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A variety of factors have been investigated with regard to the quantitation of chemical species within individual ambient aerosol particles analyzed by laser desorption time-of-flight mass spectrometry. Spectrum to spectrum differences in the interaction of the particle with the ionization laser beam, which affect the absolute peak areas in the mass spectra, can be minimized by using relative peak areas instead of absolute peak areas in each spectrum. Whereas absolute peak areas vary by an average of 59% for a given ion peak in single particle mass spectra of a monodisperse aerosol of particles formed from the same solution, relative peak areas in the same mass spectra vary only by an average of 16%. Relative sensitivity factors (RSF) relating the mass spectral ion intensity of NH(sub 4)(super +) and the alkali metal cations Li(super +), Na(super +), K(super +), Rb(super +), and Cs(super +) in single particle aerosol time-of-flight mass spectrometry to their bulk concentrations have been determined. The values for Li(super +)/Na(super +), K(super +)/Na(super +), Rb(super +)/Na(super +), Cs(super +)/ Na(super +), and NH(sub 4)(super +)/Na(super +) are found to be 0.14, 5.1, 6.0, 7.9, and 0.014, respectively. The higher response for heavier cations of the alkali metals is consistent with the periodic trends of both ionization potential and lattice energies of the species of interest. The response factor for sodium and potassium cations has been used to accurately determine the relative amounts of Na(super +) and K(super +) in sea-salt particles, by analyzing a sample of approximately 360 ambient sea-salt particles. The relative amounts of Na(super +) and K(super +) are found to be 97 and 3% in particles, respectively, whereas in seawater they are, on average, 98 and 2%.

Protein identification through peptide mass mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a standard technique, used in many laboratories around the world. The traditional methodology often includes long incubations (6-24 h) and extensive manual steps. In an effort to address this, an integrated microanalytical platform has been developed for automated identification of proteins. The silicon micromachined analytical tools, i.e., the microchip immobilized enzyme reactor (mu)-chip IMER), the piezoelectric microdispenser, and the high-density nanovial target plates, are the cornerstones in the system. The (mu)-chip IMER provides on-line enzymatic digestion of protein samples (1 (mu)L) within 1-3 min, and the microdispenser enables subsequent on-line picoliter sample preparation in a high-density format. Interfaced to automated MALDI-TOF MS, these tools compose a highly efficient platform that can analyze 100 protein samples in 3.5 h. Kinetic studies on the microreactors are reported as well as the operation of this microanalytical platform for protein identification, wherein lysozyme, myoglobin, ribonuclease A, and cytochrome c have been identified with a high sequence coverage (50-100%).

The atmospheric pressure sampling nozzle (orifice, heated capillary, or inlet) of a high mass accuracy time-of-flight mass spectrometer (TOF-MS) was modified by replacing its single nozzle with multiple atmospheric pressure nozzles. This allowed multiple streams of liquids to be introduced into the MS in parallel (an electrosprayer for each nozzle), with minimum analyte interactions between the streams. The chemical contents of all liquid streams were analyzed concurrently using a single mass spectrometer. To obtain a higher mass accuracy by providing internal reference on each scan (acquisition) and to evaluate the suitability of TOF-MS for molecular-formula confirmation, a dual-ESI-sprayer, dual-nozzle version of this design was used. The accurate masses of tens of organic compounds in the mass range of 200-3000 Da were measured, and the results were compared with those obtained using dual-sprayer, single-nozzle TOF-MS. A significant improvement in mass accuracy was observed when the former technique was used. Comparison between the mass accuracy using dual-ESI-sprayer, dual-nozzle TOF-MS and that obtained using a double-focusing mass spectrometer operating under chemical ionization (CI) and fast atom bombardment (FAB) shows the suitability of the technique for elemental-composition confirmation. Approximately 85% of samples analyzed had mass errors of less than 5 ppm, and the other 15% had mass errors less than 8 ppm. Using a high-performance liquid chromatography (HPLC) as a device for introduction of one liquid stream (sample) and a syringe pump as a device for introduction of the second liquid stream (reference standard), the accurate mass of a tryptic digest of cytochrome c was measured. The range of mass errors was from -6.1 ppm to +3.6 ppm, a significant improvement over our previously reported mass accuracy for this digest using single-nozzle TOF-MS. The interactions between analytes in the liquid streams also were investigated using a variety of sample-introduction and nozzle-design combinations, including single-ESI-sprayer, single-nozzle; dual-ESI-sprayer, single-nozzle; dual-ESI-sprayer, Y-shaped inlet; and dual-ESI-sprayer, dual-inlet. The results demonstrated that the dual-ESI-sprayer, dual-inlet design provides reference peaks on every acquisition with minimum analyte-reference interaction and, therefore, higher consistent mass accuracy.

