Gas-phase interference-free analysis of protein ion charge-state distributions: detection of small-scale conformational transitions accompanying pepsin inactivation

Analysis of protein ion charge-state distributions in electrospray ionization (ESI) mass spectra has become an indispensable tool in the studies of protein dynamics. However, applications of this technique have been thus far limited to detection of large-scale conformational transitions, which typically change the extent of multiple charging in a very significant way. However, more subtle conformational changes often elude detection, since the resulting changes of the extent of multiple charging are often smaller than the charge-state shifts caused by other external factors. Proton-transfer reactions involving protein ions and residual solvent molecules are the major extrinsic factors causing changes of charge-state distributions unrelated to conformational transitions. Since the extent of such reactions depends on the amount of various solvent components transferred to the ESI interface, profound changes of solvent composition may affect protein ion charge-state distributions not only by affecting protein higher order structure in solution but also through modulation of the efficiency of proton-transfer reactions in the gas phase. Here we demonstrate that it is possible to choose experimental conditions in such a way that the influence of gas-phase ion chemistry on protein ion charge-state distributions is not altered over a wide pH range. This methodology (gas-phase interference-free analysis of protein ion charge-state distributions, or GIFPICS) is sensitive enough to allow detection of pepsin inactivation under mildly acidic conditions. Pepsin is active and tightly folded in its native strongly acidic environment. Inactivation of pepsin at mildly acidic pH is not accompanied by global unfolding, as spectroscopic measurements suggest the protein remains compact. GIFPICS provides a means to observe this small-scale conformational transition that does not result in protein unfolding and may in fact elude detection by traditional spectroscopic techniques.     

Trace analysis of antidepressant pharmaceuticals and their select degradates in aquatic matrixes by LC/ESI/MS/MS

Treated wastewater effluent is a potential environmental point source for antidepressant pharmaceuticals. A quantitative method was developed for the determination of trace levels of antidepressants in environmental aquatic matrixes using solid-phase extraction coupled with liquid chromatography-electrospray ionization tandem mass spectrometry. Recoveries of parent antidepressants from matrix spiking experiments for the individual antidepressants ranged from 72 to 118% at low concentrations (0.5 ng/L) and 70 to 118% at high concentrations (100 ng/L) for the solid-phase extraction method. Method detection limits for the individual antidepressant compounds ranged from 0.19 to 0.45 ng/L. The method was applied to wastewater effluent and samples collected from a wastewater-dominated stream. Venlafaxine was the predominant antidepressant observed in wastewater and river water samples. Individual antidepressant concentrations found in the wastewater effluent ranged from 3 (duloxetine) to 2190 ng/L (venlafaxine), whereas individual concentrations in the waste-dominated stream ranged from 0.72 (norfluoxetine) to 1310 ng/L (venlafaxine).     

Identification of bacteriophage MS2 coat protein from E. coli lysates via ion trap collisional activation of intact protein ions

Collisional activation of the intact MS2 viral capsid protein with subsequent ion/ion reactions has been used to identify the presence of this virus in E. coli lysates. Tandem ion trap mass spectrometry experiments on the +7, +8, and +9 charge states, followed by ion/ion reactions, provided the necessary sequence tag information (and molecular weight data) needed for protein identification via database searching. The most directly informative structural information is obtained from those charge states that produce a series of product ions arising from fragmentation at adjacent residues. The formation of these product ions via dissociation at adjacent amino acid residues depends greatly on the charge state of the parent ion. Database searching of the charge-state-specific sequence tags was performed by two different search engines: the ProteinInfo program from the Protein information Retrieval On-line World Wide Web Lab or PROWL and the TagIdent program from the ExPASy molecular biology server. These search engines were used in conjunction with the sequence tag information generated via collisional activation of the intact viral coat protein. These programs were used to evaluate the feasibility of generating sequence tags from collisional activation of intact multiply charged protein ions in a quadrupole ion trap.     

