EP0986746A1 - Interface de depot en ligne d'echantillons liquides pour spectroscopie de masse a temps de vol a ionisation-desorption laser assistee par matrice (maldi-tof) - Google Patents

Interface de depot en ligne d'echantillons liquides pour spectroscopie de masse a temps de vol a ionisation-desorption laser assistee par matrice (maldi-tof)

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Publication number
EP0986746A1
EP0986746A1 EP98924853A EP98924853A EP0986746A1 EP 0986746 A1 EP0986746 A1 EP 0986746A1 EP 98924853 A EP98924853 A EP 98924853A EP 98924853 A EP98924853 A EP 98924853A EP 0986746 A1 EP0986746 A1 EP 0986746A1
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EP
European Patent Office
Prior art keywords
sample
capillary
receptor
mass spectrometer
infusion
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.)
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Application number
EP98924853A
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German (de)
English (en)
Inventor
Barry L. Karger
Frantisek Foret
Jan Preisler
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Northeastern University Boston
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Northeastern University Boston
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Application filed by Northeastern University Boston filed Critical Northeastern University Boston
Publication of EP0986746A1 publication Critical patent/EP0986746A1/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Withdrawn legal-status Critical Current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
    • H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
    • H01J49/0409—Sample holders or containers
    • H01J49/0418—Sample holders or containers for laser desorption, e.g. matrix-assisted laser desorption/ionisation [MALDI] plates or surface enhanced laser desorption/ionisation [SELDI] plates
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
    • H01J49/0431—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components for liquid samples

Definitions

  • Matrix molecules which absorb most of the laser energy, transfer that energy to analyte molecules to vaporize and ionize them. Once created, the analyte ions are analyzed in mass spectrometer, typically a TOF mass spectrometer.
  • MALDI is typically operated as an off-line ionization technique, where the sample, mixed with a suitable matrix, is deposited on the MALDI target to form dry mixed crystals and, subsequently, placed in the source chamber of the mass spectrometer.
  • solid samples provide excellent results, the sample preparation and introduction into the vacuum chamber requires a significant amount of time. Even simultaneous introduction of several solid samples into a mass spectrometer or off-line coupling of liquid-phase separation techniques with a mass spectrometer do not use TOF mass spectrometer time efficiently.
  • MALDI -MS analysis typically requires finding the "sweet spot" on the sample target, so that a reasonable signal can be obtained (5,6) .
  • liquid samples were analyzed directly inside the mass spectrometer using a variety of matrices and interfaces.
  • a nebulizer interface was used for continuous sample and matrix introduction (13-19) .
  • MALDI was then performed directly off rapidly dried droplets.
  • a continuous probe similar to a fast atom bombardment (FAB) (20) interface, was used for the analysis of a flowing sample stream with liquid matrix (21-24) .
  • Glycerol was used to prevent freezing of the sample.
  • Other attempts for liquid sample desorption were also made using fine dispersions of graphite particles (25, 26,27) and liquid matrices (2,28-40) instead of a more conventional matrices.
  • the invention is directed to a universal interface and sample load mechanism for continuous on-line liquid sample introduction directly to a mass spectrometer at subatmospheric pressure.
  • the liquid sample includes a matrix, either solid or liquid, for use in matrix- assisted-laser-desorption-ionization, most particularly in a time-of-flight mass spectrometer which can further promote throughput and utility of MALDI -TOF MS.
  • the same samples and matrices, both solid and liquid can be used as in conventional MALDI.
  • a solution of sample containing, e.g., peptide and matrix is infused directly into the source chamber of a mass spectrometer at subatmospheric pressure, deposited on a moving sample holder, such as a rotating quartz wheel, and desorbed by, e.g., a nitrogen laser.
  • the system and method of the invention are particularly amenable to multiplexing, the parallel deposition of multiple samples, e.g., from a capillary array or microchip channels, with subsequent sequential desorption with a scanned laser beam. This format is particularly useful for high throughput MS analysis.
  • Fig. 1A is a plan view of an on-line MALDI -TOF MS instrument incorporating a sample load mechanism for practicing the method of the invention
  • Fig. IB is a schematic partial side view of the sample load mechanism of the mass spectrometer of Fig. 1A;
  • Fig. 2A is a detail schematic view of Fig. IB showing the liquid deposition process on the rotating wheel within the vacuum of the mass spectrometer of Fig. 1A;
  • Fig. 2B and 2C are close up views of the liquid deposition process of Fig. 2A from a perpendicularly - cut capillary and a tipped capillary, respectively, along with the corresponding sample traces formed;
  • Fig. 3A-3C are scanning electron micrographs of deposited MALDI sample, l ⁇ M angiotensin III and 10 mM o;CHCA in 50% (v/v) methanol .