A pressure-tunable ensemble of two capillary separation columns is combined with time-of-flight mass spectral detection for the high-speed characterization and analysis of volatile organic compounds. The detector can record up to 500 complete mass spectra per second and can obtain spectral deconvolution and thus characterization of severely overlapping and unknown chromatographic peaks. The tunable capillary column ensemble consists of the series combination of a nonpolar 5% phenyl poly(dimethylsiloxane) column and a polar poly(ethylene glycol) column. Adjustment of the pressure at the column junction point is achieved by the use of an electronic pressure controller. Changes in the junction pressure result in changes in the relative contribution that each of the columns makes to overall retention. This causes changes in the retention patterns, which can be used to enhance the utility of the TOFMS detector. The use of high spectral acquisition rates allows for the deconvolution of very closely spaced peaks. Adjustment of the column junction pressure often allows for the control of peak separation for critical component pairs. Since severely overlapping chromatographic peaks can be tolerated in many cases, the time axis of the chromatogram can be compressed, thus obtaining very fast GC/MS characterization of unknown mixtures. Tunable columns are very useful for facilitating time compression since significant control of relative peak positions can be achieved.

The post-ionization time-of-flight mass spectrometry approach was adopted in an effort to assess the nature of charge transfer undergone by anthracene-picrate compounds. Experimental results suggest that samples of desorbed plume tend to generate negative ion signals when allowed to react with picric acid. These negative ions, which are indicative of charge-transfer process, may not be existent during a laser desorption of the gas phase.

The development of a system capable of the speed required for on-line capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) of tryptic digests is described. The ion trap storage/reflectron time-of-flight (IT/reTOF) mass spectrometer is used as a nonscanning detector for rapid CE separation, where the peptides are ionized on-line using electrospray ionization (ESI). The ESI produced ions are stored in the ion trap and dc pulse injected into the reTOF-MS at a rate sufficient to maintain the separation achieved by CE. Using methodology generated by software and hardware developed in our lab, we can produce SWIFT (Stored Waveform Inverse Fourier Transform) ion isolation and TICKLE activation/fragmentation voltage waveforms to generate MS/MS at a rate as high as 10 Hz so that the MS/MS spectra can be optimized on even a 1-2 s eluting peak. In CE separations performed on tryptic digests of dogfish myelin basic protein (MBP) where eluting peaks 4-8 s wide are observed, it is demonstrated that an acquisition rate of 4 Hz provides > 20 spectra/peak and is more than sufficient to provide optimized MS/MS spectra of each of the eluting peaks in the electropherogram. The detailed structural analysis of dogfish MBP including several posttranslational modifications using CE-MS and CE-MS/MS is demonstrated using this method with < 10 fmol of material consumed.

Campylobacter jejuni, Campylobacter fetus, and Campylobacter coli were compared with Helicobacter pylori and Helicobacter mustelae by direct analysis of individual cultured colonies in 50% methanol-water with a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS). H. pylori and Campylobacter species from blood agar culture produced unique, complex spectra with over 25 different ions in mass/charge (m/z) range from 2 000 to 62 000. A biomarker for H. pylori was centered around m/z 58 268, and H. mustelae was distinguished from H. pylori by its ions at m/z 49 608 and 57 231. Campylobacters could be distinguished from Helicobacters by their lack of ions around m/z 58 000 and 61 000 as well as distinguishing biomarkers of lower m/z: 10 074 and 25 478 for C. coli; m/z 10 285 and 12 901 for C.jejuni; m/z 10 726 and 11 289 for C. fetus. MALDI-TOF MS is a rapid and direct method for detection of these potentially pathogenic bacteria from culture.

MALDI mass spectrometry is explored as a method for hemoglobin characterization. To simplify and expedite the analysis, hemoglobin is obtained without purification directly from whole human blood. The use of trypsin-activated bioreactive MALDI probes is evaluated as a means to further reduce the analysis time from hours to minutes. Moreover, variations of the MALDI matrix preparation facilitate detection of the problematic tryptic peptides [Alpha]T12, [Alpha]T13, and [Beta]T12. The results reveal that MALDI-based methods are easily implemented, are rapid, and allow detection of traditionally elusive tryptic peptides.

Time-of-flight secondary ion mass spectrometry (TOFSIMS) has been utilized to detect femtomole and attomole quantities of organic species from within silicon nanovials. By using high-density arrays (10 000 nanovials/[cm.sup.2]) it is possible to chemically characterize diverse sample sets within a single chemical image. Molecular sensitivities, for the compounds investigated, vary between 85 attomoles and 25 femtomoles, and typical acquisition times are approximately 100 ms per nanovial. These vials are fabricated using photolithography and KOH etching of Si{001} wafers to create wells, with a pyramidal cross section, ranging in size from 25 to 5625 [[[micro]meter].sup.2]. The volume ranges from 30 femtoliters to 100 picoliters, respectively. A drawn glass microinjector and solenoid-driven dispenser are utilized to array picoliter volumes of organic compounds into individual silicon nanovials. Solution concentrations typically range from 1 x [10.sup.-2] to 1 x [10.sup.-4] M allowing femtomole and even attomole quantities of material to be dispensed into each vial.