Detection of protein analytes via nanoparticle-based bio bar code technology

We describe a new format for the recently introduced bio bar code technology, which improves the dose response over 10,000-fold and thereby makes this technique analytically useful. Unlike other ultrasensitive protein detection methods, such as immuno-PCR or immuno-RCA, the bio bar code technique does not employ any enzymes to achieve detection limits in the attomolar range. By sandwiching a target between a magnetic bead and an amplifier nanoparticle, a multiplicity of bar code oligonucleotides are released for each captured target analyte. These surrogate bar code targets are then hybridized to microarrays and detected with silver-amplified gold nanoparticle probes. Using PSA detection as a model, we demonstrate a linear dose response over at least 4 orders of magnitude in both target concentration and concomitant signal and a 1000-fold improvement in detection limit compared to the best ELISA system.     

Analysis of multiple endocrine disruptors in environmental waters via wide-spectrum solid-phase extraction and dual-polarity ionization LC-ion trap-MS/MS

An analytical method for the determination of 35 endocrine disrupting chemicals (EDCs) present in the aquatic environment was developed and validated. The procedure includes an off-line solid-phase extraction of 500-mL water samples using wide-spectrum polymer packing material combined with two LC-ESI-MS/MS runs, in negative and positive ionization modes. Limits of quantitation were established between 0.1 and 20.0 ng/L. Satisfactory recoveries were obtained ranging from 80.1 to 110.2%. Calibration, using deuterated internal standardization, was performed by linear regression analysis. Linearity (R(2) > 0.99) was demonstrated over individually specified ranges using seven calibration points for each analyte. Intrabatch and interbatch precision, as well as accuracy (n = 5), were investigated at low, medium, and high concentrations. Precision for all compounds, expressed as the RSD, proved to be less than 17.8 and 20.0%, respectively, for intra- and interbatch. Accuracy, expressed as the mean recovery, was between 83.1 and 108.4% at all concentrations. Stability experiments showed no significant loss or deterioration for any of the analytes. Finally, the method was applied on real samples.     

Evaluating the separation of amphetamines by electrospray ionization ion mobility spectrometry/MS and charge competition within the ESI process.

The rapid increase in amphetamine abuse for recreational purposes has created a need for fast analysis and detection methodologies. For the first time, we show the separation of six amphetamines by ESI-IMS/MS. A complete analysis can be performed in 70 s, which is faster than traditional chromatographic techniques. In addition, ESI-IMS/MS was found to provide low detection limits for the six compounds (15.4 ppb for ethylamphetamine). Charge competition between amphetamines was found to occur at high amphetamine concentrations. The degree of preferential ionization was dependent on the functional group placed on the amine. Both one-analyte and two-analyte calibration curves were evaluated on the basis of the ion evaporation model. Evaporation rates were determined for the six amphetamines, and the rates were correlated with the degree of selective ionization. Evaluation of three typical ESI solvent compositions showed that the addition of a modifier (acetic acid and formic acid) enhanced the degree of preferential ionization for some amphetamines and increased the effect of charge competition. The solvent studies show the complexity of ESI and provide possible strategies for altering the amount of charge competition between analytes. Overall, ESI-IMS/MS appears to be a promising technique because of its sensitivity and rapid separation times for the amphetamines in aqueous samples; however, further research employing biological samples is required before it can be recommended as a mainstream technique.     

Low-attomole electrospray ionization MS and MS/MS analysis of protein tryptic digests using 20-microm-i.d. polystyrene-divinylbenzene monolithic capillary columns

This work explores the use of 20-microm-i.d. polymeric polystyrene-divinylbenzene monolithic nanocapillary columns for the LC-ESI-MS analysis of tryptic digest peptide mixtures. In contrast to the packing of microparticles, capillary columns were prepared, without the need of high pressure, in fused-silica capillaries, by thermally induced in situ copolymerization of styrene and divinylbenzene. The polymerization conditions and mobile-phase composition were optimized for chromatographic performance leading to efficiencies over 100000 plates/m for peptide separations. High mass sensitivity (approximately 10 amol of peptides) in the MS and MS/MS modes using an ion trap MS was found, a factor of up to 20-fold improvement over 75-microm-i.d. nanocolumns. A wide linear dynamic range (approximately 4 orders of magnitude) was achieved, and good run-to-run and column-to-column reproducibility of isocratic and gradient elution separations were found. As samples, both model proteins and tissue extracts were employed. Gradient nano-LC-MS analysis of a proteolytic digest of a tissue extract, equivalent to a sample size of approximately 1000 cells injected, is presented.     

