Capillary electrochromatography/nanoelectrospray mass spectrometry for attomole characterization of peptides

The successful coupling of capillary electrochromatography (CEC) to an ion trap mass spectrometer via a nanoelectrospray interface (nESI) is described. Using a conductively coated tip butted to the end of a CEC column, it was possible to obtain a stable spray without any sheath liquid being employed. Selected small peptides were separated with CEC columns (100 microm i.d./25 cm long) packed with 3 microm Hypersil C8 or C18 bonded silica particles with an eluent composed of ammonium acetate/acetonitrile. Peptide mixtures of desmopressin, peptide A, oxytocin, carbetocin and [Met(5)]-enkephalin were detected in the mid-attomole range, which is the lowest amount analyzed using CEC combined with MS detection. It was also observed that sensitivity can be compromised at higher separation voltages. We demonstrate that CEC/nESI-MS, at the current stage of development, represents one of the most sensitive systems for peptide analysis.     

Spatially resolved surface enhanced Raman spectroscopy: Feasibility,intensity dependence on sampling area and attomole mass sensitivity

Spatially resolved surface enhanced Raman scattering (SR SERS) from μm sized sampling areas has been observed in the electrochemical environment for the first time. Analysis of the equation for SR SERS intensity reveals that the detected signal should be area independent. This is demonstrated experimentally for the aqueous pyridine/Ag model system using 200 kW cm⁻² peak laser irradiance. The mass detection limits for SR SERS are found to be on the order of 10⁵ molecules or 0.17 attomoles!     

Mass spectrometric detection of attomole amounts of the prion protein by nanoLC/MS/MS

Sensitive quantitation of prions in biological samples is an extremely important and challenging analytical problem. Prions are the cause of several fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs). At this time, there are no methods to diagnose TSEs in live animals or to assure a prion-free blood supply for humans. Prions have been shown to be present in blood by transfusion experiments, but based on the amount of infectivity found in these types of experiments, the amount of misfolded prion protein in blood is estimated to be only 30 to 625 amol/mL. More sensitive detection of prions in brain would allow earlier detection of disease and assure a safer food supply. We studied quantitation of the prion protein by use of nanoscale liquid chromatography coupled to a tandem mass spectrometer using the multiple reaction monitoring mode of operation. We developed a method based on the detection of VVEQMCTTQYQK obtained by reduction, alkylation, and digestion with trypsin of the prion protein. Detection of VVEQMCTTQYQK was more sensitive than for the derivative with phenylisothiocyanate (PITC) because of decreased ionization efficiency of the PITC-derivatized peptides. The VVEQMCTTQYQK method has a LOD of 20 to 30 amol for pure standards. Proof of principle is demonstrated by quantitation of the amount of PrP 27-30 in the brains of terminally ill Syrian hamsters.     

Critical concentration for colloidal crystallization determined with microliter centrifuged suspensions

We report an elegant method using centrifugal sedimentation for determining the critical particle concentration for colloidal crystallization. A small amount of a dilute suspension of monodispersed particles stored in a flat capillary cell was centrifuged to temporarily generate a nonequilibrium gradient of the particle concentration including a crystalline-noncrystalline phase boundary in the cell. In the recovering process after the centrifugation, the particle concentration of the crystalline phase at the boundary was found to always have the equilibrium value, although the global concentration distribution evolved with time. The critical concentration was determined based on spatially resolved spectrometry. The present method requires only one batch of a suspension of the order of microliters and is applicable up to high concentration regions near the closest packing without the effect of the particle aggregation.     

Two-temperature hybridization for microarray detection of label-free microRNAs with attomole detection and superior specificity

Two is better than one: Two short locked nucleic acid based probes were used to collectively capture and detect microRNAs by a simple two-temperature hybridization process. Intact microRNAs were directly measured down to attomolar concentrations with a high specificity and nearly four orders of magnitude of dynamic range. Single base mismatches in the microRNAs were potently discriminated from the perfectly matched targets.     

Improved sensitivity for quantification of proteins using triply charged cleavable isotope-coded affinity tag peptides

Isotope-coded affinity tag (ICAT) methods, in conjunction with capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS), represent a promising approach for accurate protein quantification. However, sensitivity remains a challenge for the quantification of low-copy proteins in complex biological matrices. Here we investigated the electrospray ionization (ESI) and collision-activated dissociation (CAD) behavior of peptides derivatized with the cleavable ICAT (cICAT) reagent. For cICAT-peptides that were either synthesized or obtained by digestion of model proteins, the cICAT moiety showed a tendency toward protonation under positive ESI, producing relatively intense triply charged cICAT-peptide ions ([IP+3H]3+). [IP+3H]3+ exhibited significantly higher CAD reactivity than did the doubly charged cICAT-peptide ([IP+2H]2+), and produced a greater abundance of fragments at lower collision energies. Fragmentation spectra of [IP+3H]3+ showed variable intensities of doubly charged y and b ions, and the amount of sequence information obtained was dependent on the position of the cICAT-labeled cysteine residue in the peptide sequence. However, the absolute abundances of major fragments of [IP+3H]3+ were much higher than for [IP+2H]2+. Although the efficiency of identification of cICAT-peptides was compromised by their charge distribution toward the triply charged state and by the unique CAD behavior of the [IP+3H]3+ ions, it was found that the triply charged ions provided higher sensitivity than [IP+2H]2+ for quantification using multiple reaction monitoring (MRM). ESI and CAD conditions for MRM of [IP+3H]3+ were optimized, and, for all cICAT-peptides studied, MRM using [IP+3H]3+ as precursors showed 2- to 8-fold higher sensitivity than obtained using [IP+2H]2+, without compromising quantitative accuracy. Using this approach, the time course of tyrosine aminotransferase induction by methylprednisolone was monitored in rat livers. A remarkably better signal-to-noise ratio was observed by using [IP+3H]3+ for quantification compared to [IP+2H]2+.     

