Radiant energy – Ionic separation or analysis – Ion beam pulsing means with detector synchronizing means
Reexamination Certificate
2002-02-28
2004-04-20
Nguyen, Kiet T. (Department: 2881)
Radiant energy
Ionic separation or analysis
Ion beam pulsing means with detector synchronizing means
C250S281000, C250S282000
Reexamination Certificate
active
06723983
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the operation and embodiment of a time-of-flight mass spectrometer for acquiring spectra of either primary or daughter ions with high mass precision.
BACKGROUND OF THE INVENTION
In biochemistry, it is not only the saving of time and money that makes it desirable to achieve a high analysis throughput: in many application fields, the instability of the samples makes it essential that analytic procedures are carried out rapidly. Whereas in combinatorial chemistry the saving in time when analyzing tens of thousands of samples may be the most significant factor, in proteomics it must be considered that the proteins of a proteome, following their (for example, gel-electrophoretic) separation, purification and other sample preparation processes, are susceptible to oxidative, thermal or other types of decomposition, since they are no longer protected by their former association with other proteins and by the environment of a biological solution. This means that the thousands of proteins constituting a proteome should be analyzed within 24 hours, if possible, and at most 48 hours following their separation.
It is thus not only desirable but essential to achieve a high sample throughput for biochemical analysis.
Nowadays mass spectrometers are used for many biochemical analyses, and in particular for protein analysis. Most of these are time-of-flight mass spectrometers, in which the samples are ionized by laser desorption. Although modem mass spectrometers of this type are fitted with sample inlet systems which permit a large number of samples (384, 764 or even 1536 samples, for instance) to be placed on the sample supports, diverse problems associated with the fast analysis of these samples still remain, and these problems hinder high analysis throughput. These problems include both technical difficulties associated with the mass spectrometers being used and with the procedures employed, as well as difficulties with the reproducible preparation of the samples for ionization.
In proteome research the highest priority is to identify the individual proteins as rapidly as possible, but then also to identify differences from proteins that are already known. The identification is usually achieved by measurement of the precise masses of the peptides generated by enzymatic (preferably tryptic) digestion. The mixture of digestion peptides is subjected to MALDI analysis and a so-called “fingerprint spectrum” is generated. A special search algorithm is then used to compare the list of precise masses measured with the contents of a protein sequence database, frequently already yielding definite identifications. If, however, uncertainties result from ambiguity, or from masses that do not precisely match, then the peptides in question are investigated using a daughter ion analysis, and as a rule this will provide unambiguous answers.
In the type of time-of-flight mass spectrometry most often used here, ions of an analyte substance are created in an ion source by means of a short laser pulse. The ions are accelerated to a high energy in a short acceleration path, sent through a field-free flight-section, and measured by a time-resolving ion detector. Since all the ions have the same energy, the flight time of the ions measured in this way permits the determination of the mass, m, of the ions, or, more precisely, their mass to charge ratio, m/z.
Note: for the sake of simplicity, in the following reference will only be made to the mass, m, even though in mass spectrometry always the mass to charge ratio, m/z, is measured, where z is the number of elementary charges carried by the ion. Since many methods of ionization, such as, for instance, the matrix assisted laser desorption and ionization (MALDI) that is preferably used here, predominantly produce ions with only a single charge (z=1), this distinction is of little practical relevance here.
Since a single MALDI process only generates relatively few ions, the mass spectrometric analysis of a sample based on MALDI requires the summation of between 50 and 200 individual spectra in order to obtain a useful sum spectrum. In other words, between 50 and 200 laser pulses must be separately applied, each generating ions which are independently measured as an individual spectrum for inclusion in the sum spectrum. The problems mentioned above now have three principal aspects:
Up to now, the complex sequence of voltage pulses described below, which must be triggered each by a laser pulse, is simply switched on for acquiring the spectra and switched off in order to prepare for the next sample analysis. The electrical and thermal equilibria will never really balance as a result. Only under painfully maintained equilibrium conditions, however, is it possible to accurately reproduce all the voltage pulses, and this in turn is critical for the quality of the spectra.
To acquire the spectra of daughter ions, it is at present necessary to readjust the ion source potentials between one sample and the next, and within one sample even for the several daughter ion spectra from different precursor ions, in order to achieve optimal mass resolution for the precursor ion selection. Each adjustment, however, again disturbs the equilibrium of the electronics.
The preparation of the samples on the sample supports must be so uniform, and so homogenous throughout the samples, that the process of the quasi-explosive evaporation and ionization of the samples by the laser pulse is entirely reproducible, so that the 50 to 200 individual spectra all have the same quality, and that there are no variations in the flight times. This is hard to achieve.
An almost obvious solution for the first problem would be to allow the sequence of voltage pulses to run periodically at some fundamental frequency. The sequence of pulses in the ion source (and in turn all the other sequences of pulses), however, is triggered by the laser pulse itself, in order to eliminate the relatively dramatic effects of the laser's slightly irregular ignition delay. For most lasers it is, on the other hand, inappropriate simply to allow the laser to operate continuously at a high pulse rate merely for the purpose of keeping the electronics in equilibrium. Not only might the samples on the sample support be damaged by the laser irradiation, but the laser itself only has a limited life time. The life time of the laser would be considerably reduced by such continuous operation. Thus the number of laser shots within the life time must be carefully budgeted, particularly when high sample throughput procedure is aimed for.
The ions generated by laser desorption frequently possess initial velocities that are not the same for all the ions. In order to achieve a high mass resolving power, velocity focusing by a Mamyrin ion reflector has become widely used, followed by a second field-free flight path. The ion reflector usually has two stages. In the first stage the ions are decelerated strongly, but in the second stage only gently. Faster ions penetrate further into the relatively weak linear deceleration field in the second stage of the reflector than do the slower ions, and therefore cover a greater distance. If the two deceleration fields have the correct relationship, this longer pathway compensates precisely for the higher flight speed, resulting in an increased mass resolution.
One of the most commonly used ion sources makes use of matrix assisted laser desorption and ionization (MALDI). The analyte molecules are embedded in a matrix substance, on a sample support plate. A pulse of laser light between 1 and 5 nanoseconds in length creates a cloud of molecules of both the matrix and analyte substance. The cloud expands adiabatically into the surrounding vacuum, giving the molecules in the cloud a greater spread of velocities. In this cloud, analyte molecules are continuously ionized by transfer of protons from the matrix ions, so that the analyte ions not only show a spread of velocities, their formation times are also spread.
A reflector is not able to focus this
Bruker Daltonik GmbH
Nguyen Kiet T.
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