Pulsed hydrogen/deuterium exchange MS/MS for studying the relationship between noncovalent protein complexes in solution and in the gas phase after electrospray ionization

Electrospray ionization mass spectrometry (ESI-MS) has become a standard method for monitoring noncovalent protein-protein interactions. Studies employing this approach tend to operate on the premise that the ionic species observed in the mass spectrum directly reflect the corresponding solution-phase protein quaternary structures. However, dissociation or clustering events taking place during ESI may lead to disparities between the ions observed in the mass spectrum and the protein binding state in bulk solution. Recognizing the occurrence of dissociation or clustering artifacts is not straightforward, leading to possible ambiguities in the interpretation of ESI-MS data. This work employs on-line pulsed hydrogen-deuterium exchange (HDX) for probing the origin of various species in the ESI mass spectrum of hemoglobin. In addition to the canonical hemoglobin tetramer, ESI-MS reveals the presence of monomers, dimers, hexamers, and octamers. Tandem mass spectrometry (MS/MS) is used for extracting HDX levels in a subunit-specific manner. Dimeric species exhibit exchange levels that are significantly above those of the tetramer. Monomeric hemoglobin subunits are labeled to an even greater extent. This HDX pattern implies that monomers and dimers do not represent dissociation artifacts generated during ESI. Instead, they are derived from preexisting solution-phase structures. In contrast, hexamers and octamers exhibit HDX levels that resemble those of the tetramer, thus identifying these larger species as nonspecific clustering artifacts. Overall, it appears that the pulsed HDX MS/MS approach introduced in this work represents a widely applicable tool for deciphering the relationship between ESI mass spectra and protein quaternary structures in solution.     

Role of accurate mass measurement (+/- 10 ppm) in protein identification strategies employing MS or MS/MS and database searching.

We describe the impact of advances in mass measurement accuracy, +/- 10 ppm (internally calibrated), on protein identification experiments. This capability was brought about by delayed extraction techniques used in conjunction with matrix-assisted laser desorption ionization (MALDI) on a reflectron time-of-flight (TOF) mass spectrometer. This work explores the advantage of using accurate mass measurement (and thus constraint on the possible elemental composition of components in a protein digest) in strategies for searching protein, gene, and EST databases that employ (a) mass values alone, (b) fragment-ion tagging derived from MS/MS spectra, and (c) de novo interpretation of MS/MS spectra. Significant improvement in the discriminating power of database searches has been found using only molecular weight values (i.e., measured mass) of > 10 peptide masses. When MALDI-TOF instruments are able to achieve the +/- 0.5-5 ppm mass accuracy necessary to distinguish peptide elemental compositions, it is possible to match homologous proteins having > 70% sequence identity to the protein being analyzed. The combination of a +/- 10 ppm measured parent mass of a single tryptic peptide and the near-complete amino acid (AA) composition information from immonium ions generated by MS/MS is capable of tagging a peptide in a database because only a few sequence permutations > 11 AA's in length for an AA composition can ever be found in a proteome. De novo interpretation of peptide MS/MS spectra may be accomplished by altering our MS-Tag program to replace an entire database with calculation of only the sequence permutations possible from the accurate parent mass and immonium ion limited AA compositions. A hybrid strategy is employed using de novo MS/MS interpretation followed by text-based sequence similarity searching of a database.     

