Marvin L. Vestal

Crossed-beam liquid chromatoraph—mass spectrometer combination

Abstract A new approach to combined liquid chromatograph—mass spectrometry is described which uses laser vaporization of the liquid chromatographic (LC) effluent and molecular beam techniques to transport and ionize the sample. A key feature of this approach is that samples eluting from the LC are vaporized, ionized, and mass analyzed with minimal contact with solid surfaces. Results are presented on the performance of the crossed-beam system for relatively volatile aromatic hydrocarbons and for components of nuclei acids—bases, nucleosides, and nucleotides— which provide an interesting series of biologically important compounds of decreasing volatility and corresponding increasing difficulty for mass spectrometry. Mass spectra are presented which were obtained by laser vaporization of samples from the LC effluent under both reversed-phase and ion-exchange conditions.

On the initial velocity of ions generated by matrix-assisted laser desorption ionization and its effect on the calibration of delayed extraction time-of-flight mass spectra

A novel method was developed to measure the initial velocity of ions generated by matrix-assisted laser desorption ionization (MALDI). It is shown both experimentally and theoretically that with a delayed extraction (DE) technique, the flight time of an ion changes linearly with extraction delay. The initial velocity of the ion, a consequence of the desorption process, can be determined from the slope of this linear curve. Systematic study of the initial velocity was undertaken regarding its dependence on the matrix substance, molecular weight of the analyte, ion polarity, and wavelength of irradiation. It was found that the most important factor was the matrix material. Sinapinic acid and α-cyano-4-hydroxycinnamic acid matrices ejected slower peptide and protein ions than 2,5-dihydroxybenzoic acid or 3-hydroxypicolinic acid: ∼ 300 versus ∼ 550 m/s. Matrix ions themselves exhibited a similar order of initial velocities, but these were 15–40% higher than those of insulin ions. The molecular weight of protein samples (between 5 and 25 ku) was found to have little effect on the initial velocity, but for peptides below 5 ku a gradual transition was noted toward the velocity of the matrix ions. Also decreasing velocity with increasing molecular mass was observed for DNA samples in the 4–14-ku range. In the negative ion mode slightly lower velocities were observed than in the positive ion mode. No difference was found between 337- and 266-nm irradiation. Values of the initial velocities were used to correct systematic errors in the internal calibration observed in mass spectra with delayed extraction. These velocity corrections decrease mass errors substantially in the linear mode, in particular for multicomponent mixtures.

Studies of ionization mechanisms involved in thermospray LC-MS

Abstract Earlier we reported that molecular ions were produced when aqueous solutions containing nonvolatile solutes were rapidly vaporized by forcing them through a heated nozzle. This “thermospray” process produces mass spectra for many non-volatile molecules which are very similar to those obtained by field desorption and other soft ionization techniques. Recent efforts have focused on elucidating the mechanisms involved in this ionization process. This paper summarizes our present understanding of the ionization mechanism.

Resolution and mass accuracy in matrix-assisted laser desorption ionization-time-of-flight

A mathematical model of time-of-flight mass analyzers employing uniform electric fields is presented that allows “exact” calculations of flight times as functions of mass-to-charge ratio, initial velocity and position, applied voltages, and instrument geometry. An “approximate” equation based on a series expansion of the “exact” result is derived which allows focusing conditions and limits on resolution to be determined for different instrument geometries and operating conditions. The fundamental theory is applied to predicting resolution and mass accuracy in matrix-assisted laser desorption ionization-time of flight. In this case higher order velocity focusing can provide excellent correction for the initial velocity distribution of a selected mass-to-charge ratio, but the focusing is mass-to-charge ratio dependent. There is generally a trade-off between ultimate resolution at a particular mass-to-charge ratio and resolution and mass accuracy over a broad mass range. In most practical applications the latter is more important. Calculations are compared with experimental results for a particular analyzer geometry, both at theoretical optimum velocity focus and at operating conditions where ultimate resolution is sacrificed for a broader range of relatively high resolution and better mass accuracy.

Accurate Mass Measurements Using MALDI-TOF with Delayed Extraction

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry is now an essential tool in biopolymer analysis. Sensitivity and mass range are unsurpassed, but mass measurement accuracy and resolution have been limited. With delayed extraction and a reflecting analyzer, mass measurements using MALDI-TOF can be made with an accuracy of a few parts per million (ppm). It is possible to distinguish Lys from Gln in peptides, and to determine the elemental composition of smaller molecules (mass 100–500). In database searching strategies, a smaller mass window, resulting from an increase in mass accuracy, greatly decreases the number of possible candidates. Mass measurement accuracy with errors less than 5 ppm is demonstrated on a mixture of 12 peptides ranging in mass from ca. 900 to 3700 Da. Mass measurements on 13 peaks in an unseparated tryptic digest of myoglobin gave results with an overall average error less than 3.5 ppm, with a maximum error of 7 ppm.

