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We have developed a new isotope labeling method, based on the use of isotope-coded p-dimethylaminophenacyl (DmPA) bromide as a reagent, combined with liquid chromatography--mass spectrometry (LC--MS) for high-performance metabolome analysis with a focus on profiling carboxylic acid-containing metabolites. Derivatization is simple, fast (1 h plus 30 min for quenching the reaction), and applicable to a wide range of carboxylic acids with a high yield and little or no side reaction products. This labeling method is demonstrated to be not only effective in introducing an isotope tag for accurate metabolite quantification but also improving the chromatographic retention of the metabolites in reversed-phase (RP) LC, enhancing ESI efficiency by 2-4 orders of magnitude, and facilitating the identification of metabolite peaks in LC--MS. In triplicate experiments of a 1:1 ratio of [sup.13]C-/[sup.12]C-DmPA labeled human urine, we were able to detect 2671, 2546, and 2820 ion pairs from metabolites containing one or more carboxylic acid groups. 10.1021/ac102146g

A shotgun proteome analysis method and its performance for protein identification from 500 to 5000 cells are described. Sample preparation, which was done in one tube, involved the use of a surfactant (NP-40) for cell lysis, followed by acetone precipitation of the proteins. The resulting protein pellet was washed with cold acetone to remove remaining surfactant, and the pellet was then solubilized in N[H.sub.4]HC[O.sub.3]. After trypsin digestion of the proteins, the digest was analyzed by the use of nanoflow liquid chromatography (LC) quadrupole time-of-flight mass spectrometry (QTOF MS). Sample injection and gradient speed in running LC QTOF MS were optimized. It was shown that this method could identify an average (n = 3) of 167 [+ or -] 21,237 [+ or -] 30, 491 [+ or -] 63, and 619 [+ or -] 59 proteins from 500, 1000, 2500, and 5000 MCF-7 breast cancer cells, respectively. To demonstrate the potential use of this method for generating proteome profile from circulating tumor cells (CTCs) isolated from human blood, a healthy human blood sample was spiked with MCF-7 cells, and this mixture was processed and then subjected to antibody tagging of the MCF-7 cells. The tugged cells were sorted and collected using flow cytometry. The proteome profiles of small numbers of cells isolated in this way were found to be similar to those of the original MCF-7 cells, suggesting the possibility of the use of this method for cell typing of CTCs. 10.1021/ac9023022

We report a new quantitative metabolome profiling technique based on differential [sup.12]C-/[sup.13]C-isotope dansylation labeling of metabolites, fast liquid chromatography (LC) separation and electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) detection. An isotope reagent, [sup.13]C-dansyl chloride, can be readily synthesized. This reagent, along with [sup.12]C-dansyl chloride, provides a simple and robust means of labeling metabolites containing primary amine, secondary amine, or phenolic hydroxyl group(s). It is shown that dansylation labeling offers 1-3 orders of magnitude ESI signal enhancement over the underivatized counterparts. Dansylation alters the chromatographic behaviors of polar and ionic metabolites normally not retainable on a reversed phase (RP) column to an extent that they can be retained and separated by RPLC with high efficiency. There is no isotopic effect on RPLC separation of the differential isotope labeled metabolites, and [sup.12]C-/[sup.13]C-labeled isoforms of metabolites are coeluted and detected by MS for precise and accurate quantification and confident metabolite identification. It is demonstrated that, in the analysis of 20 amino acids, a linear response of over 2 orders of magnitude is achieved for relative metabolite quantification with an average relative standard deviation (RSD) of about 5.3% from replicate experiments. A dansylation standard compound library consisting of 121 known amines and phenols has been constructed and is proven to be useful for absolute metabolite quantification and MS-based metabolite identification in biological samples. As an example, the absolute concentrations of 93 metabolites, ranging from 30 nM to 2510 [micro]M, can be determined from a pooled sample of human urines collected in 5 consecutive days labeled with [sup.12]C-dansylation and spiked with the 121 [sup.13]C-dansylated standards. Relative concentration variations of these metabolites in individual urine samples can also be monitored by mixing the [sup.13]C-dansylated pooled urine sample with the [sup.12]C-dansylated individual sample. With a 12 min fast LC separation combined with FTICR MS, 672 metabolites were detected in a human urine sample with each metabolite peak having a signal-to-noise ratio of greater than 20; the identities of most of the metabolites remain to be determined. This work illustrates that dansylation labeling and fast LC/FTICR MS can be a powerful technique for quantitative profiling of at least 672 metabolites in urine samples in 12 min.

