and a detection limit as low as 10 fM. Because of the wide availability of aptamers for numerous proteins, this platform holds great promise in ultrasensitive immunoassay.