EP1747456A1 - Chromatographie d'affinite frontale/spectrometrie de masse en tandem maldi - Google Patents

Chromatographie d'affinite frontale/spectrometrie de masse en tandem maldi

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Publication number
EP1747456A1
EP1747456A1 EP05748644A EP05748644A EP1747456A1 EP 1747456 A1 EP1747456 A1 EP 1747456A1 EP 05748644 A EP05748644 A EP 05748644A EP 05748644 A EP05748644 A EP 05748644A EP 1747456 A1 EP1747456 A1 EP 1747456A1
Authority
EP
European Patent Office
Prior art keywords
maldi
fac
column
deposition
deposited
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05748644A
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German (de)
English (en)
Other versions
EP1747456A4 (fr
Inventor
William R. Davidson
Bori Shushan
Peter Kovarik
Tom R. Covey
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
DH Technologies Development Pte Ltd
Original Assignee
MDS Inc
Applera Corp
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Application filed by MDS Inc, Applera Corp filed Critical MDS Inc
Publication of EP1747456A1 publication Critical patent/EP1747456A1/fr
Publication of EP1747456A4 publication Critical patent/EP1747456A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/10—Ion sources; Ion guns
    • H01J49/16—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission
    • H01J49/161—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission using photoionisation, e.g. by laser
    • H01J49/164—Laser desorption/ionisation, e.g. matrix-assisted laser desorption/ionisation [MALDI]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/728—Intermediate storage of effluent, including condensation on surface
    • G01N30/7286—Intermediate storage of effluent, including condensation on surface the store moving as a whole, e.g. moving wire
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography

Definitions

  • the present invention relates to methods of analyzing compounds from chromatographic analyses, in particular using mass spectrometry.
  • Bioaffinity chromatography has been widely used for sample purification and cleanup, 1 chiral separations, 2 on-line proteolytic digestion of proteins, 3 development of supported biocatalysts, 4 and more recently for screening of compound libraries via the frontal affinity chromatography (FAC) method.
  • FAC frontal affinity chromatography
  • the general approach is to deposit the FAC effluent onto a MALDI target, followed by MALDI/MS analysis.
  • MALDI analysis has the advantages of higher tolerance to buffers, lower sample consumption per analysis and reduced analysis time. Separation of the LC and MS steps also allows independent optimization of the MS detection parameters for each analyte.
  • the present inventors have integrated FAC, using newly developed sol-gel derived monolithic bioaffinity columns, 9 with MALDI-MS/MS detection, and compared the operation to FAC-ESI/MS/MS by examining the ability of small enzyme inhibitors to interact with entrapped dihydrofolate reductase (DHFR) using elution at different ionic strengths.
  • DHFR dihydrofolate reductase
  • the interfacing involves mixing the column effluent with a suitable matrix followed by continuous nebulizer-assisted electrospray deposition of the mixture onto a MALDI plate that is present on a computer controlled x-y translation stage.
  • the chromatographic trace is deposited semi- permanently onto the MALDI plate, allowing for subsequent analysis offline by MALDI/MS/MS.
  • the frontal chromatogram can be reconstructed directly to obtain breakthrough curves for each analyte. It is shown that MALDI/MS/MS has a number of benefits relative to ESI/MS/MS as a detection method for FAC, including: better tolerance to high ionic strength elution buffers,
  • the present invention includes a system for analyzing chemical samples comprising a frontal affinity chromatographic column interfaced to a MALDI mass spectrometer.
  • the present invention also includes a method of analyzing samples from frontal affinity chromatography (FAC) comprising: (a) combining effluent from a FAC column with a matrix; (b) depositing the combination in (a) on to a surface; and (c) analyzing the deposited combination using MALDI mass spectrometry.
  • FAC frontal affinity chromatography
  • Figure 1 is a schematic of a system illustrating one embodiment of the present invention that is used for FAC-MALDI/MS/MS:
  • the column outlet is connected to a mixing tee for addition of MALDI matrix solution that flows directly into nebulizer to allow spraying of the mixture onto a MALDI plate that is moved under the column outlet on a computer controlled X-Y translation stage.
