The invention relates to the fragmentation of biopolymer ions with multiple positive charges by means of electron transfer (ETD=electron transfer dissociation) in reactions with radical anions. The sequences of the basic building blocks of biopolymers and their posttranslational modifications (PTM) are nowadays predominantly determined using tandem mass spectrometers. A key technology used for this is fragmentation of the biopolymer ions. There are two fundamentally different types of fragmentation—ergodic and non-ergodic or electron-induced—and a number of methods are known for both of these.
Peptides and proteins in particular will be considered below as biopolymers. Electron-induced fragmentation of peptide or protein ions is complementary to ergodic fragmentation, first of all because it cleaves the chain of amino acids at different amino acid locations, and secondly because it does not cut off the side chains of the posttranslational modifications during fragmentation, as is done by ergodic fragmentation. Comparing the fragment ion spectra obtained from ergodic and non-ergodic, electron-induced fragmentations allows both the sequences and the modifications to be read.
The simplest electron-induced method is electron transfer dissociation (ETD), which occurs as a reaction between multiply positively charged analyte ions and special radical anions. By using particular species of negative reactant ions in order to cleave biopolymer ions with multiple positive charges, particularly peptide or protein ions, an electron is transferred to the analyte ion which immediately experiences a fracture of the backbone chain. The reactant ions are usually radical anions of the form M.−, which readily donate electrons. The prior art is described in patents US 2005/0199804 A1 (D. F. Hunt et al.) and DE 10 2005 004 324 B4 (R. Hartmer and A. Brekenfeld). Both these documents describe the fragmentation of peptide or protein ions with multiple positive charges by reactant anions using this method.
For the electron transfer dissociation method, knowledge of substances for the generation of suitable radical reactant ions is crucial. These substances must be capable of quickly and very efficiently binding electrons stably, but only weakly, in special electron attachment ion sources. Favorable ETD substances so far known have low but still positive electron affinities in the region of approximately 0.55±0.25 eV. The electrons are thus weakly bound to these substances and can easily be removed by positively charged ions and transferred to them.
The fundamental relationships between electron affinity and electron transfer are presented in the comprehensive review article by P. Kebarle and S. Chowdhury, Chem. Rev. 1987, 7, 513-534, “Electron Affinities and Electron-Transfer Reactions”.
The patent of D. F. Hunt et al. cited above explains that such substances are found in the group of polycyclic aromatic hydrocarbons. Specifically, the substances anthracene, naphthalene, fluorene, phenanthrene, pyrene, fluoranthene, chrysene, triphenylene, perylene, 2,2′-biquinoline, acridine and others are listed. As far as recorded in the NIST database (NIST chemistry webbook), these substances have electron affinities (EA) between 0.3 and 0.8 electronvolts. Fluoranthene (EA=6.7 eV) and 2,2′-biquinoline are emphasized as being particularly suitable for pure electron transfer dissociation with a high yield of fragment ions and without a significant proportion of proton transfer reactions. With the other substances (except for perylene), as can be seen in Table 1 of the cited patent, the electron attachment ion source also supplies non-radical anions of the form (M−1)−, which result in undesirable proton transfer reactions. Therefore, these polycyclic aromatics are not all equally useful for ETD.
A high effectiveness of the anions of a substance for ETD means that, on the one hand, a high yield of fragment ions of more than 50% of the ions to be dissociated is obtained and, on the other hand, the proportion of protein transfer reactions is less than 30%, preferably less than 10%. In this sense, fluoranthene and 2,2′-biquinoline are particularly effective for ETD.
However, these polycyclic aromatics, including fluoranthene, which has until now been known as very effective for ETD, have a very low vapor pressure in the order of 1 pascal at 20° Celsius, or even well below that. In electron attachment ion sources, the substances must be present with partial pressures of approximately 100 to 1000 pascal; the polycyclic aromatics therefore have to be heated in their container to between 50° and 250° C., and fed through heated lines to a heated electron attachment source. This makes the equipment difficult to design if the substance container is to be installed outside the vacuum system. The difficulties particularly concern feeding the heated gas line through the unheated wall of the vacuum system without generating a cool location where the substance vapor will condense.
In the prior art, therefore, a much simpler solution is usually applied. This involves mounting the heated substance container in the vacuum system close to the electron attachment source, or even heating the substance container by means of the electron attachment ion source, which is itself automatically heated sufficiently by the thermionic cathode required for electron emission. The disadvantage of this arrangement, however, is that in order to refill the container, not only must the container and the electron attachment source be cooled, but also the vacuum system must be vented and opened. Venting the vacuum system is, however, to be avoided whenever possible in mass spectrometry, as a considerable amount of time and effort are needed to restart the mass spectrometer, and recalibrating the mass scale and other settings are usually necessary. A further disadvantage is that it is difficult to design the equipment in such a way that the supply of substance to the electron attachment source can be interrupted during pauses in measurement or during measurements that do not use ETD. Consequently, in most cases, no method of interruption is provided; but this means continuous consumption of the ETD substances and therefore more frequent refilling.