Electrospray sample deposition for matrix-assisted laser desorption/ionization (MALDI) and atmospheric pressure MALDI mass spectrometry with attomole detection limits

Electrospray sample deposition was explored for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). In this method, nanoliter volumes of matrix/analyte mixture were electrosprayed from a high voltage biased (1-2 kV) fused-silica capillary onto a grounded MALDI plate mounted 100-500 microm from the capillary outlet. Electrospray deposition with these conditions produced sample spots 200-300 microm in diameter thus matching the laser spot size. Varying spray voltage and distance resulted in different crystal sizes and volatilization rates for alpha-cyano-4-hydroxycinnamic acid matrix. Best results were obtained when the sample was deposited as wet droplets as opposed to deposition as dried solid. Under 'wet-spray' conditions, 2-4 microm diameter crystals were formed and detection limits for several neuropeptides were 0.7-25 amol. Samples could be pre-concentrated on the plate by spraying continuously and allowing sample to evaporate in a small spot. Sample volumes as large as 580 nL were deposited yielding a detection limit of 35 pM for neurotensin 1-11. Electrospray sample deposition yielded similar results when using atmospheric pressure-MALDI coupled with a quadrupole ion trap mass spectrometer, except that the sensitivity was approximately seven-fold worse.     

A glow discharge device for atomic emission spectrometry of microliter solution samples

A novel glow discharge device designed specifically for solution analysis is described. The detection limits obtained are comparable to those obtained with demountable hollow cathode lamps, but with better precision. Rotational and excitation temperatures are examined as functions of fill gas pressure and discharge current. A sputtering constant is presented and the technique for measuring this parameter is described.     

A validated liquid chromatographic/tandem mass spectrometric method for the determination of phencyclidine in microliter samples of rat serum

A liquid chromatographic/tandem mass spectrometric method is described for the determination of phencyclidine (PCP) in small volumes of rat serum (e.g. 50 microl). Samples were extracted using a mixed-mode strong cation-exchange column and then separated isocratically using a narrow-bore (2.1 mm i.d.) 3 microm Hypersil phenyl column and a mobile phase consisting of an ammonium formate buffer (pH 2.7) with 60% (v/v) methanol. Detection was accomplished using positive ion electrospray ionization in the multiple reaction monitoring mode. Mass spectra were obtained and peaks were observed at an m/z (% abundance) of 244 (100), 159 (25), and 86 (89). Tandem mass spectra were also obtained from the m/z 244 precursor ion with peaks observed at m/z 159 (100), 86 (96), and 91 (11). Optimum serum PCP sensitivity and precision were obtained at a transition of m/z 244 --> 159. Matrix-associated ion suppression did not significantly affect the accuracy (100-112%) or precision (CV < or =8%) of the assay. The lower limit of quantitation was 1 ng ml(-1) in 50 microl of serum. The method was used to study the serum pharmacokinetics of PCP in rats after an intravenous bolus dose of PCP.     

Characterization of responses to injection of microliter samples and to peak-shaped input signals for inductively-coupled plasma/atomic emission spectrometry

The use of inductively-coupled plasma/atomic emission spectrometry for dynamic measurements, e.g., as a detector in high-performance liquid chromatography or flow injection analysis with introduction of microliter samples, requires specification of the dynamic properties of the detector. A model of the transfer function in ICP/AES, based on the Γ-distribution, is described. The two important parameters of the model are easily evaluated in practice, so that the response of an ICP/AES detector to introduction of microliter samples and the influence on peak-shaped injections are readily calculated.     

Analytical evaluation of electrothermal vaporization/low-pressure inductively coupled plasma atomic emission spectrometry for trace elemental analysis in microliter samples

A novel method for the determination of trace elements in microliter samples using the tantalum filament electrothermal vaporization/low-pressure inductively coupled plasma (ETV/LP-ICP) atomic emission spectrometry has been developed. An improved tantalum filament ETV was directly coupled with LP-ICP system for efficient vaporization of microliter samples and further quantitative analysis. The experimental parameters including ETV current, rf power and mass flow rate of argon carrier gas were optimized using the copper emission signal produced by 5 μl of standard solution (5 μg/ml). Under the optimized condition, the analytical performances including linearity, precision and detection limit for the developed system were investigated. Absolute detection limits in the range of 22–391 pg for selected eight elements (Fe, Cu, Cr, Mn, Pb, K, Zn and Mg) were obtained with satisfactory precision (<8.9% RSD). The feasibility of the developed system has been demonstrated by analyzing wheat gluten NIST standard sample.     

