Radiant energy – Ionic separation or analysis – With sample supply means
Reexamination Certificate
1999-07-29
2002-10-15
Nguyen, Kiet T. (Department: 2881)
Radiant energy
Ionic separation or analysis
With sample supply means
Reexamination Certificate
active
06465778
ABSTRACT:
The invention relates to ionization with low fragmentation and adduct formation, of high-molecular weight analyte molecules, particularly of large biopolymers, by matrix-assisted laser desorption (MALDI) from liquid matrices for mass-spectrometric analysis of the resulting ions, particularly for determination of their molecular weight.
PRIOR ART
For mass-spectrometric determination of the molecular weight of biomolecules or other high polymers, ionization by means of matrix-assisted desorption with the aid of a pulsed UV laser (MALDI) has been established as a standard method in spite of some disadvantages. MALDI is also used for structural determination, generally then with desirable fragmentation of the molecule ions by taking special measures.
Normally, lasers are used which operate within the ultraviolet light range and emit light pulses lasting a few nanoseconds, such as simple and inexpensive nitrogen lasers with 337 nanometer wavelength and two to three nanoseconds light pulse length. The pulsed generation of ions calls for the use of time-of-flight mass spectrometers (TOF-MS) for analysis, although ion storage mass spectrometers such as ion cyclotron resonance mass spectrometers (FT-ICR=Fourier-transformation ion cyclotron resonance) or high frequency quadrupole ion trap mass spectrometers can also be used successfully.
Biomolecules in this context are the oligonucleotides (i.e. the genetic material in its various forms such as DNA or RNA), proteins and polysaccharides (i.e. the essential building blocks of the living world), including their particular analogs and conjugates, such as glycoproteins or lipoproteins. Other high polymers in particular are the artificially produced polymers. In the following, these biopolymers and artificial polymers, the molecules of which are to be analyzed, are simply called the “analyte.”
The selection of matrix substance for MALDI depends on the type of analyte molecules; in the meantime, several hundred various matrix substances have become known which are each suitable for specific groups of analyte molecules. The matrix substance in particular has the following tasks: 1) to bond the sample substance in a finely distributed, very dilute form to the sample support, 2) to absorptively collect the energy from the laser beam, 3) to blow the analyte molecules individually into the gas phase by creating a vapor cloud, and 4) to ionize a portion of the analyte molecules by means of protonation or deprotonation. During this process of vaporization and ionization, neither should the analyte molecules decompose, nor should matrix or other molecules attach themselves to the analyte ions to a larger extent, since determination of the correct molecular weight is no longer possible in either case.
It has generally proven favorable to use aromatic acids as matrix substances that are crystalline in a normal state, and to integrate relatively few analyte molecules into the small matrix crystals or at least imbed them in the boundary surfaces between the small crystals. The bio-molecules are generally at least weakly water soluble so it is preferable, though not necessary, to use water soluble matrix substances. A rule of thumb has been developed which states that the selection of matrix substances becomes more difficult, the higher the molecular weight of the biosubstances is. Either fragmentation of the macromolecules increases to such an extreme degree that molecule ions can no longer be found, or adducts with matrix or other molecules are formed so that it is hardly possible to determine the molecular weight. In many cases, a mixture of adduct formation and splitting off of smaller fragments causes the mass-spectrometric signal to become a broad peak, which makes precise mass determination no longer possible.
In constantly searching for new matrix substances more favorable for certain substance categories, it has almost been forgotten that the first MALDI ionization of high-molecular substances was observed in a liquid glycerol matrix, to which a fine metallic powder was added for the absorption of UV laser beams (K. Tanaka et al., Rapid Comm. in Mass Spectrom., 2, 151, 1988). As MALDI was further developed, glycerol was used only occasionally as the matrix due to the disadvantages described below, but also in particular because glycerol cannot be excited directly by the standard UV lasers. In principle, however, this disadvantage was eliminated by a working group dissolving UV absorbents within the glycerol and thus adapting the liquid mixed matrix to the standard UV lasers, although, for unknown reasons, not all the absorbents displayed good results, and some strong UV absorbents actually prevented ionization (D. S. Cornett et al., “Liquid Mixtures for Matrix-Assisted Laser Desorption”, Anal. Chem., 65, 2608, 1993).
In a very recent article, an infrared laser was used for ionization, the radiation of which was capable of directly exciting one of the stretching vibrations of the glycerol (“Infrared MALDI Mass Spectrometry of Large Nucleic Acids”, Science 281, 2212, 1998). In particular, the radiation from an erbium-YAG laser with 2.94 micrometers wavelength excites the stretching vibrations of the OH groups. It was established that this combination of glycerol and infrared radiation with extremely low-energy photons is capable of extraordinarily sensitive, low-fragment ionization of molecules with extremely high molecular weights. Although the erbium-YAG infrared laser, most favorable for absorption of radiation, is indeed still relatively expensive and technically not yet particularly reliable, it can be expected that this type of laser will go through development similar to efficient equipment like the related neodym-YAG laser.
Glycerol is at all suitable for this type of ionization because it has a relatively low vapor pressure. Even in a vacuum, a small droplet of about one microliter evaporates quite slowly, taking about one half hour to dry out completely. This time can be utilized for MALDI analysis of several samples on a carrier plate.
Glycerol was used already one or two decades ago as a liquid medium for the ionization of dissolved substances by means of bombardment with fast neutral particles (fast atom bombardment, “FAB”). Using this method, highly sensitive, very low fragment and low adduct spectra of molecules with relatively high molecular weights were obtained.
So far there can only be speculation about the reason for the similarly high ionization effect of glycerol in these very diverse ionization methods. It appears possible that macromolecules which are almost always composed of mixed hydrophobic and hydrophilic groups (amphiphitic substances) prefer to keep their more hydrophobic side toward the surface, and project their hydrophilic side toward the very polar solution. This effect may lead to a substantial increase in the concentration of these high polymers at the surface. On the other hand, glycerol (1,2,3-propantriol) as a trihydroxylic alcohol may possibly ionize other substances very easily by means of proton donation from one of the alcohol groups. Even shockwaves propagated in the liquid with a shaking off of surface molecules has been discussed. It is also known that very polar water may be used as a matrix, but it requires extreme cooling of the carrier plates within a vacuum to prevent immediate evaporation.
Ionization by means of glycerol offers advantages, but also severe disadvantages.
Advantages: In addition to the high sensitivity for very large molecules and the relatively low fragmentation and adduct-formation, the uniform ionization yield over the entire drop surface is especially prominent on the list of advantages. Since visual control of the bombardment site is no longer necessary, and automated procedure is possible, different than the case previously for MALDI procedures where droplets are dried into solid matrices. Also considered advantageous is the fact that samples are relatively easy to prepare; a droplet with aqueous analyte solution may simply be applied to a droplet of glycerol. The pre
Franzen Jochen
Koster Claus
Bruker Daltonik GmbH
Nguyen Kiet T.
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