  • Preparation of MALDI sample (3A) dried droplet method, (3B) and (3C) trace of sample deposited at low pressure.
  • Fig. 4 shows ion gauge signals during deposition of 50% (v/v) methanol (trace A) and infusion of methanol (trace B) ,
  • Fig. 5 is a schematic partial side view of an on-line CE - MALDI -TOF MS system for practicing the method of the invention
  • Fig. 6 shows normalized MALDI -MS spectra of bovine insulin with Q ⁇ CHCA matrix.
  • Fig. 7 is a graph showing variations of angiotensin II, frag. 1-7 ion signal versus segment number.
  • Fig. 8 shows single shot MALDI mass spectra of a mixed solution of 1 ⁇ M heptapeptide EDPFLRF with (trace A) 10 mM or (trace B) 1 mM cCHCA matrix deposited on the quartz wheel at 0.33 rpm;
  • Fig. 9 is a graph showing decay of angiotensin III (m/z
  • Fig. 10 shows a single shot MALDI mass spectrum of heptapeptide EDPFLRF at a concentration of 0.1 ⁇ M, 50 attomole deposited on the desorption spot;
  • Fig. 11 is a CE-UV electropherogram of angiotensin mixture (see Table 2) ;
  • Fig. 12 is a 2 -dimensional MALDI -MS electropherogram of angiotensin mixture (see Table 2) ;
  • Fig. 13A is a plan view showing another embodiment of an on-line MALDI-TOF MS instrument for practicing the method of the invention.
  • the rotation wheel sample receptor is oriented at 90° to the orientation of the wheel sample receptor of the embodiment of Fig. 1A so that a cleaning beam 29 can access the wheel in addition to desorption beam 28;
  • Fig. 13B is a bottom perspective view of the mass spectrometer of Fig.l3A showing multiple multiplexed infusion capillaries simultaneously depositing samples on the rotating wheel .
  • the interface of the invention permits deposition of a liquid sample stream onto a moving surface inside the vacuum region of a mass spectrometer, and in particular, a time-of-flight mass spectrometer (TOF-MS instrument) .
  • TOF-MS instrument time-of-flight mass spectrometer
  • the design of the interface permits easy handling of very small (submicroliter) sample volumes and minimizes sample losses both during handling and desorption.
  • the interface and method of the invention take advantage of the significant experience available in the field of MALDI .
  • the two essential elements of a TOF mass spectrometer are the source chamber 5, within which is an acceleration (extraction) region 10, and the flight region 20.
  • the electric field in the acceleration region is given by the voltage difference between the repeller 12 and the acceleration plate 14.
  • a second acceleration plate 16 and additional ion optics may also be used.
  • ions formed at the probe tip 18 by MALDI are extracted towards the acceleration plate. Because of differences in their masses, different ions are accelerated to different velocities during their stay in the acceleration region. Thus, light ions move across the field-free (flight) region in a shorter time than do heavy ones.
  • An ion signal from the detector is recorded as a function of time and can be transformed to a function of ion mass-to-charge ratio
  • a liquid sample emerging from the probe tip 18 is deposited under vacuum onto a moving sample receptor, e.g., a rotating quartz wheel, directly in the source chamber of the mass spectrometer.
  • the analyte, premixed with the suitable matrix is deposited directly onto the quartz wheel 22 through a narrow fused silica capillary 24.
  • sample flow rate - 300 nl/min.
  • the sample immediately dries on the wheel, forming a continuous trace -40-60 ⁇ m wide and only few hundred nanometers thick.
  • the wheel rotation then brings the sample trace towards a slit 26 in the repeller plate 12, where it is irradiated and desorbed by the desorption nitrogen laser 28.
  • the subtle grooves perpendicular to the deposited trace, characteristic for the copper tape, can be seen below the deposited sample, indicating a very thin sample film. It can also be seen that the capillary tip scratched the copper and formed a groove in the middle of the trace. Assuming a rectangular profile of the 40 ⁇ m wide trace, a flow rate of 300 nL/min and a matrix density of 1.2 g/cm 3 , the thickness of the sample film was estimated as - 70 nm. The sample, however, tended to accumulate at the edges, as can be seen in Fig. 3C, with the width of the mound of 1-2 ⁇ m and several hundreds nm in height .
  • the actual sample for on-line MALDI was prepared on an unpolished DELRIN or quartz wheel, whose properties, such as thermal conductivity or surface roughness, were different from those of copper. Nevertheless, the images of the trace deposited on the copper tape could yield useful information on the potential of sample deposition and crystallization in vacuum.