  • Figure 2 is a schematic showing an exemplary embodiment of the interfacing of multiple FAC columns with a MALDI-MS plate.
  • Figure 3 is a schematic of a prior art system used for FAC-ESI/MS/MS. A switch valve is used to switch from buffer to buffer + analyte, allowing continuous infusion of analytes onto the column. The column outlet is connected to a mixing tee for addition of makeup buffer that flows directly into the PE/Sciex API 3000 triple-quadrupole mass spectrometer.
  • Figure 4 is a schematic showing the vacuum based oMALDITM ion source assembly of the API 4000, which places the MALDI sample plate within the region evacuated by the interface vacuum pump in an orientation orthogonal to the analyzer axis.
  • Figure 5 shows typical FAC-ESI/MS/MS traces obtained using protein-loaded and blank DGS/PEO/APTES monolithic columns.
  • Panel A blank column containing no protein
  • Panel B column containing 25 pmol DHFR (initial loading).
  • N-acetylglucosarnine, fluorescein, folic acid, pyrimethamine and trimethoprim were infused at 50 nM. Traces show actual ion currents to provide a clearer indication of the ion suppression effect.
  • Figure 6 shows mufti pass selection of MRM transition for folic acid using MALDI/MS.
  • Panel A shows a Ql full scan of a mixture of folic acid, fluorescein, pyrimethamine and trimethoprim (50 nM each) mixed 1:1 (v:v) with 6.2 mg/mL CHCA in MeOH and deposited on a MALDI plate.
  • the Ql spectrum has had background signals originating from the matrix removed by subtraction.
  • Panel B shows the Q3 product ion scan originating from the m/z 442 parent ion.
  • Panel C shows the Q3 product ion scan originating from the m/z 295 parent ion. All scans were obtained using medium laser translation speed (1 mm/sec) and are the average of 5 re-runs over a given sample region.
  • Figure 7 shows MALDI/MS signal intensity as a function of number of reruns of a given region for slow (0.5 mm/s, A), medium (1 mm/s, ⁇ ) and fast (3.8 mm/s, •) translation speeds.
  • the peak value refers to the number of counts obtained for the first pass over the track, the sum value refers to the total number of counts obtained from all runs over a given track at a particular speed.
  • Figure 8 shows FAC-MALDI/MS/MS traces obtained using protein-loaded and blank DGS/PEO/APTES monolithic columns.
  • Panel A blank column containing no protein
  • Panel B column containing 25 pmol DHFR (initial loading) showing breakthrough of N-acetylglucosamine, fluorescein and folic acid at early times, trimethoprim and finally pyrimethamine. All compounds were infused at 50 nM. All traces are normalized to the maximum signal obtained after compound breakthrough. Note that MALDI analysis time is 19 fold faster than LC deposition time. All FAC traces were obtained using a fast laser translation speed (3.8 mm/sec) and are the average of 5 re-runs over a given sample region.
  • Figure 9 shows the effect of ionic strength on non-specific binding of compounds to blank monolithic columns analyzed by FAC/MALDI.
  • MALDI MRM traces are shown for the first run of folic acid, trimethoprim and pyrimethamine (50 nM each) using a) 2 mM ammonium acetate buffer, b) 50 mM buffer and c) 100 mM buffer. All data was run on the same column with pre-incubation of the column in the appropriate buffer prior to introduction of compounds. All FAC traces were obtained using a fast laser translation speed (3.8 mm/sec) and are the average of 5 re-runs over a given sample region.
  • Figure 10 shows the activity of DHFR as a function of incubation time in 2 mM and 100 mM ammonium acetate buffer solutions.
  • Figure 11 shows the effect of ionic strength on the reuse of monolithic DHFR columns using FAC/MALDI.
  • Panel A shows a MALDI MRM trace obtained from the first run for folic acid, trimethoprim and pyrimemamine (50 nM each) using 100 mM ammonium acetate buffer.
  • Panel B shows MALDI MRM traces obtained from plates during deposition of eluent upon the recovery of the column using 100 mM ammonium acetate.