Coumarin tags for analysis of peptides by MALDI-TOF MS and MS/MS. 2. Alexa Fluor 350 tag for increased peptide and protein Identification by LC-MALDI-TOF/TOF MS

The goal of this study was the development of N-terminal tags to improve peptide identification using high-throughput MALDI-TOF/TOF MS. Part 1 of the study was focused on the influence of derivatization on the intensities of MALDI-TOF MS signals of peptides. In part 2, various derivatization approaches for the improvement of peptide fragmentation efficiency in MALDI-TOF/TOF MS are explored. We demonstrate that permanent cation tags, while significantly improving signal intensity in the MS mode, lead to severe suppression of MS/MS fragmentation, making these tags unsuitable for high-throughput MALDI-TOF/TOF MS analysis. In the present work, it was found that labeling with Alexa Fluor 350, a coumarin tag containing a sulfo group, along with guanidation of epsilon-amino groups of Lys, could enhance unimolecular fragmentation of peptides with the formation of a high-intensity y-ion series, while the peptide intensities in the MS mode were not severely affected. LC-MALDI-TOF/TOF MS analysis of tryptic peptides from the SCX fractions of an E. coli lysate revealed improved peptide scores, a doubling of the total number of peptides, and a 30% increase in the number of proteins identified, as a result of labeling. Furthermore, by combining the data from native and labeled samples, confidence in correct identification was increased, as many proteins were identified by different peptides in the native and labeled data sets. Additionally, derivatization was found not to impair chromatographic behavior of peptides. All these factors suggest that labeling with Alexa Fluor 350 is a promising approach to the high-throughput LC-MALDI-TOF/TOF MS analysis of proteomic samples.     

A chemometric approach to detection and characterization of multiple protein conformers in solution using electrospray ionization mass spectrometry

Protein ion charge state distributions in electrospray ionization mass spectra have a potential to provide a wealth of information on protein dynamics, because they contain contributions from all protein conformers present in solution. Such ionic contributions often overlap, limiting the amount of useful information that can be extracted from the spectra. This difficulty is overcome in the present work by using a chemometric approach, which allows spectral deconvolution to be carried out and information on individual protein conformers to be extracted. Experiments are carried out by acquiring a series of spectra over a range of near-native and denaturing conditions to ensure significant changes in the conformers' populations. A total number of protein conformers sampled in the course of the experiments is determined by subjecting the set of collected spectra to singular value decomposition. Ionic contributions of each conformer to the total signal are then determined using a supervised optimization routine. Validation of the method has been carried out by monitoring acid- and alcohol-induced equilibrium states of well-characterized model proteins, chymotrypsin inhibitor 2 (two states), ubiquitin (three states) and apo-myoglobin (four states). For each of the model proteins, a new chemometric procedure yielded a picture of protein dynamics that was in excellent agreement with their documented behavior (as studied with other biophysical tools). The new method appears to be well-suited for monitoring protein dynamics in highly heterogeneous systems consisting of multiple proteins sampling a range of conformational states.     

Resolution of multiple ssDNA structures in free solution electrophoresis

By using high concentrations of buffer, electroosmotic flow within uncoated channels of a microfluidic chip was minimized, allowing the free solution electrophoretic separation of DNA. More importantly, because of the ability to efficiently dissipate heat within these channels, field strengths as high as 600 V/cm could be applied with minimal Joule heating (<2 degrees C). As a result of the higher field strengths, separations within an 8-cm-long channel were achieved within a few minutes. However, when the electrophoretic separation of single-stranded DNA (ssDNA) less than 22 bases in length was performed, containing the fluorophore Texas Red as an end label, more than the expected single peak was observed at this high electric field. On the other hand, the free solution electrophoresis of a double-stranded DNA (dsDNA) consisting of a random sequence did exhibit the expected single peak. The appearance of these multiple peaks for ssDNA is shown to be dependent upon the base content and sequence of the ssDNA as well as on the chemical structure of the fluorophore used to tag the DNA for detection. Specifically, the peaks can be attributed to different secondary structures that result either from hydrophobic interactions between the DNA bases and an uncharged fluorescent dye or from G-quadruplexes within guanine-rich strands.     