We demonstrate a strategy of maximizing the performance of reversed-phase (RP) liquid chromatography (LC) tandem mass spectrometry (MS/MS) for efficient shotgun proteome analysis by optimizing the sample loading to the instrument in an off-line two-dimensional (2D) LC tandem MS platform. To determine the quantity of peptides present in a proteome digest or fractionated peptides from strong-cation exchange (SCX) separation, an automated system based on RPLC with a rapid step solvent gradient for peptide elution and ultraviolet (UV) detection was developed. This system also allowed the purification of the peptides by removing salts and other impurities present in a sample. It was found that controlling the amount of peptides injected into a RPLC MS/MS system was critical to achieve the maximum efficiency in peptide and protein identification. With the use of off-line 2D-LC-MS/MS, peptide fractions from the first dimension of separation were desalted and quantified, followed by injecting the opfmal amount of the sample into RPLC-MS/ MS for peptide sequencing. The application of this strategy was demonstrated in the proteome profiling of breast cancer MCF-7 cells. From the analysis of 28 SCX fractions with each injecting 1 #g of sample into a 75 [micro]m x 100 mm C18 column interfaced to a quadrupole/time-of-flight mass spectrometer, a total of 2362 unique proteins or protein groups were identfied with a false positive peptide identification rate of 0.19%, as determined by target-decoy proteome sequence searches. Replicate 2 h runs of individual fractions with the exclusion of precursor ions of peptides already identified in the first runs resulted in the identification of an additional 549 unique proteins or protein groups with a false positive identification rate of 0.60%. This example illustrated that off-line 2D-LC-MS/ MS, with maximal sample injection to the RPLC-MS, is an effective method for shotgun proteome analysis. Finally, the advantages and limitations of this method, compared to other methods, are discussed.

In shotgun proteome analysis by liquid chromatography--tandem mass spectrometry (LC-MS/MS), not all coeluting peptides at a given retention time are subjected to MS/ MS due to the limitation of spectral acquisition speed of a mass spectrometer. In this work, precursor ion exclusion (PIE) in an electrospray ionization (ESI) quadrupole time-of-flight (QTOF) mass spectrometer was explored as a means of mitigating the undersampling problem. This strategy is based on running replicates of the sample where the precursor ions detected in the initial run(s) are excluded for MS/MS in the subsequent run. Four PIE methods as well as running replicates without PIE were investigated and compared for their effectiveness in identifying peptides and proteins. In the analysis of an MCF-7 breast cancer cell lysate digest by three replicate 2 h gradient LC-ESI runs, the first PIE method used a list of precursor ions detected in the initial run(s) for exclusion and identified a total of 572 proteins from the three runs combined with an average of 3.59 peptides matched to a protein. The second PIE method involved in the generation of a list of m/z values of precursor ions along with their retention time information from the initial run(s), followed by entering these ions with retention times into the ion exclusion program of the QTOF control software for exclusion at a predefined retention time window (i.e., [+ or -] 150 s). In comparison to the first PIE method, this method reduced the possibility of excluding different peptide ions of the same m/z (within a mass tolerance window) eluted at different retention windows. A total of 657 proteins were identified with an average of 3.75 peptides matched to a protein. The third PIE method studied relied on the exclusion of the precursor ions of peptides identified through database search of the MS/ MS spectra generated in the initial run(s). This selective PIE method identified a total of 681 proteins with an average of 3.68 peptides matched to a protein. The final PIE method investigated involves the expansion of the selective PIE list by including nonidenfifiable peptide ions found in the database search. This complete PIE method identified a total of 726 proteins with an average of 3.66 peptides per protein. In the case of three replicate runs without PIE, a total of 460 proteins were identified with an average of 3.51 peptides matched to a protein. Thus, the use of an optimal PIE strategy significantly increased the number of proteins identified from replicate runs (i.e., 726 vs 460 or a 58% increase). It is further demonstrated that this PIE strategy also improves protein identification efficiency in the analysis of a yeast whole cell lysate digest--a less complex proteome digest. A total of 533 proteins identified from five replicate runs with complete PIE, compared to 353 proteins identified from the five replicate runs without PIE, representing a 51% increase in the number of proteins identified.