Tandem time-of-flight mass spectrometry.

A new tandem time-of-flight (TOF-TOF) instrument has been developed by modifying a standard matrix-assisted laser desorption ionization (MALDI)-TOF instrument to make high-performance, high-energy collision-induced dissociation (CID) MALDI tandem mass spectrometry (MS) a practical reality. To optimize fragment spectra quality, the selected precursor ion is decelerated before entering a floating collision cell and the potential difference between the source and the collision cell defines the collision energy of the ions. Standard operating conditions for tandem MS use a 1-kV collision energy with single-collision conditions and increased laser power for ion formation. Hence, both high- and low-energy fragments are observed in MALDI TOF-TOF spectra. On standard peptides, sensitivities down to 1 fmol are demonstrated. On a mixture of two solution tryptic digests at the 25-fmol level, 23 spectra were sufficient to result in proper database identification.

Modern MALDI time‐of‐flight mass spectrometry

This paper focuses on development of time-of-flight (TOF) mass spectrometry in response to the invention of matrix-assisted laser desorption/ionization (MALDI). Before this breakthrough ionization technique for nonvolatile molecules, TOF was generally considered as a useful tool for exotic studies of ion properties but was not widely applied to analytical problems. Improved TOF instruments and software that allow the full potential power of MALDI to be applied to difficult biological applications are described. A theoretical approach to the design and optimization of MALDI-TOF instruments for particular applications is presented. Experimental data are provided that are in excellent agreement with theoretical predictions of resolving power and mass accuracy. Data on sensitivity and dynamic range using kilohertz laser rates are also summarized. These results indicate that combinations of high-performance MALDI-TOF and TOF-TOF with off-line high-capacity separations may ultimately provide throughput and dynamic range several orders of magnitude greater than those currently available with electrospray LC-MS and MS-MS.

Analysis of gentamicin sulfate by high-performance liquid chromatography combined with thermospray mass spectrometry

Abstract The quantitative analysis of gentamicin sulfate by high-performance liquid chromatography (HPLC) with mass spectrometry was performed on-line utilizing thermospray mass spectrometry (TSP-MS). Chromatographic reversed-phased separation utilizing trifluoroacetic acid as an ion pair reagent resulted in the observation by TSP-MS of the major components (C1a, C2 and C1) of gentamicin sulfate as well as an additional minor component. This minor component had the identical [M + H]+ ion and fragmentation pattern of the major Method development and optimization of the mobile phase for HPLC-TSP-MS werer accomplished with a variable simplex algorithm. HPLC with electrochemical detection was utilized in conjunction with the simplex algorithm to establish a mobile phase suitable for the HPLC-TSP-MS analysis of gentamicin sulfate. Bulk preparations of gentamicin sulfate were assayed by HPLC-TSP-MS for the major components by comparison with an external standard, and by a comparison of peak areas obtained for the individual components vs. the totaled peak areas.

Field limit for ion evaporation from charged thermospray droplets

Abstract Thermospray ionization produces charged droplets from electrolyte solutions. As these droplets evaporate, the self induced field on the droplet surface increases and ion production from these droplets can take place either by ion evaporation or by Rayleigh subdivision, depending on the critical field strength required to initiate either process at that instant. An attempt was made to determine the field strength on the surface of the charged droplets in their final stage of evaporation, by measuring their reduced mobilities in a drift tube equipped with a mechanical chopper for gating the droplets. The relation between the reduced mobility and the surface field strength is discussed. The performance of the drift tube is such that in some cases the width of the mobility distributions is not significantly greater than the dispersion estimated from the apparatus. For the volatile and nonvolatile electrolytes studied, only one major peak was observed in the mobility spectra. The measured reduced mobilities are 0.44–0.53 cm 2 V −1 s −1 and the corresponding surface field strengths are 7.6–9.1 × 10 8 V m −1 . No peaks were observed with field strengths higher than 10 9 V m −1 . The observed mean value, 0.83 × 10 9 V m −1 , is interpreted as the critical field strength for ion evaporation and its implications are discussed.

The Future of Biological Mass Spectrometry

Biological applications of mass spectrometry have grown exponentially since the discovery of MALDI and electrospray ionization techniques. This growth has been further fueled by the massive volume of DNA sequence information that is now available. An ambitious goal of some of this research is to monitor the level and modification of all proteins and metabolites in a biological sample such as plasma. A major research effort in mass spectrometry and related disciplines has been expended over the past several years toward reaching this and other less ambitious goals, and considerable progress has been made; but the presently available tools are clearly not sufficient for these very difficult tasks. In this “critical insight” discussion we suggest that recent advances in time-of-flight (TOF) technology with MALDI ionization may provide some important new tools for achieving the goals of biological research.