Based on time of flight method, influence of short time vacuum arc current jump on arc plasma parameters were investigated. Superposition of the current pulse of a vacuum arc with a high operating voltage results in the appearance of ions of higher charge state in the discharge plasma and in an increase in the mean ion charge state for most of the cathode materials used in the experiment. The method of a 'short-time current jump' can be also used to investigate the parameters of a vacuum arc, in particular to estimate the ion direct velocities in vacuum arc plasmas. Our estimates show that in the presence of a current step the ion velocities are almost identical for all differently charged ions and depend only on the peak current and the ion mass. Index Terms - Charge state distribution, current jump, ion direct velocities, time-of-flight mass-spectrometer, vacuum arc.

We discuss a time-of-flight measurement of the speed of light which is inexpensive to build, has an experimental design completely accessible to students with a rudimentary knowledge of interference, and consistently produces values for c with errors of less than 1%. Students at the sophomore level can use the same apparatus to achieve uncertainties on the order of 0.25%.

Laser-induced fragmentation of mass-selected vanadium oxide cluster cations at 532 nm was studied in a time-of-flight mass spectrometer. The primary species seen in the initial mass distribution has a stoichiometry similar to bulk vanadium oxides, with some species having additional O2 units bonded to the cluster. At low laser fluence, the main photofragmentation channel seen was the loss of O2 or consecutive O2 units from clusters that are oxygen rich.

Research was conducted to examine a cation with atomic composition (N3O)+ using time-of-flight mass spectrometry. The cation was seen in the reaction products of detonated liquid nitromethane. The only possible atomic composition of the cation is (N3O)+ since the same 58 amu species was observed when protonated, deuterated and 13C-labeled nitromethane were detonated. Results provide evidence that the (N3O)+ structure is chemically bound.

The effect of spacers in modulation-doped Zn(sub 1-x)Cd(sub x)Se/ZnSe:Cl multiple quantum wells (MD-MQWs) was studied via photoluminescence (PL) and time-of-flight secondary-ion-mass spectrometry (TOF-SIMS). The diffusion of Cl and Cd was monitored by TOF-SIMS depth profiling and PL. Although TOF-SIMS did not show any substantial diffusion of Cl and Cd in both structures at temperatures up to 385 C, the PL results indicated the change in optical properties in the Zn(sub 1-x)Cd(sub x)Se/ZnSe:Cl MD-MQWs due to annealing. Findings suggested that the radiative deep-level emission may offer a more efficient channel for electron-hole recombination with increasing annealing temperature.

Research was conducted to demonstrate similarities in enzyme characteristics between UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS). Limited proteolysis results show both MurA and EPSPS exhibit similar substrate and inhibitor binding properties and open and closed conformational forms excepting EPSPS showing some difference in the catalytic loop structure.

A new, high-speed data acquisition system was tested for high storage rate time-of-flight mass spectrometry (TOFMS) detection in capillary electrophoresis (CE). For high spectral acquisition rates of 4 kHz, a spectral storage rate of 80 spectra [s.sup.-1] was achieved. The resulting detection limit was in the low amol range (10-25 amol) for continuous infusion investigations.

In this paper, we report for the first time use of laser desorption mass spectrometry for measurement of chemical cleavage sequencing products of DNA. In this method, the target DNA was labeled with biotin and subjected to chemical modification and cleavage according to the Maxam-Gilbert sequencing protocol. The biotin-containing fragments were captured by streptavidin-coated magnetic beads and separated from the other fragments. The captured fragments were released by hot ammonia treatment, and the released fragments were analyzed by mass spectrometry. Potential applications of this method in resolving sequence ambiguities and sequencing repeat sequences as well as in the analysis of DNA-protein interactions are discussed.

The dissociative dynamics of the K shell excited ozone were studied by time-of-flight (TOF) mass spectrometry of fragment ions. The TOF spectra were obtained at 0 and 90 degrees relative to the electric vector of linearly polarized soft X-ray. In the K shell excitation at 529 eV photoabsorption peak, the energetic O+ fragment ion was ejected anisotropically, while at the 536 eV band, it was isotropic. The beta values obtained from simulation of the TOF spectra were in agreement with the previous assignments that the 529 eV band is the pi*