One of the challenges associated with metabolome profiling in complex biological samples is to generate quantitative information on the metabolites of interest. In this work, a targeted metabolome analysis strategy is presented for the quantification of amine-containing metabolites. A dimethylarion reaction is used to introduce a stable isotopic tag onto amine-containing metabolites followed by LC-ESI MS analysis. This labeling reaction employs a common reagent, formaldehyde, to label globally the amine groups through reductive amination. The performance of this strategy was investigated in the analysis of 20 amino acids and 15 amines by LC-ESI MS. It is shown that the labeling chemistry is simple, fast ([less than] 10min reaction rime), specific, and provides high yields under mild reaction conditions. The issue of isotopic effects of the labeled amines on reversed-phase (RP) and hydrophilic interaction (HILIC) LC separations was examined. It was found that deuterium labeling causes an isotope effect on the elution of labeled amines on RPLC but has no effect on HILIC LC. However, [sup.13]C-dimethylation does not show any isotope effect on either RPLC or HILIC LC, indicating that [sup.13]C-labeling is a preferred approach for relative quantification of amine-containing metabolites in different samples. The isotopically labeled 35 amine-containing analogues were found to be stable and proved to be effective in overcoming matrix effects in both relative and absolute quantification of these analytes present in a complicated sample, human urine. Finally, the characteristic mass difference provides additional structural information that reveals the existence of primary or secondary amine functional groups in aminecontaining metabolites. As an example, for a human urine sample, a total of 438 pairs of different amine-containing metabolites were detected, at signal-to-noise ratios of greater than 10, by using the labeling strategy in conjunction with RP LC-ESI Fourier-transform ion cyclotron resonance MS.

An automated off-line liquid chromatography-matrix-assisted laser desorption ionization (LC-MALDI) interface capable of coupling both capillary and microbore LC separations with MALDI mass spectrometry (MS) and tandem mass spectrometry (MS/MS) has been developed. The interface is a combination of two concepts: analyte concentration from heated hanging droplets and impulse-driven droplet deposition of LC fractions onto a MALDI sample plate. At room temperature the interface allows the coupling of capillary LC separations (i.e., flow rate of

In this paper we describe a multijunction microfluidic device for the injection of a substrate into an array of preformed plugs carded by an immiscible fluid in a microchannel. The device uses multiple junctions to inject substrate into preformed plugs without synchronization of the flow of substrate and the array of preformed plugs of reagent, which reduces cross-contamination of the plugs, eliminates formation of small droplets of substrate, and allows a greater range of injection ratios compared to that of a single T-junction. The device was based on a previously developed physical model for transport that was here adapted to describe injection and experimentally verified. After characterization, the device was applied to two biochemical assays, including evaluation of the enzymatic activity of thrombin and determination of the coagulation time of human blood plasma, which both provided reliable results. The reduction of cross-contamination and greater range of injection ratios achieved by this device may improve the processes that involve addition and titration of reagents into plugs, such as high-throughput screening of protein crystallization conditions.

