Patent Application: US-3883405-A

Abstract:
a method and device for the gas - phase separation of ionic biomolecules including peptide , and protein or inorganic cluster ions or nanoparticles by ion mobility and for depositing them intact on a surface in a spatially addressable manner is described . the surface onto which the proteins are deposited can be modified for the purpose of constructing microarrays of biologically relevant materials or for promoting the growth of highly ordered protein crystals .

Description:
as used herein , “ a ” or “ an ” means one or more . the plural encompasses the singular and the singular encompasses the plural . the invention includes an instrument and method for detecting and selectively depositing gas - phase ions onto a surface . the instrument comprises an ion source for generating ions , means for separating the generated ions wherein the means for separating is fluidly coupled to said ion source ; means for selectively gating said ions based on their mobilities and means for directing said ions to a substrate , said means for selectively gating and said means for directing being fluidly coupled to said ion mobility drift cell ; and , an ion detector fluidly coupled to said ion mobility drift cell . fig1 a provides a schematic diagram of a device representing an exemplary embodiment of the present invention , instrument 1 , is comprised of five major components : an ion mobility chamber 2 , a source of ions 3 ( such as peptide and proteins ), a system of ion lenses for focusing and beam positioning 4 , a detector for determining ion mobility distributions 5 , and a surface for means of collecting peptide or protein ions 6 . briefly , a solid matrix / protein sample is deposited on a probe 7 and subsequently introduced into the ion mobility chamber via a vacuum interlock 8 . ultraviolet photons from a laser 9 directed at the probe tip 10 then preferentially generates intact gas - phase protonated molecular ions which are directed towards a differential aperture plate 11 by means of applying a nearly - linear electric field between 11 and the ion mobility backing plate 12 . a neural drift gas supplied to the ion mobility chamber via a metered port 13 impedes the progress of ions through the electric field . provided a suitable ratio of the electric field to the neutral gas number density is used , ions are nearly linearly separated based on their apparent charge - to - collision - cross section ratio . the separated peptide and protein ions then pass through the differential aperture plate 11 ( 200 to 1000 μm diameter ) and are collimated and focused by a system of ion lens elements 4 . in an exemplary embodiment of the present invention , the separated ions are then directed by electrostatic steering plates 14 to one of three final positions : an ion detector 15 ( see also fig2 a ), a contaminant collection probe 16 ( see also fig2 b ), or a peptide / protein collection surface 6 ( see also fig2 c ). when the separated ions are directed to the ion detector ( fig2 a ), the ion mobility distribution of all of the separated ions is recorded . neutral atoms or molecules that pass through the aperture plate are not electrostatically steered from their straight trajectory and are collected on the contaminant collection probe or are pumped from the detector / collector cell 17 . based on the arrival time distribution of the ions that is recorded at the detector , a timing sequence for the voltages applied to the electrostatic steering plates for the selection of a particular peptide or protein for deposition is generated . in the present invention , the ion source can be any ion source known in the art . preferably , the ion source comprises by any ionization instrumentation , including but not limited , to atmospheric pressure matrix - assisted laser desorption ionization ( maldi ), infrared maldi , laser desorption ionization ( ldi ), electrospray ionization , nanospray ionization , photoionization , multiphoton ionization , resonance ionization , thermal ionization , surface ionization , electric field ionization , chemical ionization , atmospheric pressure chemical ionization , radioactive ionization , discharge ionization , and combinations thereof . employing the ionization instrumentation on a sample generates ions . the sample can be any sample , but is preferably a chemical or biochemical sample . means for separating ions can be accomplished by any means known in the art , but is preferably performed on the basis of the ions &# 39 ; gas - phase mobility ( ion mobility ). preferably , when using ion mobility , the separating means comprises applying electric fields to the ions , and preferably , the electric fields are is selected from the group consisting of uniform electrostatic fields , periodic - focusing electrostatic fields , and