Coumarin tags for improved analysis of peptides by MALDI-TOF MS and MS/MS. 1. Enhancement in MALDI MS signal intensities

The goal of this study was the development of N-terminal tags to improve peptide identification using high-throughput MALDI-TOF MS and MS/MS. The proposed tags, commercially available fluorescent derivatives of coumarin, can be advantageous for peptide analysis in both MS and MS/MS modes. This paper, part 1, will focus on the influence of derivatization on the intensities of MALDI-TOF MS signals of peptides. Labeling peptides with tags containing the coumarin core was found to enhance the intensities of peptide peaks (in some cases over 40-fold) in MALDI-TOF MS using CHCA and 2,5-DHAP matrixes. The signal enhancement was found to be peptide- and matrix-dependent, being the most pronounced for hydrophilic peptides. No correlation was found between the UV absorptivity of the tags at the excitation wavelengths typical for UV-MALDI and the magnitude of the signal enhancement. Interestingly, peptides labeled with Alexa Fluor 350, a coumarin derivative containing a sulfo group (i.e., bearing strong negative charge), showed a 5-15-fold increase in intensity of MALDI MS signal in the positive ion mode, relative to the underivatized peptides, when 2,5-DHAP was used as the matrix. The Alexa Fluor 350 tag yielded a significantly higher signal relative to that for the CAF tag, likely due to the increased hydrophobicity of the coumarin structure. With 2,5-DHB, a decrease of MALDI MS signal was observed for all coumarin-labeled peptides, again relative to the unlabeled species. These findings support the hypothesis that derivatization with coumarin, a relatively hydrophobic structure, improves incorporation of hydrophilic peptides into hydrophobic MALDI matrixes, such as CHCA and 2,5-DHAP.     

Detection of native protein ions in aqueous solution under ambient conditions by electrospray laser desorption/ionization mass spectrometry

Liquid electrospray laser desorption/ionization (ELDI) mass spectrometry allows desorption and ionization of proteins directly from aqueous solutions and biological fluids under ambient conditions. Native protein ions such as those of myoglobin, cytochrome c, and hemoglobin were obtained. A droplet (ca. 5 microL) containing the protein molecules and micrometer-sized particles (e.g., carbon graphite powder) is irradiated with a pulsed UV laser. The laser energy adsorbed by the inert particles is transferred to the surrounding solvent and protein molecules, leading to their desorption; the desorbed gaseous molecules are then postionized within an electrospray (ESI) plume to generate the ESI-like protein ions. With the use of this technique, we detected only the protonated protein ions in various biological fluids (including human tears, cow milk, serum, and bacterial extracts) without interference from their corresponding sodiated or potassiated adduct ions. In addition, we rapidly quantified the levels of glycosylated hemoglobin present in drops of whole blood obtained from diabetic patients without the need of sample pretreatment.     

LC-atmospheric pressure chemical ionization-MS/ MS analysis of multiple illicit drugs,methadone, and their metabolites in oral fluid following protein precipitation

A quantitative LC-APCI-MS/MS method for simultaneous determination of multiple illicit drugs, methadone, and their metabolites in oral fluid was developed and validated. Sample pretreatment was limited to acetonitrile protein precipitation. LC separation was performed in 25.5 min, with a total analysis time of 35 min. Identification and quantitation were based on selected reaction monitoring. Calibration by linear regression analysis utilized deuterated internal standards and a weighing factor 1/x. Limits of detection and lower limits of quantitation (LOQ) were established between 0.25 and 5 ng/ mL and 0.5-10 ng/mL, respectively. linearity was obtained with an average correlation coefficient (R2) of >0.99, over a dynamic range from the LOQ up to maximum 500 ng/mL The method demonstrated good accuracy, intra- and interbatch precision, recovery, and stability for all compounds. No oral fluid matrix effect was observed throughout the chromatographic run. Protein precipitation provided a fast and simple sample pretreatment, while LC-APCI-MS/MS proved to be a sensitive and rugged quantitative method for multiple illicit and legal drugs in oral fluid. The method proved to be suitable for the evaluation of oral fluid as an alternative matrix to urine for monitoring illicit drug use and for determining oral fluid methadone concentrations in pregnant opiate and/or cocaine addicts.     

Detection of PCR products in solution using surface plasmon resonance

Polymerase chain reaction (PCR) products were detected using a flow injection-type sensor based on surface plasmon resonance. Asymmetric PCR was used to amplify the target DNA sequence, and two products with different length were produced. The novelty of our DNA detection system was that our target DNA was double stranded but the probe binding site, located in the 3'-terminus, was single stranded. This avoids the formation of intra- and intermolecular complexes. This novel design permitted us not only to detect PCR product but also to develop a rapid detection system for the detection of the verotoxin 2 gene of Escherichia coli O157:H7.     