In addition to electron transfer dissociation, other kinds of reaction between analyte ions with multiple positive charges and particular species of negatively charged ions can reduce the number of the charges on each of the positive analyte ions (“PTR”=proton transfer reactions, also known as “charge stripping”). This requires different kinds of anion, usually non-radical anions. In favorable cases these can be obtained from the same substances in special, switchable electron attachment ion sources (patent application DE 10 2006 049 241 A1;R. Hartmer). By reducing the number of charges, very heavy, highly-charged analyte ions can be converted into ions that are less highly charged, in order to reduce the complexity of the mass spectra from mixtures of large numbers of heavy analyte ions with high numbers of charges each. In the limiting case, the analyte ions or the fragment ions can be converted down to singly charged ions, which then yield mass spectra that are much easier to interpret. In some types of mass spectrometer it is only this charge reduction that makes it possible to resolve the isotope groups of all the ions signals into individual mass-to-charge ratios m/z, in order, as those skilled in the art know, to determine the number of charges z on the ions of this isotope group from the spacing between the ion signals, and so determine their physical mass m.
Under favorable conditions, the electron attachment ion source can be used to generate both types of anion—the radical anions for electron transfer dissociation and the non-radical anions for charge reduction. To a large extent this ion source is identical to conventional ion sources for negative chemical ionization (NCI), but is operated with a special gas with which the injected electrons are quickly thermalized. Methane is frequently used as the thermalization gas. Hydrogen radicals are also created by the electron bombardment. As disclosed in patent application DE 10 2006 049 241 A1, given a suitable substance, changing the voltage used to extract the anions is sufficient to deliver one type of anion or the other.
The reactions both for electron transfer dissociation and for charge reduction predominantly take place in reaction cells in which both positive and negative ions can be stored. Reactions in ion guide systems are also known. The reaction cells are often filled with a damping gas in which the ion movements are thermalized. The reaction cells may, for instance, consist of two-dimensional RF ion traps with special pseudopotential barriers at the ends, or three-dimensional RF ion traps. Devices with both kinds of reaction cell are available on the market, and are known to those skilled in the art. The positive analyte ions and the negative reactant ions are usually introduced one after the other into the ion trap where they are mixed together. The reactions then proceed without any further intervention.
In some cases, however, the fragmentation may be incomplete because the fragment ions formed by electron transfer dissociation remain associated. It is, however, also known that in such cases the associated fragment ions can be made to collide with the damping gas through gentle excitation of their secular oscillations, causing the associations to dissolve.
Quadrupole RF ion traps can be used as mass analyzers for the product ions created. It is then necessary to ensure that the form of the electrodes is very precisely hyperbolic in order to permit precisely resonant excitation, especially for the ions to be ejected with good mass resolution for their measurement. Measurement of the mass-sequentially ejected ions results in a mass spectrum. The accurate shape of the electrodes is necessary so that, by means of a harmonic pseudopotential field, the excitation frequencies of the oscillating ions are kept constant and independent of the oscillation amplitude during resonant excitation. The electrodes must therefore be shaped so that a well-formed quadrupole field is generated inside.
In some quadrupole mass spectrometers, a small proportion of higher-order multipole fields is deliberately superimposed onto the quadrupole field. Such deliberately generated deviations from a pure quadrupole field can, on the one hand, introduce non-linear, very strong and sharply defined resonance conditions and, on the other hand, hold the ions in resonance when a mass scan is in progress.
In three-dimensional ion traps, the ions mix of their own accord as they are introduced. In two-dimensional ion traps, a somewhat different procedure is sometimes used. If reactions between positive and negative ions are to be created in such linear ion traps, the clouds of positive analyte ions and the negative reactant ions are first collected in different sections, known as the prefilter and postfilter; then a special switching of the axis potentials sends them to be mixed in the central region of the linear ion trap. This method is disclosed in great detail in the patent application already cited above, US 2005/0199804 A1 (D. F. Hunt et al.).
The RF ion traps always have a low mass boundary for the storage of ions. Ions below a threshold mass m/z cannot be stored. The threshold mass is proportional to the amplitude of the RF field, and can be changed by altering the RF voltage. This phenomenon prevents light fragment ions from being stored after they have been created by the fragmentation reaction. On the other hand, the phenomenon can be exploited for ETD so that, after sufficient reaction time, excess reactant ions are very quickly ejected, in fractions of a millisecond, by briefly increasing the RF voltage, if the reactant ions are light enough. This method is more advantageous than ejection by means of resonant excitation, since the latter method takes longer because an entire isotope group always has to be ejected, and this makes a hole in the mass spectrum.
The multiply charged positive analyte ions are usually created in electrospray ion sources. This automatically generates ions that have, as a rule of thumb, approximately one charge for every 700 daltons of analyte molecule mass, although the number of charges shows a wide distribution. For analyte molecules with a physical mass of around 10,000 daltons, ions with a wide range of charge levels are created, extending from about seven to about 20 charges. For these ion mixtures, it is expedient to carry out PTR charge reduction—by means of reactions with suitable negative, non-radical reactant ions—before, during or after the electron transfer dissociation. It is therefore favorable if the non-radical reactant ions required for this can be created in the same electron attachment ion source, preferably from the same substance.
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