  • Panel C shows the MALDI MRM trace from a plate that was formed by deposition of column effluent during the second run of the compounds listed above through the same monolithic column. Note: total running time was 120 min at 5 ⁇ L/min, corresponding to 122 column volumes. All FAC traces were obtained using a fast laser translation speed (3.8 mm/sec) and are the average of 5 re-runs over a given sample region. DETAILED DESCRIPTION OF THE INVENTION The interfacing of bioaffinity columns to MALDI/MS as a new platform for FAC/MS studies is described herein. Capillary columns containing entrapped dihydrofolate reductase (DHFR) were used for frontal affinity chromatography of small molecule mixtures.
  • DHFR dihydrofolate reductase
  • the output from the column combined with a second stream containing the matrix molecule -cyano-hydroxycinnamic acid (CHCA) in methanol and was deposited using a nebulizer-assisted electrospray method onto a conventional MALDI plate that moved relative to the column via a computer controlled x-y stage, creating a semi-permanent record of the FAC run.
  • CHCA matrix molecule -cyano-hydroxycinnamic acid
  • the present invention therefore includes a system for analyzing chemical samples comprising a frontal affinity cliromatographic (FAC) column interfaced to a MALDI mass spectrometer.
  • FAC frontal affinity cliromatographic
  • analyzing means that information about one or more compounds in a chemical sample is obtained using the system. Such information can include, but is not limited to, compound identity (via molecular weight and fragmentation patterns), and affinity, reactivity and other kinetic constants related to the interaction of the compound with biological material in the column (i.e. the retention time on the column).
  • interfaced it is meant that the effluent stream from the FAC column is combined with a MALDI matrix material, for example from a separate stream, and the combination is deposited on any suitable surface, for example a standard MALDI- MS plate, for MALDI-MS detection.
  • the combination may be deposited as discrete spots or as a continuous track using any suitable method, for example, but not limited to, fraction collection followed by MALDI deposition; 11 nebulizer assisted direct deposition of spots 12 ' 13,14 or tracks 15 ' 16 from the capillary; electrodynamic charged droplet processing; 17 deposition using a heated droplet interface; 18 piezoelectric flow- through microdispensing; 19 ' 20 vacuum assisted deposition; 21 electric field driven droplet deposition; electrospray deposition; or capillary nebulizer spraying.
  • deposition is by nebulizer assisted direct deposition of tracks.
  • the movement of the plate during deposition is controlled by a computer.
  • An exemplary embodiment of a system of the present invention is shown in
  • Figure 1 For deposition onto a MALDI plate, effluent from column (10) may be mixed with a MALDI matrix (20). The resulting total flow may then deposited onto MALDI plate(s) (30) using any known deposition method, for example, by continuous deposition. Movement of the plate(s) in the X-Y-Z franslational stages may be controlled by computer (40). The franslational stages control the deposition motion in X-Y plane and sprayer separation from the MALDI plate along the Z axis. Along with the movement in all three axes, the application of high voltage and nebulizer gas flow may also be controlled from a single computer (40). The column flow may be combined with matrix make up flow via Tee junction (50).
  • the combined flow is carried, for example by fused silica tubing, passing through a stainless steel electrode (60) which itself is inside a nebulizer. Both the fused silica and stainless steel electrode protrude slightly from the nozzle (70).
  • Another mixing Tee (80) is used to mount the nebulizer and introduce the inert gas (90) (for example N 2 ) into it.
  • Both the electrospray voltage and nebulizer gas flow may be manually adjusted and digitally actuated.
  • deposition parameters including distance of the sprayer above the plate, nebulizer gas flow, and electric field, may be optimized to obtain maximum track homogeneity and minimum track width.
  • the translation speed with which the plate is moved under the deposition tip may also be optimized to provide optimum track thickness while maintaining the necessary chromatographic resolution.
  • the system of the present invention may be applied to the analysis of chemical samples using multiple FAC columns run in tandem.
  • a schematic showing an exemplary embodiment of an interface between multiple FAC columns and an MS plate is shown in Figure 2.