An inverse estimation of multi-dimensional load distributions in thermoelasticity problems via dual reciprocity BEM

Abstact The paper deals with inverse thermoelasticity problems. A general, robust, and numerically efficient technique, for retrieving the multi-dimensional, highly varying distributions of boundary conditions is presented. In the class of considered inverse problems, both input data and sought-for quantities are usually specified at the domain boundary, only. As a sequence of forward sub-solutions underlies the inverse analysis, the numerical method of choice for solving the field problem is the boundary element method (BEM). The derived inverse technique is capable of retrieving boundary condition distributions in steady state and transient problems. The accuracy and stability of the algorithm are verified by considering problems involving constant, functionally graded, and temperature dependent material properties. Strain components and temperatures, subject to uncertainties, are used as input data. Presented numerical examples show that the method is capable of reconstructing mechanical and thermal loads with reasonable accuracy. while on leave from Cracow University of Technology, 31-155 Cracow, Poland     

Detection of multiphosphorylated peptides in LC-MS/MS analysis under low pH conditions

An improved method of detection of multiphosphorylated peptides by RPLC-MS/MS analysis under low pH conditions (pH approximately 1.7, 3% formic acid) is demonstrated for the model phosphoproteins, bovine alpha- and beta-casein. Changes in the pH conditions from normal (pH approximately 3.0, 0.1% formic acid) to low (pH approximately 1.7, 3% formic acid) significantly improved the detection limit of multiphosphorylated peptides carrying negative (-) solution charge states. In particular, bovine beta-casein tetraphosphorylated peptide, was detected with a loading amount of only 50 fmol of trypsin-digested bovine beta-casein under low pH conditions, which is 200 times lower than necessary to detect the peptide under normal pH conditions. In order to understand the low pH effect, various loading amounts of trypsin-digested bovine alpha- and beta-caseins were analyzed by RPLC-MS/MS analyses under two different pH conditions. The question of whether the low pH condition improves the detection of multiphosphorylated peptides by increasing ionization efficiencies could not be proven in this study because synthetic multiphosphorylated peptides could not be easily obtained by peptide synthesis. Interestingly, increased hydrophilicity resulting from multiple phosphorylation events is shown to negatively affect the peptide retention on reversed-phase column material. It was also demonstrated that the low pH condition could effectively enhance the retention of multiphosphorylated peptides on reversed-phase column material. The usefulness of low pH RPLC analysis was tested using an actual phosphopeptide-enriched sample prepared from mouse brain tissues. Previously, low pH solvents have been used in SCX fractionation and TiO2 enrichment processes to selectively enrich phosphopeptides during the phosphopeptide enrichment procedure, but the improved detection of multiphosphorylated peptides in RPLC-MS/MS analysis under low pH conditions has not been reported before (Ballif, B. A.; Villen, J.; Beausoleil, S. A.; Schwartz, D.; Gygi, S. P. Mol. Cell. Proteomics 2004, 3, 1093-1101. Villen, J.; Beausoleil, S. A.; Gerber, S. A.; Gygi, S. P. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 1488-1493. Schlosser, A.; Vanselow, J. T.; Kramer, A. Anal. Chem. 2005, 77, 5243-5250.).     

Polymer encapsulation of CdE (E = S, se) quantum dot ensembles via in-situ radical polymerization in miniemulsion

Cadmium sulfide and cadmium selenide/polymer nanocomposites were prepared via in-situ radical polymerization in miniemulsion. Organically capped CdE (E = S, Se) quantum dots (QDs) were used as the starting materials and ensembles of these dots were encapsulated with no need of further surface treatment. The use of two polymer matrices was investigated: polystyrene (PS) and poly(n-butyl acrylate) (PBA). In both cases, homogenous nanocomposites were obtained and their optical properties were investigated by visible absorption and photoluminescence spectroscopy. Quantum size effects were assigned to the nanocomposites, indicating the integrity of the individual QDs upon polymer encapsulation using the miniemulsion process.