Low Femtomole Range Research Articles

Identification of 4-hydroxy-2-nonenal-modified peptides within unfractionated digests using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The lipid peroxidation product 4-hydroxy-2-nonenal (HNE) is generated as a consequence of oxidative stress and can readily react with nucleophilic sites of proteins (e.g., histidine residues), mainly via a Michael addition. The formation of such lipid-protein conjugates can alter protein properties and biological functions, thus leading to highly deleterious effects. The present work describes a rapid (very limited sample preparation) and sensitive (low-femtomole range) procedure to identify HNE-modified peptides (Michael adducts) within unfractionated tryptic digests. The protocol involves the formation of dinitrophenylhydrazones of the Michael adducts, when using 2,4-dinitrophenylhydrazine as reactive matrix, followed by analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The hydrazone derivatives present high desorption/ionization yield and can thus be preferentially detected compared to unmodified peptides. The MALDI mass spectrum obtained is therefore drastically different from the one obtained with the classical 4-hydroxy-alpha-cyanocinnamic acid matrix. Moreover, the presence of HNE, or more generally speaking carbonylated peptides, could be highlighted by 180 mass units differences (corresponding to the dinitrophenylhydrazone moiety) between these two MALDI mass spectra. Further information (e.g., localization/identification of the modified residues, peptide sequences) could be obtained by performing MALDI postsource decay (or electrospray) MS/MS experiments on the ions of interest.

Novel biosensor chip for simultaneous detection of DNA-carcinogen adducts with low-temperature fluorescence

A monoclonal antibody (MAb)–gold biosensor chip with low-temperature laser-induced fluorescence detection for analysis of DNA-carcinogen adducts is described. Optimization of the detection limit, dynamic range, and biosensing applicability of the MAb–gold biosensor chip was achieved by: (1) using dithiobis(succinimidyl propionate (DSP)) as a protein linker and (2) employing recombinant protein A to provide oriented immobilization of the MAbs. The use of DSP, which has a short methylene chain length, led to faster protein binding kinetics and higher protein surface density than a longer dithiobis(succinimidyl undecanoate) (DSU) linker. The incorporation of recombinant protein A increased the distance between the oriented MAb-bound analytes and the gold surface. The increased distance minimized fluorescence quenching, resulting in about a 10-fold increase in the fluorescence signal in comparison with a chip without protein A. The improved chip architecture was used to demonstrate that biosensing of two structurally similar benzo[ a]pyrene (BP)-derived DNA adducts, BP-6-N7Gua and BP-diolepoxide-10-N 2dG, bound to two specific MAbs immobilized from a mixture at the same address on the chip, is feasible. These mutagenic adducts are formed by one-electron oxidation and monooxygenation pathways, and are depurinating and stable DNA adducts, respectively. It is shown that the DNA adducts can be easily identified at the same address using time-resolved, low-temperature laser-based fluorescence spectroscopy. The current limit of detection is in the low femtomole range. These results indicate that a single biosensor chip consisting of a Au/DSP/protein A/MAb nanoassembly, with analyte-specific MAbs and low-temperature fluorescence detection should be suitable for simultaneous detection and quantitation of the above adducts, as well as the luminescent antigens for which selective MAbs exist.

Spectral characterization of catechol estrogen quinone (CEQ)-derived DNA adducts and their identification in human breast tissue extract.