An automated system has been developed for protein identification using mass spectrometry that incorporates sample cleanup, preconcentration, and protein digestion in a single stage. The procedure involves the adsorption of a protein or a protein mixture from solution onto a hydrophobic medium that is contained within a microcolumn. The protein is digested while still bound to the hydrophobic support. The peptides are then eluted from surface digestion to an electrospray ionization mass spectrometer for detection and sequencing. The entire system is fully automated wherein the mass spectrometer is collecting data continuously. We demonstrate that this system is capable of identifying standard protein samples at concentrations down to 100 nM. Further development of this technique may offer a potential solution for proteomics applications that require unattended operation, such as on-line monitoring and identification of microorganisms on the basis of the detection of their protein biomarkers.

A protein mass mapping approach using mass spectrometry (MS) combined with an experimentally derived protein mass database is presented for rapid and effective identification of bacterial species. A prototype mass database from the protein extracts of nine bacterial species has been created by off-line high-performance liquid chromatography (HPLC) matrix-assisted laser desorption/ionization (MALDI) MS, in which the microbiological parameter of bacterial growth time is considered. A numerical method using a statistical weight factor algorithm is devised for matching the protein masses of an unknown bacterial sample against the database. The sum of these weight factors produces a corresponding summed weight factor score for each bacterial species listed in the database, and the database species producing the highest score represents the identity of the respective unknown bacterium. The applicability and reliability of this protein mass mapping approach has been tested with seven bacterial species in a single-blind study by both direct MALDI MS and HPLC electrospray ionization MS methods, and identification results with 100% accuracy are obtained. Our studies have demonstrated that the protein mass database can be rapidly established and readily adopted with relatively less dependency on experimental factors. Furthermore, it is shown that a number of proteins can be detected using a protein sample amount equivalent to an extract of less than 1000 cells, demonstrating that this protein mass mapping approach can potentially be highly sensitive for rapid bacterial identification.

Protein phosphorylation is one of the most important known posttranslational modifications. Tandem mass spectrometry has become an important tool for mapping out the phosphorylation sites. However, when a peptide generated from the enzymatic or chemical digestion of a phosphoprotein is highly phosphorylated or contains many potential phosphorylation residues, phosphorylation site assignment becomes difficult. Separation and enrichment of phosphopeptides from a digest mixture is desirable and often a critical step for MS/MS-based site determination. In this work, we present a novel open tubtflar immobilized metal ion affinity chromatography (OT-IMAC) method, which is found to be more effective and reproducible for phosphopeptide enrichment, compared to a commonly used commercial product, Ziptip from Millipore. A strategy based on a combination of OT-IMAC, sequential dual-enzyme digestion, and matrix-assisted laser desorption/ionization (MALDI) quadrupole time-of-flight tandem mass spectrometry for phosphoprotein characterization is presented. It is shown that MALDI MS/MS with collision-induced dissociation can be very effective in generating fragment ion spectra containing rich structural information, which enables the identification of phosphorylation sites even from highly phosphorylated peptides. The applicability of this method for real world applications is demonstrated in the characterization and identification of phosphorylation sites of a [Na.sup.+]/[H.sup.+] exchanger fusion protein, His182, which was phosphorylated in vitro using the kinase Erk2.