This review of process analytical chemistry is an update to the previous review on this subject published in 1995 (A2). The time period covered for this review includes publications written or published from late 1994 until early 1999, with the addition of a few classic references pointing to background information critical to an understanding of a specific topic area. These older references have been critically included as established fundamental works. New topics covered in this review not previously treated as separate subjects in past reviews include sampling systems, imaging (via optical spectroscopy), and ultrasonic analysis. The individual review subjects are organized into their most obvious subsection. The purpose of this review is to include the more critical work from each topic area in brief summaries, rather than as protracted abstracts. For this review, an expanded definition of the eras for the implementation of process analyzers has been included. The earlier definition for process analyzers was encompassed by the terms off-line, at-line, on-line, in-line, and noninvasive (A1, A2). A more expanded descriptive list of applicable process analyzer conditions, or eras, as extracted from the various titles of published research papers, includes (in alphabetical order) the following: airborne, at-line, automatic/automated, fieldable, hyperspectral, imaging/image analysis, in situ, in-line, near-line, noninvasive/nondestructive, on-line, open path, portable/hand-held, process monitoring/production control, quality control/quality assurance, quality monitoring, rapid, real-time, and remote. These types or eras for process analyzers are included under each review topic. Analytical chemistry is a key component of measurement science that plays a valuable role in supporting the characterization of products resulting from research, product and process development, and manufacturing. Process analytical chemistry is now an established field of Analytical Chemistry, as evidenced by the growing number of publications, by sections of symposiums (e.g., FACSS, Pittsburgh Conference, ACS meetings, etc.), and by dedicated meetings such as IFPAC. The field of process analytical chemistry has developed over the past 50 years due to the growing appreciation for having process data gathered close to the production operation. This has occurred for a variety of reasons including rime and cost savings, sampling concerns, sample transport, process efficiency, and safety. These reasons contributed to process efficiency and improved process control. With the globalization of industries, productivity and quality have added to the need for effective process control while minimizing the environmental impact. This approach requires a certain level of dedication and resources from the corresponding internal research operations within the organization. This has become more difficult recently as a result of cost reduction activities driven by corporate re-engineering and subsequent resource reduction of research, production maintenance, and plant instrumentation groups. To be most meaningful, process measurement science must tie into the needs in the engineering disciplines related to process control. There is a strong need for cross discipline appreciation, understanding, and cooperation in order to effectively incorporate the new developments in analytical measurement science with the advances in process modeling, monitoring, and control. These new developments are facilitated by continued improvements in the computer-related fields (semiconductor, automation, software, etc.). An important step is not only to incorporate these computer-related developments into analytical instrumentation but to explore these computing advances for ideas in microinstrumentation. Traditional laboratory-based analytical instrumentation, as well as the process analytical technologies described in this review, will be evaluated as to whether there is value to process control in pursuing them at the microscale. There will be advantages in ruggedness, replication, and subsequent cost where multiple units are desired for useful implementation. In addition, nontraditional measurement and characterization techniques (such as imaging, acoustics, thermal, and rheology approaches) will take on additional importance. This is due to global interest in marketing, technical service and development, and sales of products where the measurement and prediction of final product properties becomes key to a successful business. Although process measurements have traditionally included temperature, pressure, and flow rate, more efficiency can be added to processes by measuring composition or structural properties in a manner allowing real-time control during a manufacturing process. This review considers the essential elements to this compositional measurement scheme. These key aspects for process analytical chemistry include chemometrics and process control algorithms, sensors, optical spectroscopy, chromatography, mass spectrometry, flow injection analysis and automated wet chemical methods, ultrasonic analysis, NMR, and other techniques.

Hyphenation of liquid chromatography (LC) techniques with electrospray ionization (ESI) orthogonal acceleration time-of-flight (oa-TOF) mass spectrometry (MS) provides both MS-based structural information and LC-based quantitative data in polymer analysis. In one experimental setup, three different LC modes are interfaced with MS: size-exclusion chromatography (SEC/MS), gradient polymer elution chromatography (GPEC/MS), and liquid chromatography at the critical point of adsorption (LCCC/MS). In SEC/MS, both absolute mass calibration of the SEC column based on the polymer itself and determination of monomers and end groups from the mass spectra are achieved. GPEC/MS shows detailed chemical heterogeneity of the polymer and the chemical composition distribution within oligomer groups. In LCCC/MS, the retention behavior is primarily governed by chemical heterogeneities, such as different end group functionalities, and quantitative end group calculations can be easily made. The potential of these methods and the benefit of time-of-flight analyzers in polymer analysis are discussed using SEC/MS of a polydisperse poly(methyl methacrylate) sample, GPEC/MS of dipropoxylated bisphenol A/adipic acid polyester resin, LCCC/MS of alkylated poly(ethylene glycol), and LCCC/MS of terephthalic acid/neopentyl glycol polyester resin.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is used for the detection of bacteriocins. A 30-s water wash of the sample on the probe is shown to be effective in removing contaminants of the analyte. The approach is utilized for rapid detection of pediocin, nisin, enterocin A and B and brochocin A and B from culture supernatants and for the detection of enterocin B throughout the purification process.

A time-of-flight mass spectrometer and ab initio calculation were used to investigate the multiphoton ionization of the hydrogen-bonded clusters C4H5N-(H20)n at 355 and 532 nm wavelengths. Results showed that a series of C4H5N-(H20)n+ and C4H5N-(H20)nH+ can be obtained at both wavelengths. The proton in the protonated products obtained at 355 nm was found to preferably link with the alpha-C of pyrrole than with the N atom. Moreover, the protonated products obtained from 532 nm seem to be influenced by an intracluster penning ionization or a charge transfer reaction.

Polyaromatic hydrocarbons ({PAHs) were detected in a National Institute of Science and Technology Standard Reference Material sample of micrometer-sized diesel particulate matter using laser desorption/laser ionization time-of-flight mass spectrometry. Among the PAHs certified to be present in the sample are phenanthrene, 9-fluorenone, fluoranthene, pyrene, 2-nitrofluorene, benz(alpha)anthracene, chrysene and 1-nitropyrene. The determination of PAHs in the sample demonstrates the suitability of time-of-flight mass spectrometry for the spatially resolved analysis of individual micrometer-sized particulates.