combinations thereof . other fields , known to those of skill in the art , are also useful in the present invention . separation of ions on the basis of their gas - phase mobility is also accomplished by reactive or unreactive collisions with species selected from the group consisting of helium , neon , argon , krypton , xenon , nitrogen , oxygen , methane , carbon dioxide , water , methanol , methyl fluoride , deuterated analogs thereof , tritiated analogs thereof , and any combinations thereof . means for selectively gating ions of particular mobility and means for directing ions to a solid substrate may be of any type know in the art , including those selected from the group consisting of direction by an electrostatic - field , direction by a magnetic - field , and combinations thereof . means for gating and deflecting are preferably performed using electrostatic or magnetic fields , however , mechanical means may be used . means for gating and directing are preferably performed by using electrostatic steering plates , however , other techniques well known in the art may be used , and these include direction by the application of magnetic fields also . mechanical means for gating through the use of shutters is also possible . the instrument and method of the present invention , in addition to its ability to selectively gate and direct analyte ions onto a substrate , can also incorporate a number of recent advances in ion mobility / mass spectrometry . for example , in u . s . pat . no . 6 , 6 , 639 , 213 to gillig et al , an improved ion mobility instrument using periodic focusing electric fields that minimize the spatial spread of the migrating ions by keeping them in a tight radius about the axis of travel is described . u . s . pat . no . 6 , 6 , 639 , 213 is incorporated by reference as though fully described herein . in u . s . application ser . no . 09 / 798 , 030 ( published as u . s . patent application publication 2001 / 0032929 a1 on oct . 25 , 2001 ), fuhrer et al , disclosed an improved ion mobility instrument using combinations of periodic and hyperbolic focusing electric fields . u . s . patent application publication 2001 / 0032929 a1 is incorporated by reference as though fully described herein . in u . s . pat . no . 6 , 683 , 299 to fuhrer et al , time - of - flight mass spectrometer instruments for monitoring fast processes using an interleaved timing scheme and a position sensitive detector are described . u . s . pat . no . 6 , 683 , 299 is incorporated by reference as though fully described herein . in u . s . application ser . no . 10 / 689 , 173 ( published as u . s . patent application publication 2004 / 0113064 a1 on jun . 17 , 2004 ), of fuhrer et al , the time - of - flight mass spectrometer instruments for monitoring fast processes using an interleaved timing scheme and a position sensitive detector was supplemented with an additional fragmentation step for additional analytical information . u . s . patent application publication 2004 / 0113064 a1 is incorporated by reference as though fully described herein . in pending u . s . application ser . no . 10 / 967 , 715 , fuhrer et al described improvements in the fast time - of - flight instrument , including photo - fragmentation of ions , the use of multiple pixel ion detectors positioned within the mass spectrometer , and the generation and analysis of one or more spatially distinct ion beamlets . it is understood that a time of flight mass spectrometer can be used as a detector stage in place of detector 5 . in pending u . s . application ser . no . 10 / 969 , 643 , schultz et al describe improved ion mobility focusing through the use of alternating high and low electric field regions . u . s . application ser . no . 10 / 969 , 643 is incorporated by reference as though fully described herein . a representative timing diagram for the voltage applied to the electrostatic steering plates is illustrated in fig3 for two cycles of ion mobility detection and peptide / protein deposition . in this example , all of the ions from the initial maldi event are directed to the detector to determine the arrival time distribution of the ion mobility separated ions ( fig3 a ( ii )). based on the elution time of the peptide or protein to be deposited which was determined in ( ii ), ions eluting at that time are then directed to the collection surface 6 for the remaining ionization events in that cycle ( fig3 a ( iii )). for elution times not corresponding to either detection or collection , no net steering is used so that undesired ions ( e . g ., from contaminants or matrix related ions ), and neutrals are collected on a contaminant collection probe . owing to the plane of symmetry between the collection probe and detector , the voltage applied to the electrostatic steering plates is the same in magnitude , but opposite