Detection and identification of pathogenic bacteria and their protein toxins play a crucial role in a proper response to natural or terrorist-caused outbreaks of infectious diseases. The recent availability of whole genome sequences of priority bacterial pathogens opens new diagnostic possibilities for identification of bacteria by retrieving their genomic or proteomic information. We describe a method for identification of bacteria based on tandem mass spectrometric (MS/MS) analysis of peptides derived from bacterial proteins. This method involves bacterial cell protein extraction, trypsin digestion, liquid chromatography MS/MS analysis of the resulting peptides, and a statistical scoring algorithm to rank MS/MS spectral matching results for bacterial identification. To facilitate spectral data searching, a proteome database was constructed by translating genomes of bacteria of interest with fully or partially determined sequences. In this work, a prototype database was constructed by the automated analysis of 87 publicly available, fully sequenced bacterial genomes with the GLIMMER gene finding software. MS/ MS peptide spectral matching for peptide sequence assignment against this proteome database was done by SEQUEST. To gauge the relative significance of the SEQUEST-generated matching parameters for correct peptide assignment, discriminant function (DF) analysis of these parameters was applied and DF scores were used to calculate probabilities of correct MS/MS spectra assignment to peptide sequences in the database. The peptides with DF scores exceeding a threshold value determined by the probability of correct peptide assignment were accepted and matched to the bacterial proteomes represented in the database. Sequence filtering or removal of degenerate peptides matched with multiple bacteria was then performed to further improve identification. It is demonstrated that using a preset criterion with known distributions of discriminant function scores and probabilities of correct peptide sequence assignments, a test bacterium within the 87 database microorganisms can be unambiguously identified.

A novel interfacing technology is described to combine solution-based separation techniques such as liquid chromatography (LC) with matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The interface includes a transfer tube having an inlet and an outlet, the inlet being adapted to accept the LC effluents and the outlet being adapted to form continuously replaced, hanging droplets of the liquid stream, and a MALDI sample plate mounted below the outlet of the transfer tube for collecting the droplets. The liquid stream in the transfer tube is heated to a temperature sufficient to cause partial evaporation of the carrier solvent from the hanging droplets. The droplets are dislodged to the MALDI plate, which is heated to above the boiling point of the carrier solvent to cause further evaporation of the carrier solvent from the collected droplets. It is found that analytes can be fractionated and deposited to a sample spot of 0.8 mm in diameter when a liquid flow rate of up to 50 [micro]L/min and a fractionation interval of 1 min/spot are used. Flow rate of up to 200 [micro]L/min can be used with a deposition sample spot of 2.4 mm in diameter on a commercial MALDI target. This heated droplet interface does not introduce sample loss, and the detection sensitivity of LC/MALDI is similar to that of standard MALDI, i.e., low femtomoles for peptide analysis with a microliter sample deposition. It is compatible with microbore and narrowbore column separation, thus allowing the injection of a larger amount of sample for separation and analysis, compared to a capillary column LC/MALDI system. The detection dynamic range is shown to be in the order of 106 for peptide mixture analysis, which is 4 orders of magnitude greater than standard MALDI. The application of this interface for combining LC with MALDI MS/MS is demonstrated in the proteome analysis of water-soluable protein components of E. coli K12 extracts.

Polyethylene is one of the most important industrial polymers and is also one of the most challenging polymers to be characterized by mass spectrometry. We have developed a substrate-assisted laser desorption/ionization (LDI) mass spectrometric method for polyethylene analysis. In this method, cobalt, copper, nickel, or iron metal powders are used as a sample substrate and silver nitrate is used as the cationization reagent. Using a conventional UV LDI time-of-flight mass spectrometer, intact oligomer ions having masses up to 5000 u can be detected. Cobalt is found to produce spectra with the highest signal-to-noise ratio and the lowest level of fragmentation. Cobalt powder size is shown to have some effect on the spectra produced. The best results are obtained with the use of cobalt powders with diameters ranging from 30 to 100 [micro]m. Fragmentation cannot be totally eliminated, but the fragment ion peaks can be readily discerned from the intact polyethylene ions in the substrate-assisted LDI spectrum. Thus, the average molecular masses of low-mass polyethylene samples can be determined by using this method. A rapid heating model is used to account for the effectiveness of using the coarse metal powders to assist the analysis of intact polyethylene molecules by LDI.