  • the next step was to implement this sample preparation technique into a MALDI -TOF mass spectrometer.
  • Time-of-Flight Instrument Although the preparation of sample for MALDI -MS by deposition in vacuum can be carried out off-line in a separate vacuum chamber, it is most preferably carried out on-line, i.e., in the source chamber of a mass spectrometer. So as to be able to introduce liquid directly into the source chamber of mass spectrometer and have greater flexibility in the design of the interface, we decided to built a mass spectrometer in house.
  • High speed pumping was used for the construction of the system in order to maintain pressure sufficiently low during continuous infusion of solvents.
  • a diffusion pump with a pumping speed higher than pumps typical in commercial mass spectrometers was chosen and found appropriate for the task.
  • the large chamber and flight tube ensured fast evacuation speed, as well as sufficient space for modification of the sample loading mechanism.
  • a liquid-nitrogen cryotrap also removed condensable vapors, such as water or methanol from the capillary outlet (pumping speed of 3000 L/s, as specified by manufacturer) .
  • the lowest pressure in the flight tube was 5xl0 "8 Torr, with a usual pressure in the low 10 "6 Torr range during the deposition of the solvent.
  • the ion gauge signal was plotted in volts, as measured, and not converted to pressure. Nevertheless, an increase of ion gauge voltage is related to an increase of pressure, e.g. an increase of IV would correspond to a 10- fold increase of pressure at constant composition of background gas. Pressure values given below were estimated from the ion gauge signal if the composition of background gas in the chamber was known.
  • the microvial with 50% (v/v) methanol was then removed from the capillary inlet at the time marked with the cross. Air began to flow into capillary, and the flow of solvent increased as the length of the solvent plug in the capillary shortened. This increasing liquid flow resulted in a pressure spike ( ⁇ 10 "5 Torr) . Finally, when all the solvent was removed, air began to flow into the chamber again, and the pressure slowly returned to its original value. This latter process was relatively long, presumably because of slow desorption of the solvent from the walls of the mass spectrometer. Similar pressure behavior was observed for methanol, 10% (v/v) methanol and water (data not shown) .
  • the width of a sample trace deposited with a tapered capillary (20 ⁇ m i.d., 150 ⁇ m o.d., 40-60 ⁇ m tip o.d.) was 40-60 ⁇ m, compared to approximately 200 ⁇ m wide sample trace deposited from the same perpendicularly-cut capillary (Fig. 2B) .
  • the wheel which was at room temperature, acted as a heat reservoir preventing the solvent in the capillary outlet from freezing. Energy necessary for evaporation and/or sublimation of the solvent was taken from the wheel as well as from the liquid solvent. Therefore, no additional heating element was necessary in contrast to other designs (21-24, 42) .
  • the solvent formed a very thin layer on the surface of the wheel making evaporation and/or sublimation fast and even. This led to thin microcrystalline or amorphous sample films, as shown in Figs. 3A-3C.
  • the capillary tube 20 ⁇ m i.d., 150 ⁇ m o.d.,
  • the wheel made of a nonconductive material, insulated the electrodes inside the mass spectrometer from the outer system.
  • a conductive metal wheel made, e.g., of aluminium or stainless steel, may be used so that the capillary tip would be connected electrically to the wheel .
  • This configuration may be useful in studies of arcing.
  • separation of the sample deposition region from the sample desorption region, as a result of mechanical transport of the sample, was advantageous.
  • the solvent, which evaporated at the deposition region did not contribute to local overpressure at the laser desorption spot. No electrical discharge at the deposition region was thus observed due to elevated local pressure.
  • separation of the two regions can allow implementation of a differential pumping scheme, in which the deposition region would be maintained under rough vacuum, at subatmospheric pressures of about 1 Torr or less.
  • Another possibility would be to enclose the deposition region by a small refrigerated trap, which would remove the solvent molecules. Frozen solvent could be released from the trap by heating when the instrument was not used.
  • the power density in the laser desorption spot of 500 MW/cm 2 would be achieved by 200 ⁇ J, 4 ns pulse of the nitrogen laser, assuming no optical losses.
  • window 42 and lens 44 absorbance, mirror 46 reflectance and non-ideal beam shape of the laser should account for up to 50% attenuation of the laser beam. Since the available desorption power density was still well above 1-10 MW/cm 2 necessary for MALDI experiments (2, 65), a neutral density filter was used to adjust the desorption power density to 10 - 20% above the threshold of the desorption power density.