  • Deposited plates may be analysed using any mass spectrometer equipped with a MALDI ion source using techniques known in the art.
  • the FAC column may be any type of column used as a solid support in any application for which FAC is used.
  • the FAC column is a bioaffinity capillary column.
  • the FAC column comprises a monolithic silica matrix.
  • the monolithic silica matrix is prepared using sol-gel techniques.
  • the monolithic silica matrix is prepared using biomolecule compatible techniques.
  • biomolecule compatible it is meant that the techniques are stabilizing to proteins and/or other biomolecules or do not facilitate their denaturation.
  • the method is used in a high through-put screen for modulators, substrates, and/or other compounds that bind to a biological molecule, for example a protein, peptide or nucleic acid (including DNA and RNA) or to biological materials, for example cells and tissues, wherein said biological molecule or material is entrapped within the matrixes of the column or otherwise immobilized onto the column.
  • a biological molecule for example a protein, peptide or nucleic acid (including DNA and RNA) or to biological materials, for example cells and tissues, wherein said biological molecule or material is entrapped within the matrixes of the column or otherwise immobilized onto the column.
  • the sample may contain for example, a library of compounds or an extract from a natural source.
  • the method may also be used to screen for putative enzymatic modulators while monitoring all chemical entities including the substrates and products of enzymatic reactions, for example in high throughput enzymatic reaction characterization, or other biomolecular reactions.
  • biomolecule or “biological material” as used herein, are interchangeable and means any of a wide variety of both naturally occurring and synthetic proteins, enzymes and other sensitive biopolymers including DNA and RNA and derivatives thereof, as well as complex systems including whole plant, animal and microbial cells that may be entrapped in silica.
  • the biomolecule may be dissolved in a suitable solvent, for example an aqueous buffer solution.
  • the biological substance is in its active form.
  • the present invention also includes a method of analyzing chemical samples from frontal affinity chromatography (FAC) comprising: (a) combining effluent from a FAC column with a matrix; (b) depositing the combination in (a) on to a surface; and (c) analyzing the deposited combination using MALDI mass spectrometry.
  • the matrix may be any material used in MALDI-MS.
  • the matrix is ⁇ -cyano-hydroxycinnamic acid (CHCA) dissolved in methanol.
  • CHCA ⁇ -cyano-hydroxycinnamic acid
  • concentration of the CHCA solution may be about 0.01 M to about 0.1 M, more suitably about 0.03 to about 0.05 M.
  • the effluent and matrix can be combined in about a 1:5 to about 5:1 volume ratio, suitably about a 1 :2 to about 2:1 volume ratio. In an embodiment the effluent and matrix are combined in about a 1 :1 volume ratio.
  • the effluent from the FAC column will comprise the eluent and optionally, one or more compounds from the sample. Any eluent suitable for FAC and the particular column being used may be employed. It is a particular advantage of the present invention that the eluent may comprise high ionic strength elution buffers, for example buffers with an ionic strength greater than 10 nM.
  • TEOS Tetraethylorthosilicate
  • APTES 3- aminopropyltriethoxysilane
  • Diglycerylsilane precursors were prepared from TEOS as described elsewhere. Trimethoprim, pyrimethamine, folic acid, poly(ethyleneglycol) (PEG / PEO, MW 10 kDa) and fluorescein were obtained from Sigma (Oakville, ON). MALDI matrix solution (6.2mg/mL oc-cyano-hydoxycinnamic acid, CHCA, in methanol) was obtained from Agilent (part no. G2037A). Recombinant dihydrofolate reductase (from E. coli), which was affinity purified on a methotrexate column, was provided by Professor Eric Brown (McMaster University).
  • Fused silica capillary tubing 250 ⁇ m i.d., 360 ⁇ m o.d., polyimide coated
  • Polymicro Technologies Panoenix, AZ
  • All water was distilled and deionized using a Milli-Q synthesis A10 water purification system. All other reagents were of analytical grade and were used as received.