Time-of-flight measurements were performed on a system for laser deposition of high-quality diamond-like carbon (DLC) thin films. It was found that energetic plasma containing high-energy carbon ions is needed to produce high quality DLC thin films by laser deposition. The energy distribution became broader with increasing laser fluence. Results suggested that shallow implantation or subplantation of impinging energetic ions is needed for the formation of diamond-like bonding.

A study was conducted to analyze cross sections of mixtures of tryptic digest peptide ions established by electrospray ionization using a novel ion mobility/time-of-flight spectrometry method. The peptide mixtures were electrosprayed into the gas phase. The cross sections were found to be dependent on the amino acid structure of each peptide. The reduced cross sections correlated with the fractions of nonpolar or polar aliphatic residues.

Aerosol particles have received significant public and scientific attention in recent years due to studies linking them to global climatic changes and human health effects. In 1994, Prather et al. (Prather, K. A.; Nordmeyer, T.; Salt, K. Anal. Chem. 1994, 66, 1403-1407) developed aerosol time-of-flight mass spectrometry (ATOFMS), the first technique capable of simultaneously determining both size and chemical composition of polydisperse single particles in real time. ATOFMS can typically analyze between 50 and 100 particles/min under typical atmospheric conditions. This significant volume of data requires automated data analysis for efficient processing. This paper reports the successful analysis of ATOFMS data acquired during a 1996 field study in Southern California using an adaptive resonance theory-based neural network, ART-2a. The ART-2a network revealed particle categories consistent with those obtained previously by manual analysis. The classification was accomplished in less time than the acquisition, rendering it possible to develop a data acquisition system using an on-line ART-2a that classifies particles as they are acquired.

The potential of capillary electrophoresis (CE) with off-line matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has been demonstrated for the examination of a glycoprotein factor associated with cancer cachexia. A comparison of CE profiles of a healthy volunteer and a cancer patient shows the presence of additional peaks in the electropherogram of the cancer patient that could be associated with cachexia. Micropreparative CE was performed with 180-[[micro]meter] fused silica capillary columns with tapered ends to collect CE fractions for further identification by MALDI-TOF-MS. The analysis of crude urine samples of cancer patients exhibiting cachexia, as well as CE fractions, with MALDI-TOF-MS using ferulic acid as the matrix shows a number of characteristic ions at m/z values of [approximately] 24 and [approximately] 67 kDa. The 24-kDa peak may be identified as the cachectic factor, a glycoprotein, whereas the peak at 67 kDa is identified as albumin, which is present in urine of most patients, and to which the cachectic factor is non-covalently bound. The combined use of CE and MALDI-TOF-MS was successful in detecting cachexia in all of the patients in this study, including one patient that was in an early phase of the disease.

To purify and characterize low-abundance proteins in complex biological mixtures, we used a novel strategy that combined preparative two-dimensional liquid-phase electrophoresis (2D-LPE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS). Preparative 2D-LPE is based on the same isoelectric focusing and gel electrophoresis principles as the widely used analytical 2D gel electrophoresis, except that analytes remain in solution throughout separation. This novel approach shows many improvements compared to analytical 2D gel electrophoresis for the separation of proteins in biological fluids. For example, larger volumes/amounts of samples can be loaded, yielding sufficient amounts of low-abundance proteins for further characterization. Since proteins remain in liquid phase during the entire procedure, extra steps such as electro-elution, extraction, or transfer to membranes from the gels prior to mass spectrometric analysis are obviated. We report the usefulness of 2D-LPE combined with MALDITOF MS for the purification and characterization of cystatin C and [Beta]-2 microglobulin in human cerebrospinal fluid. This method should be applicable to a wide range of biological fluids, such as cerebrospinal fluid, serum, tissue extracts, cell media, whole cells, and bacterial lysates.

Exoglycosidase digestion combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been demonstrated to be an effective method for the structural characterization of glycoconjugates and oligosaccharides in picomolar amounts. A sample preparation method is described, in which 6-aza-2-thiothymine (ATT) in water is used as matrix and enzymes are dialyzed before use against a low concentration of volatile buffer such as ammonium acetate. Under these conditions, a series of sequential on-target exoglycosidase treatments was carried out in one single analyte spot in the presence of ATT matrix. Subsequent mass spectrometric analysis of the resulting products yielded information on both the completeness of the reaction and structural features of the glycoconjugates such as monosaccharide sequence, branching pattern, and anomeric configurations of the corresponding glycosidic linkages. The results show that all exoglycosidases used retain their activity in the presence of ATT matrix. Hence, structural analysis of carbohydrates or mixtures thereof can be performed very fast, without intermediate desalting steps or sample splitting. This approach is illustrated by the analysis of underivatized glycans, oligosaccharide derivatives, glycopeptides, and glycolipids. Depending on the analyte, amounts of sample required could be limited to a few picomoles.

