Radiant energy – Ionic separation or analysis – With sample supply means
Reexamination Certificate
2002-02-28
2003-09-09
Lee, John R. (Department: 2881)
Radiant energy
Ionic separation or analysis
With sample supply means
C250S281000, C250S300000, C250S443100, C250S442110, C250S441110, C250S440110, C250S42300F
Reexamination Certificate
active
06617575
ABSTRACT:
TECHNICAL FIELD
This invention pertains generally to the field of matrix-assisted laser desorption/ionisation (MALDI) and MALDI mass spectrometry (MS). More specifically, the present invention pertains to modified targets suitable for use with liquid matrices (e.g., glycerol and lactic acid) in liquid MALDI methods, as used, for example, in infrared (IR) liquid MALDI MS, preferably using time of flight (TOF) instruments. The present invention also pertains to ion sources, mass spectrometers, methods of MALDI and methods of mass spectrometry using such modified ion source targets.
BACKGROUND
Traditional mass spectrometric methods are extremely useful for the analysis of low molecular weight compounds. However, for high molecular weight compounds, for example, biopolymers such as proteins and carbohydrates, the problem to be solved was to convert relatively non-volatile macromolecules into intact, isolated, and ionised molecules in the gas phase. A number of so-called desorption/ionisation techniques have been developed to solve this problem. In field desorption methods, a strong electric field is applied to the sample. In fast atom bombardment and
252
Cf plasma desorption, the sample is bombarded by highly energetic ions or atoms. In thermospray ionisation and electrospray ionisation methods, ions are generated directly from small, charged liquid droplets. Laser desorption/ionisation (LDI) and the newly developed variant of this method, “matrix assisted laser desorption/ionisation” (MALDI), make use of short, intense pulses of laser light to induce the formation of intact gaseous ions.
Two factors dominate in the choice of laser for desorption methods. First, efficient and controllable energy transfer to the sample requires resonant absorption of the molecule at the laser wavelength. Consequently, lasers emitting in the ultraviolet (UV), which can couple to electronic states, or in the mid-infrared (mid-IR), which can excite rovibrational states, have so far shown the best results. Second, to avoid thermal decomposition, the energy must be transferred within a very short time. Typically, laser pulses or “shots” with durations on the order of 1 to 200 ns are employed. Given the short pulse durations, and the fact that laser beams can easily be focussed to spot sizes that are small compared with the other dimensions of the ion source, the ions are generated essentially at a point source in space and time, as a “packet” of ions. This pulsed desorption of ions favours the use of a time-of-flight (TOF) mass analyser, which makes it possible to record a complete mass spectrum for each laser shot. However, LDI methods may also be adapted for other mass spectrometers, including magnetic sector, quadrupole, Fourier transform ion cyclotron resonance (FT-ICR), and ion trap instruments.
In a time-of-flight (TOF) mass analyser, the velocity of an ion is used to determine its mass-to-charge ratio (m/z). A packet of ions is accelerated to a fixed kinetic energy by an electric potential, typically 1-30 kV. The velocity of a particular ion within the packet will then be proportional to (m
i
/z
i
)
−½
, where m
i
/z
i
is the ion's mass-to-charge ratio. The ions are then allowed to pass through a field-free region, typically 0.1 to 3 m in length, where they are separated into a series of spatially discrete individual ion packets, each travelling with a velocity characteristic of its mass and charge. A detector at the end of the field-free region produces a signal as each ion packet strikes it. A recording of the detector signal as a function of time is a TOF mass spectrum. The difference between the start time, common to all ions, and the arrival time of an individual ion at the detector is proportional to (m
i
,z
i
)
+½
and therefore can be used to calculate the ion's mass. Such a calculation can then be used to convert the axis of the spectrum from time into a mass-to-charge ratio axis, yielding a conventional mass spectrum.
The performance of mass spectrometers is typically described in terms of mass accuracy and mass resolving power. Mass accuracy is a measure of the error involved in assigning a mass to a given ion signal. It is typically expressed as the ratio of the mass assignment error divided by the mass o Of the ion and is frequently quoted as a percentage. Mass resolving power (also known as “mass resolution”), m/&dgr;m, is a measure of an instrument's capability to produce separate signals from ions of similar mass. For TOF instruments, it is typically expressed as the mass, m, of a given ion signal divided by the full width of the signal, &dgr;m, which is measured between the points of half-maximum intensity (FWHM). Factors which determine mass resolving power for a TOF instrument include the ion production time, initial velocity distribution, and extraction time. For example, conventional or “linear” TOF mass spectrometers may be adapted to include an “ion mirror,” to yield a “reflectron” TOF mass spectrometer (reTOF), which permits correction for the peak width contribution arising from the initial energy distribution. Reflectron configurations effectively increase the ion's path length during separation, and therefore analysis time, and so increase susceptability to metastable effects.
The term “metastable” is used herein in the conventional sense to describe ions which fragment at some time after formation and before detection, typically during mass analysis. Since most mass spectrometers rely on the separation of species according to mass and charge, the fragmentation of a large ion into two or more smaller species will change the separation parameters mid-flight. Consider, for example, a packet of ions, M
1
+
, some of which decay in flight (in the field free region of a TOF instrument) to form (lighter) daughter ions, M
2
+
, and neutral daughter species, M
3
0
. If, after fragmentation, the ions are subjected to an accelerating potential (e.g., an ion mirror in a reflectron instrument), then the parent and daughter ions will have different velocities. Both the parent ions, M
1
+
, and the daughter ions, M
2
+
, will be detected, but the latter at a mass intermediate between that of M
1
+
and M
2
+
, according to the precise time of fragmentation and the accelerating potential. This can result in a smear or tail of intensity to lower mass (from M
1
+
), and a consequent loss of resolving power. Metastable effects are largely dependent on the particular parent ion (e.g., greater metastable effects for labile and high mass ions), and the quantity and distribution of internal energy. Thus, ionization methods which deposit a large proportion of internal energy in levels which lead to bond-breaking and fragmentation often suffer from substantial metastable effects. For TOF instruments, metastable effects increase with increasing mass, since heavier ions have longer analysis times, and thus more opportunity to fragment before detection. For reflectron TOF (reTOF) instruments, the path length (and flight time) is also increased, again providing more opportunity for fragmentation prior to detection. Increasing background pressure, for example, in the field free region of a TOF instrument, have also been shown to increase metastable effects. See, for example, Berkenkamp, 1997.
Efforts to improve mass resolving power in TOF instruments have typically relied on focussing methods, to minimize the dependence of flight time on initial conditions. Examples of such methods include “velocity focussing” (typically used when the spatial distribution is narrow) and “space focussing” (typically used when the velocity distribution is narrow). See, for example, Vestal, 1998.
One method of “velocity focussing” employs a delayed extraction of ions, as compared to a immediate and constant (static) extraction of ions. In “static extraction” methods, ions are subject to a large constant accelerating potential (e.g., 10-20 kV) from the instant they are formed. In “delayed extraction” (DE) methods, also known as “time lag
Hashmi Zia R.
Lee John R.
Ludwig Institute for Cancer Research
Nixon & Vanderhye P.C.
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