The availability of a suitable database is critical in a proteomic approach for bacterial identification by mass spectrometry (MS). The major limitation of the present public proteome database is the lack of extensive low-mass bacterial protein entries with masses experimentally verified for most bacteria. Here, we present a method based on mass spectrometry to create protein mass tables specifically tailored for bacterial identification. Several issues related to the detection of bacterial proteins for the purpose of database creation are addressed. Three species of bacteria, namely, Escherichia coli, Bacillus megaterium, and Citrobacter freundii, which can be found in the ambient environment, were chosen for this study. Direct matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis of each bacterial extract reveals 20-29 protein components in the mass range from 2000 to 20 000 Da. HPLC fractionation of bacterial extracts followed by off-line MALDI-TOF analysis of individual fractions detects 156-423 components. Analysis of the extracts by HPLC/electrospray ionization MS shows the number of detectable proteins in the range of 46-59. Although a number of components were common to the three detection schemes employed, some unique components were found using each of these techniques. In addition, for E. coli where a large proteome database exists in the public domain, a number of masses detected by the mass spectrometric methods do not match with the proteome database. Compared to the public proteome database, the mass tables generated in this work are demonstrated to be more useful for bacterial identification in an application where the bacteria of interest have limited protein entries in the public database. The implication of this work for future development of a comprehensive mass database is discussed.

Chemical structures of polymer end groups play an important role in determining the functional properties of a polymeric system. We present a mass spectrometric method for determining end group structures. Polymeric ions are produced by electrospray ionization (ESI), and they are subject to source fragmentation in the ESI interface region to produce low-mass fragment ions. A series of source-fragment ions containing various numbers of monomer units are selected for high-energy collision-induced dissociation (CID) in a sector/time-of-flight tandem mass spectrometer. It is shown that high-energy CID spectra of source-induced fragment ions are very informative for end group structure characterization. By comparing the CID spectra of fragment ions with those of known chemicals, it is possible to unambiguously identify the end group structures. The utility of this technique is illustrated for the analysis of two poly(ethylene glycol)-based slow-releasing drugs where detailed structural characterization is of significance for drug formulation, quality control, and regulatory approval. Practical issues related to the application of this method are discussed.

A method for generating peptide mass maps from dilute protein samples is presented. The method involves the concentration of proteins from aqueous solution by adsorption onto reversed-phase polymeric microbeads. These beads are then washed extensively to remove contaminants, after which the bound proteins are digested with trypsin. Analysis of the digestion products is performed by MALDI-TOF mass spectrometry following direct deposition of the beads on a MALDI target, along with the matrix solution. The procedure is demonstrated using solutions of cytochrome c, lysozyme, and bovine serum albumin. The results of these digests are compared to trypsin digestions of the protein samples without sample preconcentration. Comparative results are also presented for protein solutions contaminated with 2 M NaCl, 2 M urea, or sodium dodecyl suffate at concentrations up to 0.02%. These results reveal that, with the microbead preconcentration procedure, peptide mass maps can routinely be generated from highly contaminated samples with a protein concentration of only 100 nM.

The analytical performance of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for direct analysis of peptide and protein mixtures is strongly dependent on the sample and matrix preparation. A two-layer sample preparation method is demonstrated to be very effective for analyzing complex mixtures. In this method, the first layer on the MALDI probe is the densely packed matrix microcrystals formed by fast solvent evaporation of a matrix solution. A mixture solution containing both matrix and sample is then deposited onto the first layer to form uniform analyte/matrix micrococrystals. It is found that the addition of matrix to the second-layer sample solution proves to be critical in analyzing mixtures of peptides and proteins covering a broad mass range. The effect of solvent conditions for preparing the second-layer solution is discussed. The application of this method is demonstrated for the analysis of cow's milk where milk proteins as well as peptide fragments produced from proteins by indigenous proteinases are detected. Direct analyses of peptides and proteins from a bacteria extract and crude egg white are also illustrated.