Reproducible time-of-flight cluster static secondary ion mass spectra (ToF-SSIMS) were obtained for various standard calcium phosphate (CP) powders, which allowed for phase identification. X-ray diffraction was not able to detect signals from microscopic amounts of CP ([approximately]15 mmol [m.sup.-2]). The phases studied were [Alpha]-tricalcium phosphate [[Alpha]-[Ca.sub.3][(P[O.sub.4]).sub.2]], [Beta]-tricalcium phosphate [[Beta]-[Ca.sub.3]-[(P[O.sub.4]).sub.2]], amorphous calcium phosphate [[Ca.sub.3][(P[O.sub.4]).sub.2] [multiplied by] x[H.sub.2]O], octacalcium phosphate [[Ca.sub.8][H.sub.2][(P[O.sub.4]).sub.6][center dot][H.sub.2]O], brushite (CaHP[O.sub.4] 2[H.sub.2]O), and hydroxyapatite [[Ca.sub.10][(P[O.sub.4]).sub.6][(OH).sub.2]]. The SIMS spectra were obtained via bombardment with (CsI)[Cs.sup.+] projectiles. X-ray photoelectron spectroscopy (XPS) core levels of the P 2p, Ca 2p, and O 1s orbitals and the relative O 1s loss intensity were examined. The P[[O.sub.3].sup.-]/P[O.sub.2]- ratios from ToF-SSIMS spectra in conjunction with XPS of the CP powders showed much promise in differentiating between these phases at microscopic CP coverages on the metal oxide surface.

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.

Hydrophobic peptides, especially those with acid-labile protecting groups, are difficult to characterize using mass spectrometric methods. We have developed a new procedure for matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis of such samples. Hydrophobic peptides, which are insoluble in aqueous solutions, are dissolved in chloroform and combined with matrixes prepared in chloroform or chloroform/methanol solutions. The use of a common solvent for the matrix and the analyte improves the analyte isolation step in MALDI mass spectrometry. The lack of acidic solutions previously used for electrospray ionization or MALDI mass spectrometry of hydrophobic peptides extends this methodology to cyclic or protected hydrophobic peptides. Conventional peptide matrixes, such as 2,5-dihydroxybenzoic acid and sinapinic acid, as well as 3-indoleacrylic acid are shown to be suitable for hydrophobic peptides. Cyclic hydrophobic peptides and linear hydrophobic peptides with blocked termini are detected as the cation-adducted pseudomolecular ion due to the lack of suitable sites of protonation on the analyte.

Acidic peptides have been studied by negative ion postsource decay (PSD) matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The peptides contained from 5 to 16 residues and were chosen on the basis of their patterns of the acidic residues. Using typical MALDI sample preparation techniques employing an acidic matrix, gastrin I (1-14), and epidermal mitosis inhibiting pentapeptide yielded much larger deprotonated ion signals, [[M - H].sup.-], than protonated ions, [[M + H].sup.+]. This may be due to their absence of basic residues, coupled with their arrays of acidic residues. The PSD fragmentation of the peptide negative ions showed that an array of acidic residues, as in gastrin I (1-14), yielded simple spectra containing mainly backbone cleavage ions from the C-terminus. Hirudin (54-65), which contains two sets of two consecutive Glu residues, and fibrinopeptide A and fibrinopeptide B, with isolated acidic residues, also showed backbone cleavages as common fragment ions. In addition, the two sets of isolated consecutive amino acid residues in [Cys(Bzl).sup.84]-C[D.sub.4] (81-92) and hirudin (54-56) yielded internal ions from the cleavages at the (O=C) - NH bond between the acidic residues. Also observed were ions with unique side chain losses, such as the loss of [C.sub.6][H.sub.4]O from a tyrosine residue and S[CH.sub.2][C.sub.6][H.sub.5] and [CH.sub.2][C.sub.6][H.sub.5] from a benzylated cysteine residue. Compared to the positive mode, the negative-ion PSD yielded fewer fragments which usually involved only one type of backbone cleavage (e.g., [[[y.sub.n] [H.sub.2]O].sup.-]). These simple spectra aided interpretation. Overall, the acidic peptides studied yielded negative ion PSD spectra that were useful for peptide sequencing.

A new method for the sampling and off-site analysis of hemoglobin variants by mass spectrometry is reported. This technique uses a nonporous polyurethane membrane as the collection device and transportation medium of a blood sample for analysis. The same membrane is then used as the MALDI-TOF MS sample support for mass spectrometric analysis. Minimal invasive sample collection is afforded by collecting less than 1 [[micro]liter] of blood using a common lancet device. MALDI-TOF MS is performed directly on the membrane, after washing off the interfering plasma components, followed by the addition of matrix. This reduces the time of analysis and prevents sample loss. Enzymatic digestion can be performed directly on the membrane, using in this case trypsin, allowing for further characterization of the sample. The method is much less invasive compared to drawing blood with a syringe. The sample may be transported to the laboratory by regular mail, and thus the method can serve remote locations. We demonstrate the procedure by characterizing the Hb Shepherds Bush hemoglobin variant, b74-(E18)Gly[right arrow]Asp.

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.

