This invention relates to ion sources, and particularly to a Matrix Assisted Laser Desorption/Ionization ion source for use with an associated mass analyzer.
Different ion sources can be used in conjunction with mass analyzers. These include pulsed ion sources and continuous ion sources. Common examples of ion sources include the matrix assisted laser desorption ionization (MALDI) ion source and the surface enhanced desorption ionization (SELDI) ion source.
Matrix Assisted Laser Desorption/Ionization (MALDI) mass spectrometry is a useful technique for analysis of labile molecules, like proteins and nucleotides, and is widely employed in biochemistry and the life sciences. For a number of years, this ionization technique was used under purely vacuum conditions. Analyzed sample (analyte) is dissolved in a suitable matrix that absorbs in a selected UV wavelength range. The sample is deposited onto the sample plate, positioned in a vacuum chamber and irradiated by a pulsed laser, most commonly by a nitrogen laser at 337 nm. The matrix absorbs UV light, experiences rapid disintegration, and emits a microscopic plume of matrix vapors and a pulse of analyte ions. Conventionally, an ion plume extracted by a DC electric field and analyzed by a time-of-flight mass spectrometer. Recently, examples of MALDI using IR wavelengths have been described.
MALDI mass spectrometry was further improved with the introduction of the Delayed Extraction (DEMALDI) technique, in which a small delay of about 100 ns is incorporated between the laser pulse and the subsequent application of the extracting field. This substantially reduces collisions between analyte ions and matrix vapors and allows the extraction of molecular ions. With a proper choice of time delay and pulsed extracting field, the DE technique preserves pulsed properties of the ion plume and provides superior results. Nevertheless, the technique is still sensitive to sample preparation and requires searching for a sweet spot on a matrix crystal. The technique also requires fine tuning of laser energy near the threshold of ion production, depending on a particular crystal spot and, thus, can be difficult to automate for a high throughput mass spectrometric analysis, as is currently demanded by proteomics applications and single nucleotide polymorphism (SNP) screening.
With the introduction of MALDI at an intermediate gas pressure, ion source conditions were fully decoupled from mass analysis. In this technique, the MALDI ion source operates at a pressure in the range of 0.01 to 0.1 torr, and the ion beam is extracted into an RF-only ion guide. Collisions with neutral gas in the RF guide damp ion kinetic energy and convert ion pulses into a continuous or a quasi-continuous ion beam. The ion beam is subsequently analyzed by an orthogonal time-of-flight mass spectrometer (oa-TOF MS), again converting the ion beam into ion pulses, which are extracted orthogonal to the initial direction of the beam. In spite of the dual conversion between the pulsed and continuous beam, and in spite of associated ion loss, the technique appears to provide excellent sensitivity.
Damping by gas collisions was brought to the extreme by introduction of atmospheric pressure MALDI. In this technique, a single MALDI sample is positioned in the conventional electrospray interface in front of the nozzle, sampling ions into a differentially pumped interface. The sample is irradiated by a nitrogen laser, producing analyte ions. Ions are extracted by a strong electric field and are blown from the sample by a gas jet. All these means are necessary to suppress cluster formation with matrix molecules. Even so, cluster-formation severely limits merits of the technique, and so far the method has only been applied to peptide samples with a single matrix—alpha-cyano-hydrocinnamic acid. Operation at atmospheric pressure seems to be convenient, since the samples can be changed without loading into a vacuum chamber. However, in a high throughput ion source, sample loading time becomes negligible compared to analysis time, since sample plates accommodate from 100 to 400 samples. In addition, ion extraction at atmospheric pressure is accompanied by severe ion losses which limit sensitivity. Considering the sacrifice in performance, atmospheric pressure MALDI appears less practical than MALDI at intermediate gas pressure.
Looking at a wider range of operating conditions, Verentchikov et al. (WO 00/77822 A2) have found that MALDI at intermediate gas pressure provides substantially better results at gas pressures between 0.1 and 1 torr. Gas collisions at such pressure rapidly cool ion internal energy and allow soft ionization in a wide range of laser energy. Further increase in gas pressure is limited by formation of ion clusters with matrix molecules. At pressures up to about 1 torr, gas heating the RF ion guide to about 200° C. can resolve this problem. At higher gas pressures, however, cluster formation becomes so abundant that at any harsh conditions in the interface it is more likely to fragment ions than to clear them out of cluster shell.
Collisional cooling of internal energy allows using laser energy about three to five times higher than the threshold of ion production, which enhances ion intensity by several orders of magnitude. The technique is also compatible with high repetition rate lasers at a kilohertz range. As a result, ion intensity in MALDI at intermediate gas pressure can be several orders of magnitude higher than in DE MALDI the latter being compatible with high repetition rate lasers because of technical difficulties associated with high repetition rate of the extraction pulse. High signal intensity in MALDI with collisional cooling allows rapid analysis—a desirable feature in proteomics and SNP applications. Ease of automation makes the technique particularly suitable for high throughput analysis. However, using high intensity ion beam creates a new problem contamination of the source electrodes by matrix vapors emitted due to the high repetition rate of the laser pulses at elevated energy. At a rate of screening of 1 to 10 samples per second, approximately 1 mg/s of matrix is emitted from the sample plate and deposited on the source electrodes. Some of that material is emitted as hot matrix vapors and some as small droplets or clusters. Matrix layers deposit to form dielectric films. Charging of those films by the ion beam distorts the electric fields and eventually repels the ion beam. Heating of the front electrodes and the RF ion guide as in Verentchikov et al. (WO 00/77822 A2) merely leads to matrix deposition onto subsequent electrodes of the transport interface and mass spectrometer.
Similar problems of interface protection have been addressed in applications using the electrospray (ESI) technique. In that technique, a sample, dissolved in a solvent, is pumped through a small-bore capillary into an ion source that is filled with a gas at atmospheric pressure. Applying a few kilovolts to the capillary atomizes the solvent into a fine aerosol. The charged aerosol evaporates solvent and produces a mixture of vapors, micro-droplets, and ions of the sample. Ions are sampled into a nozzle and transported into the vacuum chamber of a mass spectrometer via a differentially pumped interface. If no protection is used, the salts and buffers from real pharmaceutical samples quickly clog the interface.
In the so-called ‘ion spray’ technique, nebulization is assisted by a coaxial gas jet. The jet entrains the aerosol and directs it at a spot displaced from the nozzle entrance. Ions are attracted towards the nozzle by an electric field and travel across the gas jet faster than charged droplets. By adjusting the spray position, source clogging can be reduced with limited reduction in signal intensity. Interface ruggedness is also improved with the introduction of so-called ‘curtain gas’ devices. In these devices, an additional annular electrode is installed in front of the sampling nozzle. Gas is introduced between the nozzle and the electrode, and flows through the electrode aperture towards the spray capillary, repelling vapors and small droplets. An electric field between the spray capillary, electrode, and nozzle assists ion extraction through the gas counter flow.
In the so-called Z-spray and M-spray interfaces, interface ruggedness is further improved by providing a bent channel so that direct carryover of droplets is impeded while ion transport is facilitated by a combination of gas flows and electric fields. Such systems can be used for high flow rates of solvent, up to 2 mL/min.