  • Instrumentation FAC/MS System The system used for FAC/ESI-MS studies is shown in Figure 3. Syringe pumps (Harvard Instruments Model 22) were used to deliver solutions, and a flow-switching valve was used to toggle between the assay buffer and the solution containing the compound mixture. This solution was then pumped through the column to achieve equilibrium.
  • Effluent was combined with a suitable organic modifier to assist in the generation of a stable electrospray and detectability of the sprayed components using a triple-quadrupole MS system (PE/Sciex API 3000TM).
  • a Rheodyne 8125 injector valve was used to switch from buffer to buffer+analyte streams during operation.
  • Columns were interfaced to the FAC system using Luer- capillary adapters (Luer Adapter, Ferrule and Green Microtight Sleeve from Upchurch (P-659, M-100, F-185X)). All other connections between components were achieved using fused silica tubing. Instrumentation for FAC/MALDI/MS/MS is shown in Figure 1.
  • the translation stage is a part of a three-axis positioning system consisting of a 404 series axis, Aries controllers and ACR PCI control card from Parker Hanifin and Compumotors, respectively, that controls the deposition motion in X-Y plane and sprayer separation from the MALDI plate along the Z axis. All three axes as well as application of high voltage (custom built digitally controlled high voltage power supply, 4kV) and nebulizer gas flow (Clippard minimatics valve ET- 2M) were controlled from a single Dell Precision 340 computer through the ACR control card. The column flow was combined with CHCA make up flow in a stainless steel Tee junction from Valco.
  • the combined flow was carried by fused silica tubing (200 ⁇ m/100 ⁇ m o.d./i.d.) passing through a stainless steel electrode which itself was inside a nebulizer. Both the fused silica and stainless steel electrode protrude slightly (lmm) from the nozzle (0.6 mm i.d.).
  • a mixing Tee was used to mount the nebulizer and introduce the N 2 gas into it. Both the electrospray voltage and nebulizer gas flow were manually adjusted and digitally actuated. Deposition parameters, including distance of the sprayer above the plate, nebulizer gas flow, and electric field, were optimized to obtain maximum track homogeneity and minimum track width.
  • the translation speed with which the plate was moved under the deposition tip was also optimized to provide optimum track thickness while maintaining the necessary chromatographic resolution.
  • the optimal height of the electrospray tip was 8 mm above the sample plate, while a combination of gas flow (Nitrogen at 1.5 L/min) and electric field (3 kV between the electrospray tip and MALDI plate) was used to deposit the traces.
  • the MALDI plate was moved at 0.2 mm/sec relative to the stationary deposition tip.
  • the deposited plates were analyzed using an AB/Sciex API 4000TM triple quadrupole mass spectrometer equipped with an AB/Sciex oMALDITM ion source and high repetition rate (1.4 kHz) PowerChip NanoLaser (355 nm) from JDS Uniphase.
  • the vacuum based oMALDITM ion source replaced the normal orifice/interface assembly of the API 4000TM and its Turbo VTM source, thus placing the MALDI sample plate within the region evacuated by the interface vacuum pump in an orientation orthogonal to the analyzer axis, as shown in Figure 4. Normal source parameters were used to set-up and control the oMALDITM ion source.
  • the MALDI plate was held on an X-Y translation stage in front of an orifice and skimmer that separate it from the analyzer.
  • the modified API 4000TM retained its full capability of scan modes and scan speeds.
  • the deposited track (plate) was moved relative to the desorbing laser beam at a constant speed of 3.8mm/sec by the MALDI source X-Y stage, unless otherwise stated.
  • the desorbing laser beam was focused to a 180 x 230 ⁇ m spot on the track surface.
  • This aqueous solution also contained ca. 20 ⁇ M of DHFR.
  • 100 ⁇ L of the Buffer/PEG/APTES/DHFR solution was mixed with 100 ⁇ L of hydrolyzed DGS and the mixture was immediately loaded via syringe pump into a fused silica capillary (ca. 2 m long).
  • the final composition of the solution was 8% w/v PEO (10 kDa), 0.3% v/v APTES and 10 ⁇ M DHFR in 25 mM HEPES buffer.