Estrogens, including the natural hormones estrone (E(1)) and estradiol (E(2)), are thought to be involved in tumor induction. Catechol estrogen quinones (CEQ) derived from 4-hydroxyestrone (4-OHE(1)) and 4-hydroxyestradiol (4-OHE(2)) react with DNA and form depurinating N7Gua and N3Ade adducts that might be responsible for tumor initiation (Cavalieri, E. L., et al. (2000) J. Natl. Cancer Inst. Monogr. 27, 75). Current detection limits for the CEQ-derived DNA adducts by high-performance liquid chromatography with multichannel electrochemical detection are in the picomole range. To improve the limit of detection (LOD) for CEQ-derived DNA adducts, spectrophotometric monitoring was investigated. Spectroscopic studies of 4-OHE(1)-1-N3Ade, 4-OHE(1)-1-N7Gua, 4-OHE(2)-1-N3Ade, and 4-OHE(2)-1-N7Gua adduct standards were performed at 77 and 300 K. Upon laser excitation at 257 nm, the 4-OHE(1)- and 4-OHE(2)-derived N7Gua and N3Ade adducts are strongly phosphorescent at T = 77 K. No phosphorescence was observed at 300 K. Both N3Ade and N7Gua adduct types have weak phosphorescence origin bands near 383 and 385 nm, respectively. The corresponding phosphorescence lifetimes are 1.11 +/- 0.05 and 0.37 +/- 0.05 s. The LOD, based on phosphorescence measurements, is in the low femtomole range. The concentration LOD is approximately 10(-9) M, i.e., similar to that recently obtained for CEQ-derived N-acetylcysteine conjugates (Jankowiak, R., et al. (2003) Chem. Res. Toxicol. 16, 304). The LOD in capillary electrophoresis (CE) with field-amplified sample stacking and absorbance detection is about 3 x 10(-8) M. To verify whether CEQ-derived DNA adducts are formed in humans or not, tissue extracts from two breast cancer patients were analyzed by CE interfaced with room temperature absorption and low temperature (laser-excited) phosphorescence spectroscopies. For the first time, formation of CEQ-derived DNA adducts is shown in humans. For example, the level of 4-OHE(1)-1-N3Ade in the breast tissue extract from a patient with breast carcinoma (8.40 +/- 0.05 pmol/g of tissue) is larger by a factor of about 30 than that in the breast tissue sample from a woman without breast cancer (0.25 +/- 0.05 pmol/g of tissue). In contrast, similar amounts of 4-OHE(2)-1-N3Ade were observed in both types of tissue. Although more breast tissue samples from women with and without breast cancer need to be studied, these results suggest that the N3Ade adducts could serve as biomarkers to predict the risk of breast cancer.

Fourier transform ion cyclotron resonance mass spectrometry with nanolc/microelectrospray ionization and matrix-assisted laser desorption/ionization: analytical performance in peptide mass fingerprint analysis

Protein identifications by peptide mass fingerprint analyses with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) were performed using microelectrospray ionization coupled to nano liquid chromatography (NanoLC), as well as using matrix-assisted laser desorption/ionization (MALDI). Tryptic digests of bovine serum albumin (BSA), diluted down to femtomole quantities, have been desalted by fast NanoLC under isocratic elution conditions as the high resolving power of FT-ICR MS enables peptides to be separated during the mass analysis stage of the experiment. The high mass accuracy achieved with FT-ICR MS (a few ppm with external calibration) facilitated unambiguous protein identification from protein database searches, even when only a few tryptic peptides of a protein were detected. Statistical confidence in the database search results was further improved by internal calibration due to increased mass accuracy. Matrix-assisted laser desorption/ionization and micro electrospray ionization (ESI) FT-ICR showed good mass accuracies in the low femtomole range, yet a better sensitivity was observed with MALDI. However, in higher femtomole ranges slightly lower mass accuracies were observed with MALDI FT-ICR than with microESI FT-ICR due to scan-to-scan variations of the ion population in the ICR cell. Database search results and protein sequence coverage results from NanoLC FT-ICR MS and MALDI FT-ICR MS, as well as the effect of mass accuracy on protein identification for the peptide mass fingerprint analysis are evaluated.

Spectroscopic characterization of the 4-hydroxy catechol estrogen quinones-derived GSH and N-acetylated Cys conjugates.