Hydrophobic peptides, especially those with acid-labile protecting groups, are difficult to characterize using mass spectrometric methods. We have developed a new procedure for matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis of such samples. Hydrophobic peptides, which are insoluble in aqueous solutions, are dissolved in chloroform and combined with matrixes prepared in chloroform or chloroform/methanol solutions. The use of a common solvent for the matrix and the analyte improves the analyte isolation step in MALDI mass spectrometry. The lack of acidic solutions previously used for electrospray ionization or MALDI mass spectrometry of hydrophobic peptides extends this methodology to cyclic or protected hydrophobic peptides. Conventional peptide matrixes, such as 2,5-dihydroxybenzoic acid and sinapinic acid, as well as 3-indoleacrylic acid are shown to be suitable for hydrophobic peptides. Cyclic hydrophobic peptides and linear hydrophobic peptides with blocked termini are detected as the cation-adducted pseudomolecular ion due to the lack of suitable sites of protonation on the analyte.

Acidic peptides have been studied by negative ion postsource decay (PSD) matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The peptides contained from 5 to 16 residues and were chosen on the basis of their patterns of the acidic residues. Using typical MALDI sample preparation techniques employing an acidic matrix, gastrin I (1-14), and epidermal mitosis inhibiting pentapeptide yielded much larger deprotonated ion signals, [[M - H].sup.-], than protonated ions, [[M + H].sup.+]. This may be due to their absence of basic residues, coupled with their arrays of acidic residues. The PSD fragmentation of the peptide negative ions showed that an array of acidic residues, as in gastrin I (1-14), yielded simple spectra containing mainly backbone cleavage ions from the C-terminus. Hirudin (54-65), which contains two sets of two consecutive Glu residues, and fibrinopeptide A and fibrinopeptide B, with isolated acidic residues, also showed backbone cleavages as common fragment ions. In addition, the two sets of isolated consecutive amino acid residues in [Cys(Bzl).sup.84]-C[D.sub.4] (81-92) and hirudin (54-56) yielded internal ions from the cleavages at the (O=C) - NH bond between the acidic residues. Also observed were ions with unique side chain losses, such as the loss of [C.sub.6][H.sub.4]O from a tyrosine residue and S[CH.sub.2][C.sub.6][H.sub.5] and [CH.sub.2][C.sub.6][H.sub.5] from a benzylated cysteine residue. Compared to the positive mode, the negative-ion PSD yielded fewer fragments which usually involved only one type of backbone cleavage (e.g., [[[y.sub.n] [H.sub.2]O].sup.-]). These simple spectra aided interpretation. Overall, the acidic peptides studied yielded negative ion PSD spectra that were useful for peptide sequencing.

A new method for the sampling and off-site analysis of hemoglobin variants by mass spectrometry is reported. This technique uses a nonporous polyurethane membrane as the collection device and transportation medium of a blood sample for analysis. The same membrane is then used as the MALDI-TOF MS sample support for mass spectrometric analysis. Minimal invasive sample collection is afforded by collecting less than 1 [[micro]liter] of blood using a common lancet device. MALDI-TOF MS is performed directly on the membrane, after washing off the interfering plasma components, followed by the addition of matrix. This reduces the time of analysis and prevents sample loss. Enzymatic digestion can be performed directly on the membrane, using in this case trypsin, allowing for further characterization of the sample. The method is much less invasive compared to drawing blood with a syringe. The sample may be transported to the laboratory by regular mail, and thus the method can serve remote locations. We demonstrate the procedure by characterizing the Hb Shepherds Bush hemoglobin variant, b74-(E18)Gly[right arrow]Asp.

The differential cross section of the S(1D) + D2 -> SD + D reaction at a collision energy of 5.3 kcal/mol was mapped through the Doppler-selected time-of-flight technique. The angular distribution was highly forward/backward peaking with very slight asymmetry in favor of the forward direction. Vibrational structures of the SD product were resolved in the angle-specific translational energy distribution. A strong relationship between the product angular and speed distributions was observed.

Time-of-flight spectroscopy under crossed molecular beams has been used in measuring the collision energy dependence of the Ca*((super 1)D(sub 2)) + HBr --> CaBr*(A,B) + H reaction over the 210 to 290 meV range. Results show there is a pronounced maximum at 215 meV followed by a post-maximum decline in the reaction cross section of the A and B excited channels. Some oscillations are also more noticeable in the reaction cross section for the formation of B rather than for that leading to the A state.

A series of liquid O,S-dialkyl dithiocarbonates (yankLate esters) have been synthesized, and time-of-flight (TOF) mass spectra were recorded for their vapors using both ultraviolet (UV) and vacuum ultraviolet (VUV) laser excitation. These compounds are chemical derivatives of low vapor pressure xanthate salts which have found important commercial application as collectors in mineral sulfide flotation circuits. Our experiments demonstrate that esters ionized by short-wavelength VUV light can be detected by parent mass with high efficiency and minimal fragmentation. In contrast, the mass spectra of the same compounds obtained by UV light excitation exhibit a large number of low molecular mass peaks. A preliminary quantitative analysis of the composition of a gas-phase mixture of xanthate esters has also been achieved, which indicates possible subfemtomole detection limits.