A nanoliter-chemistry station combined with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was developed to characterize proteins at the attomole level. Chemical reactions including protein digestion were carried out in nanoliter or subnanoliter volumes, followed by microspot sample deposition of the digest to a MALDI-TOF mass spectrometer. Accurate mass determination of the peptides from the enzyme digest, in conjunction with protein database searching, allowed the identification of the proteins in the protein database. This method is particularly useful for handling small-volume samples such as in single-cell analysis. The high sensitivity and specificity of this method were demonstrated by peptide mapping and identifying hemoglobin variants of sickle cell disease from a single red blood cell. The approach of combining nanoliter chemistry with highly sensitive mass spectrometric analysis should find general use in characterizing proteins from biological systems where only a limited amount of material is available for interrogation.

Mixtures of dye-labeled, M13-forward DNA primers were separated by capillary gel electrophoresis and detected on-the-fly, using fluorescence lifetime measurements, to evaluate four-decay detection for multiplex DNA sequencing. Three different four-dye systems were used, two that were excited at 488 nm and one that was excited at 514 nm. Each dye-labeled primer was identified on the basis of the lifetime of the conjugated dye using nonlinear least squares or the maximum entropy method to analyze the lifetime data. Overlapping electrophoretic peaks were generated by making multiple injections of mixtures of the dye-labeled primers. The overlapping peaks were resolved by fitting the data to two-, three- or four-component lifetime models used in nonlinear least-squares analysis in which each lifetime component was fixed to the predetermined lifetime of the corresponding dye-labeled primer. In two of the dye systems, the lifetimes of the four dye-labeled primers were sufficiently different to allow peak resolution. In the other dye system, addition of 10% DMSO to the run buffer changed the lifetime of one dye-labeled primer, allowing it to be resolved from another dye-labeled primer with similar lifetime.

Dyes of several classes were investigated as candidates for use in a multiplex, four-decay fluorescence detection scheme for DNA sequencing. The dyes include nitrobenzofuran dyes, rhodamine dyes, fluorescein dyes, cyanine dyes, Nile Red, and BODIPY dyes. Based on the results of fluorescence spectral and lifetime studies, an initial set of four dyes was selected for further study: NBD-amino-hexanoic acid (NBD-HA, [Tau] = 1.1 ns), tetramethyl-rhodamine, methyl ester ([Tau] = 2.2 ns), rhodamine green ([Tau] = 4.3 ns), and BODIPY 505/515 ([Tau] = 5.9 ns). Limits of lifetime detection of the four dyes were investigated, and lifetime resolution was demonstrated for mixtures of the free dyes in batch solution. Lifetimes of dye-labeled DNA primers also were determined in batch solution and detected on-the-fly in capillary electrophoresis (CE). Conjugation of the dyes to DNA improved the resolution of their individual lifetimes in mixtures in batch measurements. When attached to the primer, tetramethyl-rhodamine exhibited biexponential decay with a dominant lifetime of 3.8 ns, making it unsuitable for four-decay sequencing. Contact with the CE gel lengthened the lifetime of NBD-HA-labeled primer from 1.3 to 2.1 ns but did not affect the lifetimes of the other dyes. Lifetime detectability of labeled primers at individual points along an electrophoretic peak is in the attomole range.

A strength of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is its ability to analyze mixtures without separation. MALDI mass spectrometers capable of providing a linear mass calibration over a broad mass range should find wide use in these applications. This work addresses issues pertinent to mass measurement accuracy of a time-lag focusing MALDI time-of-flight instrument and presents a new approach to improving mass accuracy by using a functional wave extraction pulse, instead of a square wave, for time-lag focusing. A model is described of an ideal extraction pulse shape that provides constant total kinetic energy for all ions. If total kinetic energy is constant, then there is an exact linear correlation between ion mass and flight time raised to the second power. Using a descending wave extraction pulse, it is demonstrated that mass accuracy of better than 30 ppm using two internal calibrants and better than 70 ppm using external calibrants can be obtained over a 25 ku mass range. The practical aspects of an instrument needed to obtain consistent mass accuracy is discussed. It is found that ion flight time shows a small dependence upon laser flux; flight times increase slightly as the flux increases. But this dependence is much smaller than is observed in continuous-extraction MALDI.

