Protein profiles with atmospheric pressure ionization
View Patent ↗The invention relates to the acquisition of mass spectra of complex protein mixtures, often called protein profiles, for example to search for biomarkers which indicate stress situations, or to identify microbes. Up to now protein profiles have been acquired using ionization by matrix-assisted laser desorption with high detection sensitivity in linear time-of-flight mass spectrometers, but these display very poor mass resolution and a very poor reproducibility of the mass values. The invention provides methods which produce surprisingly similar mass spectra, but with far higher mass resolution and mass accuracy. Ionization takes place outside the vacuum at ambient pressure, preferably by means of laser desorption and CI post-ionization. Analysis of the ions takes place in a high-resolution mass spectrometer, for example a reflector time-of-flight mass spectrometer with orthogonal ion injection.
1. Method for acquiring mass spectra of complex analyte mixtures from biomaterial, comprising the following steps:
(a) providing a high-resolution mass spectrometer capable to analyze ions generated at atmospheric pressure,
(b) preparing samples containing the analyte mixtures on a sample carrier,
(c) desorbing sample molecules from a sample on the sample carrier in an inert gas at atmospheric pressure,
(d) creating reactant ions inside the plume of desorbed sample molecules and thereby ionizing the analyte molecules by reactions with the reactant ions,
(e) feeding at least a part of the analyte ions into the vacuum system of the mass spectrometer, and
(f) acquiring high-resolution mass spectra of the analyte ion mixture.
2. Method according to claim 1 , wherein the high-resolution mass spectrometer is a reflector type time-of-flight mass spectrometer with orthogonal ion injection.
3. Method according to claim 1 , wherein the sample on the sample carrier contains a mediator substance beside the analyte molecule mixture.
4. Method according to claim 3 , wherein the mediator substance is selected from the group of matrix substances used hitherto in ionization by matrix-assisted laser desorption.
5. Method according to claim 3 , wherein molecules of the mediator substance are transformed into reactant ions by electrons shot into the desorption plume.
6. Method according to claim 5 , wherein the electrons are generated by beta radioactivity, by a corona discharge, or by a surface under irradiation.
7. Method according to claim 3 , wherein molecules of the mediator substance inside the desorption plume are transformed into reactant ions by photo ionization.
8. Method according to claim 7 , wherein the photons for the photo ionization are created by an UV lamp or by an UV laser diode.
9. Method according to claim 1 , wherein the sample contains a substance which decomposes into small molecules during desorption.
10. Method according to claim 1 , wherein desorption of the analyte molecules is produced by bombardment with a continuous light beam from a CW laser or a CW laser diode.
11. Method according to claim 1 , wherein desorption of the analyte molecules is produced by bombardment with light pulses from a pulse laser or a pulsed laser diode.
12. Method according to claim 11 , wherein the pulse frequency of the desorption light pulses is sufficiently high to produce a continuous flow of gasified sample material.
13. Method according to claim 12 , wherein the pulse frequency of the light pulses is higher than 500 Hertz.