Oligosaccharides derivatized with 4-aminobenzoic acid 2-(diethylamino) ethyl ester (ABDEAE) can be analyzed by ESI (Yoshino, K.; et al. Anal. Chem. 1995, 67, 40284031) and MALDI (Takao, T.; et al. Rapid Commun. Mass Spectrom. 1996, 10, 637-640) mass spectrometry. In this study, oligosaccharides derived from the enzymatic cleavage of the sugar chains of glycoproteins ribonuclease B, erythropoietin, and transferrin were subjected to ABDEAE derivatization, prior to analysis on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF Ms) for high-resolution mass measurement and a postsource decay (PSD) experiment. In the mass measurement of ABDEAE derivatives, quasi-molecular ion species have been observed in monoisotopic resolution using 2,5-dihydroxybenzoic acid as the matrix from spots that contain 50-200 fmol of sample; in the PSD analyses from the spots contained 500 fmol-1 pmol of sample, the predominant backbone ion series which covers the entire mass range for all the derivatives, the internal ion series which reflect the branched trimannosyl core structure of N-glycans, and the low m/z fingerprint ion of ABDEAE were consecutively observed, permitting structure elucidation of the oligosaccharides. Given the effectiveness of this derivatization in terms of its high sensitivity and resolution with respect to MALDI-TOF MS, current methodology is clearly applicable to the sensitive detection and accurate structural analysis of N-glycans.

Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOFMS) has several advantages in the study of biomolecules and synthetic polymers. The technique's usability is attributed to innovations in infrared techniques, improved lasers and digitizers and the availability of pulsed extraction. The important criteria in selecting a MALDI-TOFMS instrument include mass resolution, sensitivity and mass accuracy. Another advantage of a MALDI-TOFMS is its need for low maintenance.

Research was conducted to examine the ion-molecule chemistry of boron trifluoride using electron impact ionization of molecular clusters. Time-of-flight mass spectrometry was used to analyze the resulting ion distribution. Results based on the ab initio modeling of the ions show that mass distribution is the result of reactions in and fragmentation of the clusters. Findings demonstrate that mass spectrometry is an efficient probe of ion-molecule chemistry within ionized clusters.

Research was conducted to examine the photoionization of SiBr4 in the valence shell and the Br(3d) and Si(2p) inner shell excitation/ionization regions using a time-of-flight mass spectrometer and synchrotron radiation over the range 30-133 eV. Photoelectron-photoion coincidence and photoion-photoion coincidence techniques were employed to investigate the dissociation processes of SiBr4. Results provide information on energy dissipation processes of the core-excited states.

Ablation of polyethersulfone (PES) films at 266 nm has been studied by using laser ionization time-of-flight (TOF) mass spectrometry. PES is made up of two phenylene groups, a sulfone group and an ether oxygen with carbon-carbon bonds within the phenylene rings. Neutral products arriving at an ion extraction position of a TOF mass spectrometer were detected by delaying a post-ionization laser pulse. Results indicate that the decomposition of PES occurs due to the scission of the chain itself and the cleavage of some of the phenylene rings.

A protocol including 2D SDS-PAGE, electroblotting proteins onto nitrocellulose membranes, and CNBr cleavage, followed by MALDI-MS analysis of intact proteins and peptide fragments and a database search, has been optimized and applied to the rapid identification of the Escherichia coli response to hypochlorous acid. The methodology has proved to be efficient from the point of view of sensitivity (picomole range) and selectivity. In particular, MALDI analysis of proteins and CNBr fragments by directly dissolving the membrane in an acetone solution of matrix, without previous elution, is reliable and reproducible. The accuracy of the MW determination is somewhat reduced compared to that of methods involving elution and purification of proteins and digests; nevertheless, the utilization of large MW windows combined with the pI entry in database searches had allowed, for most of the spots, the selection of only one protein candidate. Finally, 19 proteins exhibiting a response to hypochlorous acid stress have been confirmed or identified on the basis of this protocol.

In matrix-assisted laser desorption/ionization of proteins, there exists a certain amount of fast metastable decay immediately after laser irradiation. The fragment ions thus formed can be resolved and their m/z values measured accurately by employing delayed extraction linear time-of-flight mass spectrometry. At higher than threshold laser fluences, proteins exhibit a series of fragment ions providing useful sequence information. We also observe that when moderate amounts of salts are present in the sample with sinapinic acid being the matrix, the intensifies of [c.sub.n] ions (N-terminal fragments) are enhanced compared to other types of fragment ions. This enhancement in [c.sub.n] ion signals allows direct sequencing of proteins. The [c.sub.n] ions are completely absent when Xxx-Pro bonds are encountered and are of lower intensity when Xxx-Gly bonds are involved. Further, the [c.sub.n] ion series is interrupted at Xxx-Cys, when the cysteine is involved in a disulfide bond. Upon reduction of the disulfide bonds, the series continues and information is available for longer stretches. Using 10-20 pmol of recombinant proteins, sometimes contiguous sequence information up to 70 residues is obtained in a matter of minutes. Applications of the technique to some recombinant proteins with intra- or interchain disulfide linkages are presented.