Alexandre V. Loboda

A tandem quadrupole/time-of-flight mass spectrometer with a matrix-assisted laser desorption/ionization source: design and performance

A matrix-assisted laser desorption/ionization (MALDI) source has been coupled to a tandem quadrupole/time-of-flight (QqTOF) mass spectrometer by means of a collisional damping interface. Mass resolving power of about 10,000 (FWHM) and accuracy in the range of 10 ppm are observed in both single-MS mode and MS/MS mode. Sub-femtomole sensitivity is obtained in single-MS mode, and a few femtomoles in MS/MS mode. Both peptide mass mapping and collision-induced dissociation (CID) analysis of tryptic peptides can be performed from the same MALDI target. Rapid spectral acquisition (a few seconds per spectrum) can be achieved in both modes, so high throughput protein identification is possible. Some information about fragmentation patterns was obtained from a study of the CID spectra of singly charged peptides from a tryptic digest of E. coli citrate synthase. Reasonably successful automatic sequence prediction (>90%) is possible from the CID spectra of singly charged peptides using the SCIEX Predict Sequence routine. Ion production at pressures near 1 Torr (rather than in vacuum) is found to give reduced metastable fragmentation, particularly for higher mass molecular ions. Copyright 2000 John Wiley & Sons, Ltd.

ORTHOGONAL INJECTION OF MATRIX-ASSISTED LASER DESORPTION/IONIZATION IONS INTO A TIME-OF-FLIGHT SPECTROMETER THROUGH A COLLISIONAL DAMPING INTERFACE

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

A useful binary matrix for visible-MALDI of low molecular weight analytes

In this work, a new absorbing candidate, rhodamine (R) 575, is described, which forms the basis of a binary matrix operating at 532 nm. Analyte ionization is found to be much more efficient when the dye is combined with a proton donor such as hydrochloric acid or α-cyano-4-hydroxycinnamic acid, or a proton acceptor such as sodium hydroxide. This makes the matrix more generic than many others that have been tried. Furthermore, under visible illumination R575 produces very few chemical fragments, making it useful for small molecular weight analyte detection. Spectra for a variety of analytes are shown. Insight into the MALDI mechanism was obtained by comparing the similarities and differences of visible-MALDI with the more common UV and IR-MALDI strategies.

Measurements of Protein Structure and Noncovalent Interactions by Time-of-Flight Mass Spectrometry with Orthogonal Ion Injection

Genomic sequences are becoming available with increasing speed as a result of the advances in gene technology. Nevertheless, both covalent and noncovalent modifications can occur in the corresponding protein sequence before the protein is fully functional, so it is important to know the actual protein sequence to understand its function [1, 2]. It is equally important to determine the higher order protein structure, and its interaction with other biological components. Noncovalent interactions of this type play a key role in molecular recognition phenomena such as enzyme-substrate interaction, receptor-ligand binding, formation of oligomeric proteins, assembly of transcription factors, and formation of cellular structures themselves [3–4].