Laser desorption combined with photoelectron ionization in a time-of-flight mass spectrometer has been developed for surface analysis of bulk polymers. In this technique, a C[O.sub.2] laser beam is focused onto a polymer surface to induce fast thermal dissociation and/or photodissociation of the polymeric materials. The resulting neutral species are then ionized using photoelectron ionization (PEI). The PEI process involves a laser - metal interaction in which a low-power, pulsed UV laser beam is directed to an appropriate metal surface exterior to the source region of a Wiley - McLaren time-of-flight mass spectrometer. The photoelectrons generated are accelerated as a narrowly distributed beam by the fringing fields of the source region. The electron beam, traveling in a direction almost parallel to the extraction grid, is used to ionize the desorbed neutrals that are entrained into the ionization region. A number of polymers of different molecular weight and monomer structure are examined using this technique. It is found that this technique can produce readily interpretable mass spectra for structural analysis and chemical identification. The application of this technique for terminal group analysis in chemically modified polymers is illustrated. A comparison of this technique with other mass spectrometric methods is presented. In addition, it is shown that the detection sensitivity can be very high, with a detection limit of

Matrix-assisted laser desorption ionization mass spectrometry has been developed for direct mass analysis of enzymatic reaction products of oligosaccharides in human blood serum without the use of extraction or chromatographic separation. Molecular labeling of the substrate is used to achieve beth the detection sensitivity and selectivity required in rapid analysis of reaction products in serum. It is found that tetramethylrhodamine (TMR)-labeled oligosaccharides provide 100-fold sensitivity enhancement over the corresponding underivatized oligosaccharides. In order to selectively retain the TMR-labeled molecules on the sample probe while washing away contaminants in a serum sample, a sample/matrix preparation method is developed. This technique provides detection sensitivity of labeled oligosaccharides in the range of hundreds of femtomoles per microliter. The mass measurement accuracy is better than 0.01% when a linear time-of-flight mass spectrometer is used. The application of the technique is illustrated for the subpicomole detection and quantitation of the conversion of the disaccharlde [Alpha]Fuc(1 [yields] 2)[Beta]Gal-TMR to the blood group B active trisaccharide [Alpha]Fuc(1 [yields] 2)[[Alpha]GaI(1 [yields] 3)][Beta]Gal-TMR, catalyzed by the blood group B galactosyltransferase present in human serum.

A time-lag focusing method is developed for the improvement of mass resolution in a linear time-of-flight mass spectrometer for matrix-assisted laser desorption/ionization (MALDI). In this technique, the ions generated by the MALDI process are extracted by a pulsed voltage. A short time delay (280 ns) is inserted in between the laser desorption/ionization event and the ion extraction. The region between the repeller and extraction grid is field-free during the delay. The time-lag extraction allows the ions generated in the region between the repeller and the extraction grid to separate according to their velocity (energy). Application, to the repeller, of the appropriate pulse voltage provides the energy correction necessary to simultaneously detect all ions of the same mass/charge regardless of their initial energy, resulting in improved mass resolution. It is demonstrated that mass resolution in the range of 3000-6000 fwhm can be obtained. With this mass resolution, isotopically resolved mass spectra are observed for peptides with masses up to 3000 Da. For proteins, such as bovine insulin, cytochrome c, and apomyoglobin, resolution in the range of 800-1000 fwhm is observed with a mass measurement accuracy better than 0.01%.

A pulsed sample introduction (PSI) interface can be applied to integrate flow injection analysis with time-of-flight mass spectrometry in the identification of volatile and nonvolatile substances. The PSI interface exhibits reproducible results but does not present any memory effect and severe peak tailing. The compound-dependent detection limits obtained from aniline, tryptamine and indole-3-acetic acid are 3, 4 and 140 ng, respectively. Finally, the interface system does not induce clustering in supersonic jet expansion.