Hollow waveguide quantum cascade laser spectrometer as an online microliter sensor for gas chromatography

An optical absorption sensor for gas chromatography (GC) is presented. It consists of a quantum cascade laser along with a long piece of Hollow Waveguide for Infrared (HWIR) transmission inserted into the GC line. It measures the infrared absorption in each individual gas peak after separation by the GC column, and maintains the shapes of gas peaks after the HWIR sensor, making the gas samples further available for other sensors. By adding an inline combustion module before the HWIR sensor, the concentrations of many carbon containing compounds can be acquired by measuring CO2 absorption in their peaks. The HWIR sensor detects isotopologues of CO2 separately, and therefore can be used to measure carbon isotope ratios of heavy compounds. Application of the HWIR sensor to the detection of 13CO2 and CDH3 is described.     

A Detection System Based on Giant Magnetoresistive Sensors and High‐Moment Magnetic Nanoparticles Demonstrates Zeptomole Sensitivity: Potential for Personalized Medicine

Zeptomole detector: A highly sensitive giant‐magnetoresistive chip and FeCo nanoparticles can be used to linearly detect 600–4500 copies of streptavidin. Under unoptimized conditions, this system also detects human IL‐6 with a sensitivity 13‐times higher than that of standard ELISA techniques.

     

Mechanism elucidation for nonstochastic femtosecond laser-induced ionization/dissociation: from amino acids to peptides

Femtosecond laser-induced ionization/dissociation (fs-LID) has been demonstrated as a novel ion activation method for use in tandem mass spectrometry. The technique opens the door to unique structural information about biomolecular samples that is not easily accessed by traditional means. fs-LID is able to cleave strong bonds while keeping weaker bonds intact. This feature has been found to be particularly useful for the mapping of post-translational modifications such as phosphorylation, which is difficult to achieve by conventional proteomic studies. Here we investigate the laser-ion interaction on a fundamental level through the characterization of fs-LID spectra for the protonated amino acids and two series of derivatized samples. The findings are used to better understand the fs-LID spectra of synthetic peptides. This is accomplished by exploring the effects of several single-residue substitutions.     

A sensitive high-pressure liquid chromatographic method for the determination of propranolol in microliter serum samples

Summary A high-pressure liquid chromatographic assay capable of quantitating subnanogram per milliliter concentrations of propranolol in microliter volume of serum was developed. Propranolol was extracted from serum with hexane at pH 9.7. Further sample clean up was achieved by back extraction. Separation of propranolol from its metabolites and normal serum constituents was performed on 10 m C₁₈ reversed-phase column packing. High sensitivity was attained with fluorescence detection using a low UV excitation wavelength. This assay is suitable for use in pharmacokinetic studies involving small laboratory animals.     

A novel derivatization method for peptides to increase sensitivity and backbone fragmentation in liquid secondary-ion mass spectra.

Derivatization is used to increase both negative-ion sensitivity and positive-ion sequence information in the liquid secondary-ion mass spectra (LSIMS) of a series of peptides. The derivatization method involves acylation with pentafluorobenzoyl fluoride in a single-step reaction, and the reaction mixture is applied directly to the probe tip for analysis. Acylation takes place at the unprotected N-terminus, tyrosine, and lysine. The derivatives exhibit increased signal-to-noise ratio for [M-H]- ions, especially where there is not already an acidic amino acid residue in the peptide. In positive-ion LSIMS, the N-terminal group acts to retain the charge at the N-terminus, simplifying the fragmentation by producing N-terminal fragment ions. It also increases positive-ion fragmentation, sometimes very dramatically, making sequence determination more straightforward. The simplicity of the process, together with the enhancements it provides, make this a generally useful method for obtaining peptide structural information.     

PLGA-based implants for sustained delivery of peptides/proteins: Current status,challenge and perspectives

Peptide and protein drugs with therapeutic effects suffer from their short half-life and low stability, albeit their high efficiency and specificity. To overcome these demerits, long-acting drug delivery systems have been developed, wherein poly(lactic-co-glycolic acid) (PLGA) implants are most preferred owing to their excellent biodegradability and biocompatibility. Dozens of PLGA based products have been approved since 1986, when the first product, named Decapeptyl^Ⓡ, successfully marched into market. To meet the increasing demand for delivering various peptides and proteins, different kinds of technologies have been developed for lab-scale fabrication or industrial manufacture. This review aims to introduce recent advances of PLGA implants, and give a brief summary of fundamental properties of PLGA, fabrication technologies of peptides/proteins-loaded PLGA implants as well as factors influencing the drug release processes. Moreover, challenges and future perspectives are also highlighted.