  • the mixture became cloudy due to spinodal decomposition (phase separation) over a period of 1-3 sec about 2-3 min prior to silica polymerization ( ⁇ 10 min) to generate a hydrated macroporous monolithic column containing entrapped protein.
  • the monolithic columns were aged for 2-5 days at 4 °C and then cut into 5 cm lengths before use. The columns had an initial loading of 25 pmol of active DHFR in 5 cm, of which ⁇ 6 pmol was active and accessible in the column.
  • the makeup flow (used to assist in the generation of stable electrospray ionization) consisted of methanol containing 10% (v/v) NH 4 OAc buffer (2 mM) and was delivered at 5 ⁇ L.min "1 , resulting in a total flowrate of 10 ⁇ L.min 1 entering the ESI mass spectrometer.
  • the makeup flow was replaced with a flow of matrix (CHCA 6.2mg/mL in methanol) at 5 uL.min l .
  • the ESI mass spectrometer was operated in MRM mode with simultaneous detection of m/z 222 ⁇ m/z 204 (N-acetylglucosamine CE 15eV); m/z 249 ⁇ m/z 233 (pyrimethamine CE 42eV); m/z 291 ⁇ m/z 230 (trimethoprim CE 35eV); m/z 333 ⁇ m/z 202 (fluorescein CE 15eV) and m/z 442 ⁇ m/z 295 (folic acid).
  • MALDI MS/MS analysis was also performed using MRM scan mode but due to fragmentation during the MALDI desorption process the transitions for N-acetylglucosamine and folic acid were changed to m/z 204 ⁇ m/z 138 (CE 18eV) and m/z 295 ⁇ m/z 176 (CE 30eV), respectively.
  • the much shorter analysis times achievable with MALDI makes necessary a reduction in signal accumulation bin duration (dwell time) in order to maintain sufficient sampling frequency.
  • the ESI based MRM analysis used 1000 ms dwell while the MALDI MRM dwell was reduced to 40 ms per transition.
  • DHFR Stability in Ammonium Acetate DHFR was diluted to 40 nM in 2 mM or 100 mM ammonium acetate, (which contained 3 ⁇ M HEPES and 2 ⁇ M ⁇ aCl) and was incubated for various periods of time up to 24 hours.
  • DHFR activity was measured by monitoring the decrease in absorbance at 340 nm using a Tecan Satire microplate reader. Activity data is reported relative to the activity obtained from a DHFR sample that was diluted in 50 mM Tris.HCl, pH 7.5, containing 2 mM DTT.
  • FAC/ESI-MS/MS Figure 5 shows FAC/ESI-MS/MS traces obtained for elution of mixtures of DHFR inhibitors and control compounds through DGS/PEO/APTES columns containing no protein (Panel A) or an initial loading of 25 pmol of active DHFR (Panel B).
  • the blank column shows the expected breakthrough of all compounds in the first few minutes (between 1 and 4 min), although both pyrimethamine and trimethoprim, which are cationic, are retained slightly longer than the anionic compounds fluorescein and folic acid.
  • the retention which is present when using 2 mM ammonium acetate buffer, is indicative of non-selective interactions between the cationic compounds and the anionic silica column, showing that normal-phase silica chromatography is not fully suppressed at low ionic strength.
  • trimethoprim 4 nM, elution time of 22 min
  • Figure 6 shows a MALDI Ql spectrum of a mixture of the four target analytes (folic acid, pyrimethamine, trimethoprim and fluorescein) after appropriate background subtraction to reduce CHCA background signals. Peaks are evident for each of the four compounds; however, the primary peak for folic acid occurs at m/z 295 rather than at m/z 442, indicative of a fragment ion being the primary species present for this compound. Focusing on folic acid, product ion scans obtained from the same track using the m/z 295 parent ion clearly show a maximum peak at m/z 176, with an intensity of 5.5 x 10 5 cps.
  • a question that arises is the number of times that a particular region of a track can be re-run, as this determines how to best utilize the ability to re-run an already sampled portion of the track and hence increase the efficiency of the detection process.
  • the number of times a track can be re-run depends on the laser fluence and the speed with which the laser is translated over the sample.