Estrogens, including the natural hormones estrone (E(1)) and estradiol (E(2)), are thought to be involved in tumor induction. Specifically, catechol estrogen quinones (CEQs) derived from the catechol estrogens 4-hydroxyestrone (4-OHE(1)) and 4-hydroxyestradiol (4-OHE(2)) react with DNA and form DNA adducts (Cavalieri, E. L., et al. (1997) Proc. Natl Acad. Sci. U.S.A. 94, 10037). CEQs are also conjugated with GSH, a reaction that prevents damage to DNA, providing biomarkers of exposure to CEQs. Current detection limits for these analytes by HPLC with multichannel electrochemical detection are in the picomole range (Devanesan, P., et al. (2001) Carcinogenesis 22, 489). To improve the detection limit of CEQ-derived conjugates, spectrophotometric monitoring was investigated. Fluorescence and/or phosphorescence spectra of the 4-OHE(1), 4-OHE(2), Cys, N-acetylcysteine (NAcCys), 4-OHE(1)-2-SG, and 4-OHE(2)-2-SG conjugates and their decomposition products 4-OHE(1)-2-NAcCys and 4-OHE(2)-2-NAcCys were obtained at 300 and 77 K. It is shown that (i) 4-OHE(1)- and 4-OHE(2)-derived SG and NAcCys conjugates are weakly fluorescent at 300 K (with the emission maximum at 332 nm) but strongly phosphorescent at 77 K; (ii) Cys and NAcCys exhibit fluorescence and phosphorescence only at 77 K; and (iii) 4-OHE(1) and 4-OHE(2) are weakly fluorescent at 300 and 77 K and not phosphorescent. The phosphorescence spectra of SG and NAcCys conjugates are characterized by a weak origin band at approximately 383 nm and two intense vibronic bands at 407 and 425 nm. After they are cooled from 300 to 77 K, the total luminescence intensity of SG and NAcCys conjugates increases by a factor of approximately 150 predominantly due to phosphorescence enhancement. Theoretical calculations revealed, in agreement with the experimental data, that the lowest singlet (S(1)) and triplet (T(1)) states of 4-OHE(2)-2-NAcCys are of n,pi* and pi,pi* character, respectively, leading to a large intersystem crossing yield and strong phosphorescence. The limit of detection (LOD) for CEQ-derived conjugates, based on phosphorescence measurements, is in the low femtomole range. The concentration LOD is approximately 10(-9) M. Therefore, we propose that capillary electrophoresis interfaced with low temperature phosphorescence detection can be used to test for human exposure to CEQs by analyzing urine.

Recent developments in atmospheric pressure MALDI mass spectrometry

A new atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) ion source that includes an extended capillary for delivery of AP-MALDI ions toward a mass spectrometer (MS) atmospheric pressure interface inlet aperture, an optical fiber for laser energy transfer to a target, and x– y travel stages for high throughput analysis of multiple samples, is described. The new source has been interfaced with a Thermo Finnigan LCQ ion trap mass spectrometer bringing powerful high throughput tandem MS capabilities to MALDI analysis. The resulting AP-MALDI-LCQ instrument demonstrated an absolute sensitivity in the low femtomole range for simple peptide mixture analysis, a mass range up to 2000–4000 (limited by LCQ), and an extensive fragmentation of singly-charged AP-MALDI ions in MS/MS experiments. The potential strength of this technique has been illustrated by numerous applications ranging from fragile molecule analysis to tryptic digest analysis for protein identification.

A robust approach for the analysis of peptides in the low femtomole range by capillary electrophoresis-tandem mass spectrometry.

A capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) approach has been developed for routine application in proteomic studies. Robustness of the coupling is achieved by using a standard coaxial sheath-flow sprayer. Thereby, greater stability than nanoelectrospray ionization-mass spectrometry coupling of sheathless capillary electrophoresis or nanoliquid chromatography (nano-LC) is achieved, resulting in stable operation for several weeks and unattended overnight sequences. The applied sheath flow is reduced to 1-2 microL/min in order to increase sensitivity. Standard peptides and those of digests of standard proteins and gel-separated proteins can be detected in the low femtomole range (full scan and MS/MS). Detection limits are found to be as low as 500 amol. Low femtomole amounts are required for unequivocal identification by MS/MS experiments in the ion trap and subsequent database search. By applying a simple pH-mediated stacking the concentration sensitivity can be lowered to some tens of fmol/microL (nM), depending on capillary size. This sensitivity is close to published values for sheathless CE-MS and nano-LC-MS, respectively (a comparison to reference values is presented). Moreover, with capillaries of about 50 cm in length separations in less than 10 min are possible resulting in a throughput of up to four analyses per hour. This is a factor of 4-12 times faster than nano-LC separation, being the state-of-the-art techniques for proteomic studies.