in polarity , depending on the desired ultimate trajectory of the ions ( i . e ., detector or collector , fig3 b ). by changing the magnitude of the voltage applied , the spatial position of the ion deposition can be tuned . it should be understood that the dimensions of the deflections and the distances between the detector 5 , collector 6 , and contaminant collection probe 16 can be very small ( miniaturized ). in the present exemplary embodiment , steering plate voltage polarity is determined by the electronic state of fast - switching circuitry ( fig4 ). the state of the switch is changed by the application of a tunable waveform (± 5 v ) constructed on the basis of the timing diagram as illustrated in fig3 . when the inverted (−) or non - inverted (+) output state is “ high ” the switch delivers a voltage equal to that supplied by an external power supply (± 0 to 35 v ) to the inverted or non - inverted electrostatic steering plate , respectively . concurrently , the inverted or non - inverted output in the “ low ” state is connected to ground . the current circuitry has a rise / fall time of ca . 1 ns and can operate up to a switching frequency of approximately 100 khz . arrival time distribution selectivity ( i . e ., mobility separated ion selectivity ) by using the switching circuitry described is illustrated in fig5 . the top panel illustrates the arrival time distribution observed by gating all mobility - separated ions ( atomic ions ablated from the steel probe tip , matrix related ions ( α - cyano hydroxycinnamic acid ), and the peptide gramicidin s ) to the detector . in subsequent panels , the steering plates are gated to transmit ions from the peak eluting at 220 μs , but in successively more selective timing windows ranging from 150 to 20 μs . note that owing to the symmetry of the instrument , the selected ions in fig5 are directed to the surface by simply switching the electronic state of the switching circuitry . in terms of selectivity , there is little to no evidence for ions reaching the detector from rejected portions of the arrival time distribution . an instrument was built based upon the exemplary embodiment of the present invention for proof - of - concept experiments . the peptide gramicidin s ([ pvolf ] 2 ) was soft - landed ( 7 . 5 ev kinetic energy ) onto a hydrocarbon coated - stainless steel collection surface for 15 hours at a maldi repetition rate of 30 hz . the deposited peptide was then washed into 10 μl of deionized water , spotted onto a maldi sample plate with a co - matrix of 2 , 5 - dihydroxybenzoic acid , and then analyzed by maldi - time - of - flight - ms resulting in the spectrum illustrated in fig6 . this spectrum clearly indicates that gramicidin s is deposited on the collection surface intact . in another embodiment , the utilization of high repetition rate maldi ( i . e ., 0 . 5 to 10 khz ) will reduce these deposition times to several minutes . in the present exemplary embodiment , the collection surface is positioned at a point equidistant from the steering region as that from the detector plane to said region . the collection surface may consist of a static probe , plate , or microwell plate and may be surface modified . the spatial addressability of ion deposition can be accomplished by one or more pairs of electrostatic steering plates ( or other ion optical geometries ). manipulation of the direction of ion deposition can be used to pattern the deposition . in an alternate embodiment , the ion beam position remains static and the collection surface is translated relative to the ion beam via x - y micropositioners for spatial addressability . the ultimate spatial resolution of deposited peptides or proteins on the collection surface , in the present exemplary embodiment , is limited by the diameter of the differential aperture plate 12 ( ca . 200 μm ), but could be conservatively improved to 10 to 100 μm 2 spot sizes by utilizing ion optical methods well known in the art . in alternate embodiments of the present invention ( fig1 b ), on - line analysis of the amount of analyte deposited will be accomplished by in situ analysis of the native - state fluorescence of the gas - phase ions by means of using a pulsed - laser source of uv photons for excitation 18 ( e . g ., 266 nm by using a frequency - quadrupled nd : yag laser ) and measuring emission by means of an avalanche photodiode or photomultiplier tube detector 19 situated 90 ° with respect to the fluorescence laser propagation . in the presently - described exemplary embodiment , the focused beam of ions passes through a fluorescence interaction region 20 for non - destructive detection prior to soft - landing at the collection surface 21 . excess excitation photons and undesired ions deflected by means of the steering plates are collected at a photon / ion beam dump 22 