  • the laser fluence used for the MALDI process was set to 3 ⁇ J/pulse. This value optimized the signal-to- noise ratio while minimizing thermal degradation of the track surface, thus allowing maximum sample utilization.
  • the effect of sampling speed on the number of possible re-runs over the same region of the track is shown in Figure 7.
  • sample consumption depends on the speed with which laser traverses the track, with greater speed causing less sample consumption and allowing more re-runs.
  • the maximum speed of the MALDI source stage (3.8 mm/sec) allows about 30 re-runs prior to sample exhaustion occurring in a given region of the track, where the majority of the signal is desorbed during the first 15 passes.
  • a small portion (10%) of the total track is sampled, it is likely that up to 7-8 different regions could be sampled per track, and thus in practice a single deposited track could be sampled over 100 times.
  • Figure 8 shows the FAC traces obtained upon desorption from MALDI plates onto which the eluent from either blank (Fig 8a) or DHFR columns (Fig 8b) had been deposited using 2 mM ammonium acetate as the running buffer.
  • Fig 8a the compounds elute in the first two traces that are deposited onto the MALDI plate (arrows show the traces that have been analyzed).
  • the bottom scale of Figure 8 shows MALDI analysis time, which can be converted into LC elution time using ratio of deposition speed to laser read out speed as a multiplication factor, which is 19 in this case.
  • the fluorescein, N-acetylglucosamine and folic acid elute first (1.5 min LC time) followed by trimethoprim (3 min LC time) and pyrimethamine (3.5 min LC time), again showing non-specific binding of the analytes when using low ionic strength buffers.
  • trimethoprim 3 min LC time
  • pyrimethamine 3.5 min LC time
  • Table 2 compares the signal-to-background levels obtained from ESI and MALDI MS/MS methods using 2 mM and 100 mM ammonium acetate (AA) buffer levels for MALDI and 2 mM for ESI, and provides a means for conversion of the normalized plots to absolute counts. It should be noted that even though the ESI and MALDI experiments were each made using a different mass analyzer, API 3000TM and API 4000TM respectively, a general comparison (intended as a guide only) is possible since by converting the API 4000TM for MALDI operation by fitting an oMALDITM source its normal orifice/interface and Turbo VTM source have been removed.
  • the MALDI process offers the ability to reduce its noise by combining signal from numerous re- runs of a track. The resulting noise reduction through signal averaging can be applied until a desired level required for data interpretation is reached.
  • FIG. 11 shows the effects of high ionic strength on the reusability of the monolithic DHFR columns.
  • Panel A shows the FAC-MALDI/MS/MS trace obtained for the initial run of the column using 50 nM of folic acid, pyrimethamine and trimethoprim in 100 mM ammonium acetate
  • Panel B shows the recovery run obtained using 100 mM ammonium acetate
  • Panel C shows the second run of the same column under identical conditions to those used in Panel A.
  • the overall performance of the column when using 100 mM ionic strength is far superior to that obtained when using 2 mM ionic strength.
  • the retention time for both trimethoprim and pyrimethamine decreases by only 20% (11.5 to 10 min for trimethoprim, 16.5 to 13.5 min for pyrimethamine) at 100 mM ionic strength, whereas decreases close to 85% in retention time were obtained at 2 mM ionic strength. 9
  • the retention time for all compounds at 100 mM ionic strength was significantly shorter than was obtained at 2 mM. In part this was due to the use of a shorter column for the latter experiments (5 cm vs.
  • the monolithic columns may find use in screening of compound mixtures against a wide variety of useful targets.
  • Another advantage of the low i.d. monolithic columns is the ability to interface the capillary columns directly to an ESI or MALDI mass spectrometer, which is likely to make them suitable for HTS of compound mixtures using FAC/MS.
  • the low i.d. of the present monolithic columns allows them to deposit a relatively thin stream of analyte on a MALDI plate, allowing for high density deposition (up to 12 traces per plate).