Concentrations of Seven Iodothyronine Metabolites in Brain Regions and the Liver of the Adult Rat

The concentrations of the iodothyronine metabolites T4, T3, 3,5-diiodothyronine (3,5-T2), 3,3′-diiodothyronine (3,3′-T2), reverse T3 (rT3), 3,3′-T2 sulfate (3,3′T2S), and T3 sulfate (T3S) were measured in 12 regions of the brain, the pituitary gland, and liver in adult male rats. Quantification of iodothyronine was performed by RIA following a newly developed method of purification and separation by HPLC. 3,5-T2, 3,3′-T2, rT3 and T2S were detectable in the low femtomolar range (20–200 fmol/g) in most areas of the rat brain. T3S was detectable only in the hypothalamus. The concentrations of T3 and T4 were approximately 20- to 60-fold higher, ranging between 1 and 6 pmol/g. There was a significant negative correlation between the activities of inner-ring deiodinase and T3 concentrations across brain areas. In the liver, 3,5-T2, rT3, and T3S were measurable in the low femtomolar range, whereas 3,3′-T2 and 3,3′T2S were not detectable. 3,5-T2 and 3,3′-T2 were not detectable in mitochondrial fractions of the brain regions. Tissue concentrations of 3,5-T2 exhibited a circadian variation closely parallel to those of T3 in the brain regions and liver. T3 was not a substrate for outer-ring deiodination under different experimental conditions; thus, it remains unclear which substrate(s) and enzyme(s) are involved in the production of 3,5-T2. These results indicate that five iodothyronine metabolites other than T3 and T4 are detectable in the low femtomolar range in the rat brain and/or liver. The physiological implications of this finding are discussed.

Concentrations of seven iodothyronine metabolites in brain regions and the liver of the adult rat.

The concentrations of the iodothyronine metabolites T(4), T(3), 3,5-diiodothyronine (3,5-T(2)), 3,3'-diiodothyronine (3,3'-T(2)), reverse T(3) (rT(3)), 3,3'-T(2) sulfate (3,3'T(2)S), and T(3) sulfate (T(3)S) were measured in 12 regions of the brain, the pituitary gland, and liver in adult male rats. Quantification of iodothyronine was performed by RIA following a newly developed method of purification and separation by HPLC. 3,5-T(2), 3,3'-T(2), rT(3) and T(2)S were detectable in the low femtomolar range (20-200 fmol/g) in most areas of the rat brain. T(3)S was detectable only in the hypothalamus. The concentrations of T(3) and T(4) were approximately 20- to 60-fold higher, ranging between 1 and 6 pmol/g. There was a significant negative correlation between the activities of inner-ring deiodinase and T(3) concentrations across brain areas. In the liver, 3,5-T(2), rT(3), and T(3)S were measurable in the low femtomolar range, whereas 3,3'-T(2) and 3,3'T(2)S were not detectable. 3,5-T(2) and 3,3'-T(2) were not detectable in mitochondrial fractions of the brain regions. Tissue concentrations of 3,5-T(2) exhibited a circadian variation closely parallel to those of T(3) in the brain regions and liver. T(3) was not a substrate for outer-ring deiodination under different experimental conditions; thus, it remains unclear which substrate(s) and enzyme(s) are involved in the production of 3,5-T(2). These results indicate that five iodothyronine metabolites other than T(3) and T(4) are detectable in the low femtomolar range in the rat brain and/or liver. The physiological implications of this finding are discussed.

A 20 kV orthogonal acceleration time-of-flight mass spectrometer for matrix-assisted laser desorption/ionization

A compact time-of-flight mass spectrometer has been constructed with the reflecting analyzer region orientated to be orthogonal to the desorption axis of a matrix-assisted laser desorption/ionization source. The instrument is designed with a detector that is large enough to accommodate a correspondingly large desorption velocity spread without the need for collisional cooling. Laser desorbed positive ions from a positively biased probe enter a field free fill region of an orthogonal accelerator, initially at ground potential. The ions are then orthogonally accelerated to reach a final potential of −20 kV and a kinetic energy of ∼21 keV. The space focus of the reflectron geometry is adjusted to lie in the ion mirror to allow easy conversion to the linear mode. The detector of the current configuration is a 70 mm diameter microsphere plate. The spectrometer has a total length of less than 1 m and, in good agreement with simulations, it provides a typical resolution of 8000 (full-width at half-maximum). Detection limits determined with ∼10–50 laser shots were in the low femtomole range up to m/z ∼ 10000. Beyond this limit, sensitivity appears to be lowered by decreasing detector efficiency and the increasing velocity spread of the desorbed ions. Mass calibration of the instrument is very simple and reproducible. Measured mass accuracy with external calibration is better than 100 ppm over several days. Limitations in mass accuracy are attributed to the drift of the power supplies and timing jitter. A focusing method for improving high mass sensitivity by a factor of typically 20 is briefly described and demonstrated with the detection of molecular ions of myoglobin (m/z ∼ 17000).