positioned in - line with the excitation laser . in either embodiment , the collection surface may consist of a variety of materials , for example : steel , gold , glass , h - sam , f - sam , glycerol , or condensed - phase materials . the collection surface can further be functionalized using alkanethiol - gold or silanization chemistries ( e . g ., reaction with primary amines for peptides and proteins ) to immobilize and the deposited analyte . by immobilizing the analyte with covalent cross - linking agents , the deposited material can be patterned on the collection surface to generate microarrays of desired material . the present invention may alternatively be used for promoting the growth of highly ordered protein crystals from said gas - phase purified analytes . the ordering of such protein crystals may be enhanced by carefully cooling the mobility separated ions by application of rf gas phase cooling procedures and further controlling the beam energy of the thus cooled mobility separated proteins by use of carefully designed deceleration optics located in front ( upstream ) of the collector surface . instrumentation and method for rf cooling are well known to those of skill in the art ; see for example , u . s . pat . no . 6 , 6 , 639 , 213 , u . s . patent application publication 2001 / 0032929 a1 , u . s . pat . no . 6 , 683 , 299 , u . s . patent application publication 2004 / 0113064 , and u . s . application ser . no . 10 / 969 , 643 ; these patents and published patent applications are incorporated by reference as though fully described herein . this crystallization may also be desirably influenced by the choice of collector surface morphology which might include , for example , an ordered single crystalline substrate . the use of a two dimensional mobility spectrometer could also be used ( with or without gating techniques ) for simultaneously spatially separating and depositing the entire output of the maldi ( or laser ablation ) ionization process . the generation of the analyte ions is not restricted to the maldi process . the practice of this invention would easily include the use of a pulsed or trapped output from a continuous soft ionization source . alternatively the use of a continuous generation of the ions by , for example , electrospray ionization followed by a differential ion mobility spectrometer for selecting the desired analyte ions for deposition . one application of any of these embodiments will be to crystallize proteins or epitopes of drug binding sites of proteins so that their atomic structure can be determined by synchrotron generated x - ray diffraction techniques so that the atomic composition and geometric orientation of the drug binding site can be determined . one additional application is in drug screening which is made possible by the combination of soft ionization , mobility separation , gating and soft landing for the growth of bio - crystals which contain a small molecule drug candidate already interacting with the protein which contains the targeted binding site . the growth of these single crystal proteins or peptide which containing the small molecule can thus enable the structure determination by x - ray diffraction . this allows the direct determination of whether or not the potential drug candidate reaches the binding site and if so how it interacts with the binding site . a further application is the soft landing of semiconductor or small metal particulates so that combinatorial analysis of physical properties ( e . g . chemical reactivity , electron emission , catalytic activity , photoemission of electrons ) can be rapidly determined . all patents and publications referenced herein are hereby incorporated by reference . it will be understood that certain of the above - described structures , functions , and operations of the above - described embodiments are not necessary to practice the present invention and are included in the description simply for completeness of an exemplary embodiment or embodiments . in addition , it will be understood that specific structures , functions , and operations set forth in the above - described referenced patents and publications can be practiced in conjunction with the present invention , but they are not essential to its practice . it is therefore to be understood that the invention may be practiced otherwise than as specifically described without actually departing from the spirit and scope of the present invention as defined by the appended claims . u . s . pat . no . 4 , 822 , 466 j . w . rabalais and s . r . kasi , chemically bonded diamond films and method for producing same ( apr . 18 , 1989 ). u . s . pat . no . 5 , 374 , 318 j . w . rabalais and s . r . kasi , process for the deposition of diamond films using low energy , mass - 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