  • the time capacity of a MALDI plate is determined by the width of the deposited track as well as its deposition speed. Reducing the deposition speed will increase the plate capacity but it will also degrade the LC resolution as material eluted at any instant in time is deposited over a finite area, given by the spray diameter, and the overlap of two adjacent events increases. Since the spray diameter directly affects both the capacity of a plate and fidelity of the chromatography record, it is important to keep it as small as possible. In practical terms, the loss of chromatographic resolution that can be tolerated dictates the lowest deposition speed.
  • the ratio between deposition and interrogation speed determines how many re-runs and different analysis experiments can be performed over a track at a time, saving significant time over an LC re-run.
  • Certain parameters with the FAC-MALDI/MS/MS method reported herein may be optimized to enhance performance. For example, deposition methods that can produce narrower, less disperse traces would provide a higher density of analyte on the plate. 35 This should lead to a higher analyte concentration in the laser beam and thus a better LOD. Lower diameter columns may allow faster LC separations with lower flowrates that are compatible with deposition of thin tracks on the MALDI target.
  • MALDI/MS In addition to thinner columns, methods to suppress the inherent background from the MALDI matrix would minimize the need for subtraction of matrix background signals from analyte signals. While this is less of a problem when using MRM mode, and indeed was not required in the current study, such methods could be used with drug compounds that have product ions that are similar in structure to commonly used MALDI matrix species.
  • An advantage of MALDI/MS relative to ESI/MS for FAC studies is the ability to use much higher ionic strength buffers during the FAC run. The activity of proteins is known to be highly dependent on factors such as solution pH and ionic strength, and in most cases maximum activity is obtained using buffers that mimic physiological conditions (i.e., 20-50 mM buffer, 100 mM KC1, pH -7.4).
  • high ionic strength provides a more effective double layer, which better screens the charge of the anionic silica surface, and thus reduces electrostatic interactions between the charged analytes and the silica surface.
  • Na + and K were avoided to minimize issues with adduct ion formation.
  • ammonium acetate which is a volatile buffer, was chosen to adjust ionic strength. The use of this buffer did not lead to the formation of adduct ions, and provided conditions that were amenable to LC deposition even at 100 mM concentrations. It is possible that even higher levels of ammonium acetate could be used for FAC/MALDI, but such levels were not examined in this study.
  • MALDI/MS/MS provides better tolerance of high ionic strength buffers, less ion suppression, faster MS analysis times, access to more modes of MS analysis per LC run, and potentially offers the ability to acquire data using different mass analyzers (triple-quadrupole, TOF, TOF- TOF, Q-TOF, Ion Trap, FT-MS) from the same sample, which could be- beneficial in cases where higher molecular weight species are analyzed.
  • the ability to perform multiple MS analyses per LC run can be used advantageously to optimize detection of low concentration analytes or to identify unknown compounds that might be present in a natural product library or similar compound mixture.
  • ESI/MS the MRM transitions, and hence the identity, of compounds must be know prior to the FAC run. Otherwise, unknown compounds must be identified indirectly using an indicator compound in "roll-up" mode, with compound identification done off-line. As shown herein, such roll-up effects can be confused with ion-suppression when using ESI/MS/MS, leading to difficulties in identifying true "hits" when using indicator mode.
  • MALDI/MS/MS minimizes these problems, making the indicator mode more reliable, and also allows full MS analysis of deposited analytes, aiding in identification of unknowns.

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Abstract

Des colonnes de silicium nonocristallin sol-gel contenant une dihydrofolate réductase piégée ont été utilisées dans la chromatographie d'affinité frontale de mélanges de petites molécules. Le résultat de la colonne a été combiné à un second flux contenant la molécule matricielle (HCCA) puis directement déposé sur une plaque MALDI classique déplacée par rapport à la colonne via un étage x-y contrôlé par ordinateur, ce qui a permis de créer un enregistrement semi-permanent de l'essai FAC. En outre, l'utilisation de MALDI MS a permis de séparer les procédés FAC et MS, et par conséquent, d'obtenir des tampons à force ionique nettement plus élevée devant servir dans des études par FAC et de parvenir à une meilleur rétention de l'activité protéique après de nombreux essais successifs.
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