The chemistry of protein sequence analysis.

N-terminal sequence analysis by Edman chemistry continues to play an important role in the structural analysis of proteins and peptides. Improvements in the sensitivity of the method have been achieved mainly at the level of increasing the sensitivity of the on-line analysis of PTH amino acids by RP-HPLC (reverse phase high performance chromatography). Using microbore columns (0.8-1.0 mm), it is possible to run standards at the 0.5-1.0 pmol level and to sequence samples in the 1-5 pmol range. Due to constraints in current chromatographic methods, it is unlikely that further improvements in sensitivity will be achieved by this approach alone. Although alternative Edman reagents, including fluorescent chemistries, have promised to increase the sensitivity of sequencing into the low femtomole range, none of the methods have progressed into routine usage. These reagents and chemistries are critically evaluated in this review, and the problems which have prevented their further development discussed. Instrumental constraints are also considered. It is concluded that the development of more sensitive methods requires further research into both the chemistry and the instrumentation, and that alternative separation and detection methods may also play a role.

Coupling of capillary zone electrophoresis to mass spectrometry (MS and MS/MS) via a nanoelectrospray interface for the characterisation of some β-agonists

Capillary zone electrophoresis (CZE) was coupled to mass spectrometry (MS) via a nanoelectrospray interface (nESI), using conductively coated tips (8 μm orifice) butted to the end of a fused-silica capillary. The CZE/nESI-MS system was carefully optimised to achieve a stable operation of the arrangement in order to run also MS/MS experiments. Mixtures of β-agonists were successfully separated using 5 mmol/L ammonium acetate in 80% MeOH as a buffer. The limit of detection was 500 attomole of a substance injected. The system was also investigated from the point of view of quantification of the substances. Careful measures were taken to avoid any systematic errors (evaporative loss during the electrokinetic injection and reduction of migration times due to the electrospray pull during the CE run). The data show that the response of a mass spectrometric detector in the low-femtomole range not only depends on the concentration of the analyte but also on the properties of the nanoelectrospray process itself, that is why the linearity of the response can be compromised.

Detection of Protein Tyrosine Kinase Activity Using a High-Capacity Streptavidin-Coated Membrane and Optimized Biotinylated Peptide Substrates

A protein tyrosine kinase (PTK) assay system is described that uses a series of optimized biotinylated peptide substrates in conjunction with a streptavidin-coated matrix (SAM2) biotin capture membrane. The SAM2biotin capture membrane provides low backgrounds and high linear binding capacity (up to ∼3.6 nmol of biotinylated PTK peptide/cm2), resulting in high signal-to-noise ratios and greater reproducibility. Capture of the phosphorylated peptide substrates onto the SAM2membrane is rapid and occurs independent of the amino acid sequence of the peptide, thereby overcoming difficulties commonly encountered with other methodologies. Two broad-specificity biotinylated PTK peptide substrates were identified with optimum kinetic properties, allowing members from eight distinct classes of enzymes, including transmembrane (epidermal growth factor receptor (EGFR), fibroblast growth factor receptor, insulin receptor, and platelet-derived growth factor receptor) and cytoplasmic (p43abl, p56lck, p60src, and p93fes) PTKs, to be analyzed. A third biotinylated peptide substrate, shown to be highly selective for the EGFR, was used to illustrate the versatility of this system for both broad specificity and highly selective detection of PTK activity. The ability to accurately detect activity under optimum conditions and with crude cell extract samples, including kinetic analysis and with enzyme detection limits in the low femtomole range, supports the utility of this assay system for studying PTK enzymes.

Orthogonal injection of matrix-assisted laser desorption/ionization ions into a time-of-flight spectrometer through a collisional damping interface

