Time-of-flight mass spectrometer with multiplex operation

Details Time-of-flight mass spectrometer with multiplex operation Time-of-flight mass spectrometer with multiplex operation

2002-03-06

2004-05-11

Nguyen, Kiet T. (Department: 2881)

Radiant energy

Ionic separation or analysis

Ion beam pulsing means with detector synchronizing means

C250S288000

Reexamination Certificate

active

06734421

ABSTRACT:

FIELD OF THE INVENTION
The invention concerns a time-of-flight mass spectrometer for the analysis of a large number of samples on a sample carrier using laser desorption and associated analytical procedures.
BACKGROUND OF THE INVENTION
Time-of-flight mass spectrometers in which the samples are ionized by pulsed laser desorption operate as follows: the laser beam is focused onto a sample that is held on a sample carrier. A laser desorption pulse generates ions from the analyte molecules. An applied voltage accelerates the ions into a field-free flight tube. Because of their different masses, the ions in the ion source are accelerated to different velocities. The smaller ions reach the time-resolving ion detector earlier than the larger ions. From the measured time-resolved ion currents the flight times of the ions can be determined; from their flight times their masses are calculated. An energy-focusing reflector and a delay in acceleration (often called delayed extraction) are used to increase resolution.
There is a strong trend in biochemistry towards processing and analyzing very large numbers of samples in parallel. The chip technique has become particularly well known. This technique works with large arrays of samples in a very small space. For certain analytical procedures using such sample arrays, e.g. those using fluorescence spectrometry, devices (“chip readers”) are already available which permit parallel analyses of all the samples on the chip at the same time; if not strictly simultaneously, they use a laser scanning procedure to read sequentially in only a few milliseconds per sample. In contrast to this, mass spectrometric analysis at present requires a few seconds for each sample, so that it is currently slower than the chip reader by a factor of about 1,000.
The chip technique is primarily used in genetics, but also starts in protein analysis. In genetics, various types of oligonucleotide are usually bonded to the surfaces in the individual compartments of the array. These oligonucleotides serve as detection sequences for amplified DNA samples; they are often referred to as probes. If the DNA in the sample contains a strand that is complementary to the oligonucleotide, then the DNA will attach itself to the oligonucleotide. This attachment is referred to as “hybridization”. The attached strands of DNA can, in this process, bring fluorescent dyes with them, or can alter the fluorescence wavelength of fluorescent dyes that are already located at the bonded oligonucleotides. Chips charged in this way can then be measured in the chip readers mentioned above, and the fluorescence of a compartment indicates the presence of the DNA sequence in question.
Mutations such as, for instance, point mutations can also be examined in this way. Point mutations consist of the modification of a single nucleotide. They are nowadays referred to as SNPs (single nucleotide polymorphisms). Because, however, the hybridization temperature (also known as the melting temperature) for mutation sequences and wild type sequences are only minimally different, detection through simple hybridization is always liable to error, because mutation DNA and wild type DNA undergo mixed hybridization in specific ratios, where the ratio depends very strongly on the temperature. In contrast to this kind of mutation detection through simple hybridization, mass spectrometric mutation detection is very reliable and unambiguous.
Mass spectrometric mutation detection is usually based on enzymatic modification of the probes that have been attached to the chips. The modification is controlled by the hybridized template, so that information about the mutations is transferred from the templates to the probes. One of these enzymatic modifications is known as limited primer extension. Preparation of the samples requires more time than simple hybridization, and the measurement also takes longer, because there is as yet no known method of analyzing a large number of samples simultaneously in the way that can be done in those chip readers. Mass spectrometric measurement of mutations has, however, the advantage of high analytic reliability, something which plays a significant role in diagnostic procedures as well as, for instance, in association with police records.
In protein analysis, antibody proteins can be bonded to the array compartments, and these can capture specific proteins from a sample. Other types of protein-specific affinity bonding can also be used for the protein chip technique. Further detail will not be given here of protein analysis, of which an extraordinarily wide variety exists.
As has already been stated, mass spectrometry offers the advantage of high analytic reliability, but this is countered by the disadvantage of the long time needed for the measurement, being some 1,000 times slower than chip readers. This raises the questions of whether and how mass spectrometry can be accelerated, and in particular of whether spectrometry can be carried out on entire arrays of samples.
There is an odd discrepancy in the mass spectrometric analysis of samples with ionization by laser desorption:
It is, on the one hand, possible to manufacture pulsed lasers with high repetition rates in the region of 10,000 pulses per second; this would permit a mass spectrum to be recorded in only about 100 microseconds, which in turn would mean that 10,000 individual spectra could be measured each second. If 10 individual mass spectra (from 10 laser pulses) are required for a sum spectrum that can be effectively analyzed, the analysis could be complete within one millisecond, which compares favorably with fluorescence chip readers.
On the other hand, however, there is a threshold energy for the generation of ions by laser desorption, below which the laser desorption process does not generate any ions at all. This threshold energy significantly heats the sample. For this reason, a laser frequency greater than about 20 Hertz results in excessive heating and degradation of many samples. Each laser desorption process, moreover, causes the samples to become electrically charged, since only particles of one polarity are withdrawn, while those with the opposite polarity are accelerated back onto the sample. Time is therefore necessary, particularly for MALDI samples with their insulating matrix crystals, so that the charges can disperse. Dissipation of the charges is also in many cases made slower because the chips that are used as a substrate for the samples are made of poorly conducting semiconductor material. Electrical charge causes the quality of the spectra to deteriorate, because the ions generated by the sequence of laser desorptions are given different energies. So if the 10 individual spectra required are to have good quality, at least about a half a second has until now been required. In addition to this, further time is needed to move the new sample into the laser focus, and for the data to be read from the transient recorders. These times are considerably longer than a millisecond; they limit the frequency with which individual samples can be measured, even if the laser pulse frequency could be significantly raised.
In theory, therefore, a minimum period of about one second is required for the analysis of a sample, although in practice the analysis times for a sample are longer still, and still require two to three seconds even in laboratories used to the routine.
Forty years ago, however, there were already developments in mass spectrometry in which a micrometric/analytic representation of a small area was created through ionization by secondary ions (SIMS=secondary ion mass spectrometry). The leader in the field was the French firm Cameca, whose devices were ion-optical imaging magnetic sector-field mass spectrometers. They were used for surface analysis, usually of metal surfaces. Light/dark images were used to determine the presence of specific elements or compounds, and their distribution over the surface.
Time-of-flight mass spectrometers have also already been used for the “chemical” representation of su

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