Time of flight mass spectrometry apparatus

Details Time of flight mass spectrometry apparatus Time of flight mass spectrometry apparatus

2002-11-25

2004-08-24

Lee, John R. (Department: 2881)

Radiant energy

Ionic separation or analysis

Ion beam pulsing means with detector synchronizing means

C250S282000

Reexamination Certificate

active

06781122

ABSTRACT:

The invention relates to time of flight mass spectrometry apparatus.
There are growing needs in biological fields for the characterisation of biomolecules available in minute quantities (picomolar, femtomolar, attomolar levels), and in general for structural characterisation of macromolecules including polymers.
Peptides formed by enzymatic digestion of proteins constitute one example in the field of proteomics. Analyses of synthetic polymers constitute a related challenge, although here the constraint of limited availability of sample is normally less relevant. The ionisation method of matrix-assisted laser desorption/ionisation (MALDI) in combination with time-of-flight (TOF) analysers has become one of the standard approaches to characterisation by mass spectrometry of non-volatile, thermally labile substances such as peptides, proteins and polymers. Electrospray ionisation (ESI) is the other method of ionisation of importance for TOF mass spectrometry of peptides, proteins, polymers and other non-volatile, thermally labile substances.
Established methods combine MALDI with electrostatic ion mirrors, incorporating either one homogeneous reflecting field (one-stage) or two homogeneous reflecting fields (two-stage). The optical axis of an ion mirror may be either approximately in line with or approximately normal (orthogonal) to that of the ion source. These mirrors afford limited capability for compensation of differing ion energies. An intermediate time-focus has been proposed as being advantageous creating effectively a tandom mass spectrometer, and the time-focus being a position for the location of an ion-dissociation cell. MALDI has been combined with so called curved-field ion mirrors and gradient field ion mirrors in which the potential gradient changes steadily with distance. These mirrors have been found to give modest resolution. High resolution could in principle be achieved in a combination of a quadratic field and laser desorption source through restricting ions entering the field to near-zero angles in order to conform to the one-dimensional approximation. The problem, which has been long recognised, is that there are transverse forces because the potential distribution is a saddleback. The sensitivity of such a device is too low to be practical for biological and other application, although adequate for measurement of atoms and small molecules in space research where sample consumption is not an issue.
According to the invention there is provided apparatus for the structural characterisation of large molecules by time-of-flight mass spectrometry, the apparatus comprising an ion source, bunching means, an ion mirror and a large area detector, wherein the ion mirror is arranged to produce a reflecting field, the bunching means is arranged to compress or bunch ions to provide spatial focusing concomitant with time focusing of ions at or near the entrance to the ion mirror, and the detecting surface of the detector is mounted in the focal surface of the ion mirror.
The ion mirror may be arranged to produce a substantially quadratic field. The quadratic nature of the field is required if entering ions possess a range of translational energies, but other suitable fields could be employed in the case of ions formed in the ion source prior to significant acceleration and with near-common translational energies.
There is also provided a method for the structural characterisation of large molecules (masses typically in the range 10 to 10
4
Da) by time-of-flight mass spectrometry at high-sensitivity and high-resolution by means of the combination of an ion mirror characterised by a quadratic field (including the parabolic case), or substantially quadratic field, ionisation of sample by laser desorption or electrospray ionisation and ion bunching (including time-lag focusing which is also known as “delayed extraction” or “delayed acceleration”) to provide concomitant time-focussing and space-focussing at or near to the entrance of the ion mirror either of a single parent mass (specifically m/z) or of a significant number of masses composing the parent and fragment ions formed therefrom by dissociation in a field free region. These fragment ions share a common time-focus with their parent ion. The time and space focus acts as the object of the ion optical system that is the ion mirror leading to a time-focus on an image focal surface characteristic of the mirror and independent of the ion energy. The aforementioned-fragment ions and their parent ions are time-resolved and their time-foci are characteristic of their masses (specifically m/z). A large area detector is placed in this focal surface.
Thus the invention includes the combination of ion bunching, for example, time-lag focusing in a MALDI source. biomolecules and other molecules with masses often of the order of 10
3
Da and almost always in the range 10
2
Da to 10
4
Da and ion mirrors with substantially quadratic fields. Such heavy ions are formed by MALDI with initial velocities close to 1000 ms
−1
, and his initial velocity is not critically dependent upon mass over the mass range in question (few hundred Da to a few tens of thousands of Da). MALDI contrasts with other methods of laser desorption and other methods of ionisation where ions possess common initial kinetic energies. Thus the ions formed by MALDI constitute beams comparable in regard to common velocity to supersonic molecular beams, and in this regard MALDI is distinct from some other methods of ionisation. The mass-independent velocity (and hence mass dependent initial kinetic energy) allows ion bunching techniques including time lag focusing (also known as pulsed extraction) to be used to give advantageously sharp common time-foci and spatial focus for a range of ion masses (specifically m/z's).
The invention includes spatial focusing of the ion beam at a focal point at the entrance of an ion mirror, concomitant with time focusing. The time-focus may be in a fringing field of the mirror or even inside the mirror, depending upon the nature of a fringing field. A detector placed at this common position of time and space focusing records a time-of-flight spectrum with high resolution.
A distinguishing feature of the invention is that the time and space focus is not destroyed by fragmentation in field-free regions between source and ion mirror. The fragmentation is the result of energisation of the parent ions by collisions in the source, in the dense plume at and above the sample surface in the case of MALDI, and/or along the flight path from source to ion mirror. Preferably fragment ions are mass analysed by the ion mirror. Suitably, fragment ions are formed by decomposition of parent ions in a free-field region between the ion source and the ion mirror and are mass analysed by the ion mirror. Because of the masses of the parent ions (typically in the range 10
2
-10
4
Da) and because of the conditions (dissociation in field-free regions), translational energy release in fragmentations of the ions are not large (≦100 meV), and the perturbation of the velocity of the dissociating system is not significant. The mean velocity of a given fragment will be identical or very close to that of its parent ion. The directional characteristics of a parent ion will be retained by its fragments because of the small energy releases and large fragment masses. Thus the space-focussing and time-focussing characteristics of a parent ion will be retained by its fragments, when fragmentation takes place in the field-free region between source and ion mirror. Sharp space-focus and time-foci are achieved for all parent ions and for all fragment ions with all of the envisaged ion optical arrangements. Fragment ions from a given parent ion share the same space-focus and time-focus as their parent ion. Different parent ions share the same space-focus but possess different time-foci depending upon their mass-to-charge ratios.
The concomitant space-focussing and time-focussing may be achieved through pulsed electric potentials applied in the ion source either

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