Ions are produced from a conventional matrix-assisted laser desorption/ionization (MALDI) target by irradiation with a nitrogen laser pulsed at 20 Hz. After being cooled by collisions in an RF-quadrupole ion guide, the ions enter an orthogonal-injection TOF mass spectrometer, already used for electrospray. The collisional cooling spreads the ions out along the axis of the quadrupole to produce a quasi-continuous beam, which is then pulsed into the mass spectrometer at a repetition rate of about 4 kHz. Approximately five ions enter the mass spectrometer with each injection pulse, and these are detected using single-ion counting and registered in a TDC with 0.5 ns resolution. The performance of the instrument is similar to that obtained with an ESI source. A uniform mass resolution of about 5000 (full width at half maximum definition) is routinely obtained for molecular weights up to about 6000 Da, with mass accuracy around 30 ppm. The sensitivity for peptides is in the low femtomole range. The mass range is currently limited by the low energy (5 keV) of the ions at the detector, although ions of cytochrome C (12 359 Da) have been detected. The performance of the instrument for peptides is competitive with delayed-extraction MALDI in the usual axial geometry, but with the advantage of mass-independent focusing conditions, and a simple two-point calibration procedure. However, the most important advantages result from the nearly complete decoupling of the ion production from the mass measurement. In the usual MALDI experiment the instrument must be carefully adjusted for optimum performance, and the optimum parameters depend on the matrix and the method of sample preparation. As a result of the decoupling, the performance of the instrument is independent of source conditions. This allows much greater flexibility to experiment with different matrices, different substrates (including insulating substrates), and different laser wavelengths, pulse widths and fluences. Because of the decoupling, the design also allows convenient use of both ESI and MALDI sources (and possibly others) on the same spectrometer. © 1998 John Wiley & Sons, Ltd.

MALDI mass spectrometry of biomolecules and synthetic polymers using alkali hexacyanoferrate (II) complexes and glycerol as matrix

K 4[Fe(CN) 6]/glycerol and Na 4[Fe(CN) 6]/glycerol have been investigated as liquid matrix systems for UV-MALDI MS applying a N 2 laser. Analyte molecules were detected as sodium or potassium adduct ions and, in the case of proteins, as well as protonated molecular ions. Mass accuracies were comparable to those found with standard solid matrix systems with −0.06 to +0.05% deviation in the reflectron mode and with −0.24 to +0.13% in the linear mode. Useful results could be obtained within a mass range of 15 000 Da for single-charged proteins and 8000 Da for potassium cationized polyethylene glycols. Detection limits were found for hydrophilic compounds in the low picomol range and for lipophilic compounds as triacylglycerols or peracetylated and partially benzylated carbohydrates in the low femtomol range. As shown by scanning electron microscopic investigations, the generation of a thin homogenous matrix layer was essential for a successful mass spectrometric experiment. A very careful cleaning of the target surface with glacial acid prior to matrix deposition improved the formation of such a matrix film that maximum sensitivity as well as good reproducibility of the experiments could be achieved.

Surface plasmon resonance biomolecular interaction analysis mass spectrometry. 1. Chip-based analysis.

The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) in concert with surface plasmon resonance-based biomolecular interaction analysis (SPR-BIA) is reported. A chip-based biosensor unit was used to simultaneously monitor biomolecular interactions taking place on four different regions of the sensor chip (flow cells). Species retained during SPR-BIA were then identified by performing MALDI-TOF directly from within the area of the flow cells. Analyses were performed on an antibody/antigen/antibody system with detection limits in the low-femtomole range. The combined assay demonstrates the use of SPR-BIA to evaluate the relative stability of sequential solution-phase interactions, as well as, upon MALDI-TOF analysis, the ability to unambiguously confirm the presence of species retained during the interaction analysis.

Quantitative analysis of fluorinated ethylchloroformate derivatives of protein amino acids and hydrolysis products of small peptides using chemical ionization gas chromatography-mass spectrometry

The derivatization and extraction efficiencies of 16 protein amino acids were investigated using trifluoroethanol+ethylchloroformate+pyridine as the derivatization reagents and chloroform as the solvent. The derivatization efficiencies ranged from 90% to 99% for all the amino acids studied except aspartic acid (79%). The extraction efficiencies of the derivatized amino acids using chloroform were close to 100%. In addition, the detection limits and linear dynamic ranges of trifluoroethanol ethylchloroformate (TFE-ECF) derivatives of these amino acids were studied using positive-ion chemical ionization gas chromatography-mass spectrometry (GC-MS). The detection limits of the protein amino acids were mostly in the low femtomole range. The linear dynamic ranges of these amino acids were in the range of zero to three orders of magnitude. The GC-MS analysis of the derivatized amino acids was applied to the hydrolysis products of Equal, a sugar substitute containing aspartame and diprotin B, a tripeptide. The results demonstrate that the TFE-ECF derivatization of the hydrolysis products of small peptides followed by GC-MS analysis can be used for the identification of their amino acid composition (except arginine) and for their complete amino acid analysis.

Use of an ion trap storage/reflectron time-of-flight mass spectrometer as a rapid and sensitive detector for capillary electrophoresis in protein digest analysis.

An ion trap storage (IT)/reflectron time-of-flight mass spectrometer (reTOFMS) has been coupled to capillary electrophoresis (CE) via a sheathless microelectrospray ionization method. This hybrid mass spectrometer has proved to be a rapid and sensitive detector for CE, where mass spectra could be acquired at a speed sufficient to maintain the high-resolution capabilities of CE separations. The nonscanning property of the time-of-flight mass analyzer can provide a full mass range spectral acquisition speed of up to 25 spectra/s with a data system developed in our laboratory. For the work reported herein, a spectral acquisition speed of 4 spectra/s was found to be optimal for maintaining the quality of the separation while achieving high sensitivity. Tryptic digests of bovine cytochrome c and beta-lactoglobulin A were analyzed using the CE/IT/reTOFMS combination, resulting in total ion electropherograms similar to those obtained using UV absorption detection. Taking advantage of the ion storage capability of the ion trap, a detection limit in the lowfemtomole range was routinely obtained for these digests using the total ion electrophoretic mode and CE capillaries of typical dimensions (41 microns i.d.). This high sensitivity was achieved while maintaining a resolution of approximately 1500 for mass identification using the capabilities of the IT/reTOF device. Due to the high acquisition speed and the mass discrimination capabilities of the mass detector, all the peaks in the total ion electropherograms, including some totally or partially unresolved peaks, could be unambiguously identified.

Nonradioactive monitoring of organic and inorganic solute transport into single Xenopus oocytes by capillary zone electrophoresis

Transport of organic and inorganic solutes into and out of cells requires specialized transport proteins. Given a sufficiently sensitive analytical method for measuring cellular solute concentrations, it should be possible to monitor solute transport across the plasma membrane at the level of single cells. We report a capillary zone electrophoresis approach that is generally applicable to monitor solute transport into Xenopus laevis oocytes, requires only nanoliters of sample, and involves no radioactive materials. The sensitivity of capillary electrophoresis with UV detection is typically on the order of 10(-5)-10(-6) M, resulting in the mass detection limits in the low femtomole range. We show that capillary zone electrophoresis serves as a simple technique to measure solute transport into oocytes. Studies of the mammalian oligopeptide transporter PepT1 and the Na(+)- and K(+)-coupled epithelial and neuronal glutamate transporter EAAC1 expressed in oocytes demonstrate that transport of the dipeptide Trp-Gly via PepT1 and transport of Na+ and K+ via EAAC1 across the oocyte plasma membrane can be monitored by measuring intracellular tryptophan absorption and by indirect UV detection of inorganic ions, respectively. The CZE method allowed the simultaneous detection of changes of intracellular Na+ and K+ concentrations in response to EAAC1-mediated Na+ cotransport and K+ countertransport. This is the first report of a capillary zone electrophoresis-based quantitative analysis of intracellular components of a single cell in response to transport activity.

Thin-layer immunoaffinity chromatography with bar code quantitation of C-reactive protein.

A rapid thin-layer immunoaffinity chromatographic method for quantitation in serum of an acute phase reactant, C-reactive protein (CRP), which can differentiate between viral and bacteria] infections, is described, where material and reagent costs are minimal. The analysis is based on the "sandwich" assay format using monoclonal antibodies directed against two sites of CRP. One of the antibodies is covalently bound to defined zones on a thin-layer immunoaffinity chromatography membrane, while the other antibody is covalently bound to deeply dyed blue latex particles. After incubation (CRP sample and latex particles), the CRP-latex immunocomplex is allowed to migrate along the immunoaffinity chromatography membrane. In the presence of antigen, a sandwich is formed between the CRP-latex immunocomplex and membrane-bound antibodies, which results in the appearance of blue lines on the membrane. Antibody immobilization on the TLC membrane is made with a redesigned piezoelectric-driven ink-jet printer. The time required for the analysis is less than 10 min. Quantitation is achieved either by counting the lines visually, with scanning reflectometry, or with a modified bar code reader. The limit of detection was estimated in the low femtomolar range using the naked eye as detector.