  • the range of rotation speed of the wheel 22 given by the stepper motor 48 was 0 - 12 rpm; however, the usable range of rotation speed was narrower. Clogging of the capillary with frozen solvent and solutes was possible at low speeds, e.g. below - 0.1 rpm.
  • the desorption laser could not irradiate all the sample at high rotation speed, because some segments of the trace would pass the slit in the repeller between laser shots. This means that the number of steps of the motor per second should not be higher than the maximum repetition rate of the laser (30 Hz) . Defocusing the laser in order to irradiate a larger area of the sample trace, corresponding to several steps of the motor is not desirable in some cases.
  • a large laser desorption spot could reduce resolution of the separation method.
  • several shots (10 - 50) of the desorption laser should generally be applied to each 87 ⁇ m long segment on the sample trace to analyze more of the deposited sample.
  • the ratio between the velocity at which analytes enter the capillary and the circumference velocity of the wheel provides a potential means of concentration of the analytes infusing either directly or after separation.
  • concentration factor of a given analyte is equal to the ratio of the velocity with which the analyte would exit the separation column and the circumference velocity of the wheel .
  • Variation of the rotation velocity of the wheel could change the concentration factor for different compounds during an analysis. For example, broader peaks of slower migrating compounds could be focused by gradual deceleration of the wheel rotation during CE analysis.
  • the concentration factor may be decreased because of analyte losses in the liquid junction.
  • Some characteristics of the sample infusion/deposition are shown in Table 1. Even at the lowest rotation speed of the wheel, 0.17 rpm, the number of laser shots per segment of the trace was 6 at maximum repetition rate of the laser. In order to apply more shots to each segment and to use more sample to obtain a better signal-to-noise ratio, a laser with a higher repetition rate would be needed or the rotation speed of the wheel would be lowered after the deposition finished.
  • a linear Wiley-McLaren type TOF mass spectrometer (46) with a i m long drift region was constructed.
  • the 20 cm cubic source chamber 5, sample load mechanism, acceleration optics, 10 cm diameter flight tube 20 and detector 19 were purchased from R. M. Jordan Co., Grass Valley, CA.
  • Original sample load mechanism was used only to analyze conventional MALDI samples (prepared by dried droplet method) .
  • the distances between the repeller plate 12 and the first grid 14 and between the first 14 and the second 16 grids were each 12.7 mm. Ion transmission for each of the two grids, which were grounded, was 90%.
  • the voltage on the repeller plate (+15 kV) was controlled by a power supply (Model CZE1000R/X2263 , Spellman, Hauppauge, NY) .
  • a 40 mm dual microchannel plate (MCP) with extended dynamic range (Galileo, Sturbridge, MA) served as ion detector.
  • the ion transmission of the detector input grid was 82%, leading to a total ion transmission of the three grids of 66%.
  • the instrument was evacuated by a diffusion pump (Model VHS-6, Varian, Lexington, MA) with a maximum pumping speed of 2,400 L/s.
  • a refrigerated recirculator (Model CFT- 75Neslab, Portsmouth, NH) was used to cool the diffusion pump. Oil contamination of the mass spectrometer was prevented by a cryotrap (Model 326-6, Varian) and an electropneumatic gate valve (Model GV-8000V-ASA-P, MDC, Hawyard, CA) .
  • the diffusion pump was backed by a two-stage mechanical pump (Model Pascal 2015, Alcatel, Annecy, France) equipped with a molecular seive trap (Model KMST-150-2, MDC) .
  • a vacuum controller (Model 307) with two convectron gauges and two ion gauges was purchased from Granvill -Philips, Boulder, CO. The convectrons were located in the foreline and in the source chamber, and the working ion gauge was in the detector region.
  • a laboratory-built TOF MS controller operated the diffusion pump, electropneumatic gate and HV power supplies. The controller protected the instrument and its components from damage due to an accidental pressure increase or a cooling malfunction. It also contained the voltage supply for the MCP detector.
  • the laser beam was attenuated with a stepped neutral density filter 21
  • the small cell did not contain high voltage electrodes because it was designed only for monitoring of the deposition process.
  • the cell was evacuated by a mechanical pump (Model DD 20, Precision Scientific, Chicago, IL) .
  • a mixed solution of analyte and matrix was deposited via a fused silica capillary 24 (Polymicro Technologies, Phoenix, AR) , 20 ⁇ m i.d., 150 ⁇ m o.d. and 12.0 cm in length, on a quartz wheel 22 (Optikos,
  • the capillary was accommodated in a probe 18 made of a stainless steel tube, 9.53 mm o.d., 6.7 mm i.d., 7 cm length.
  • a pipe adapter with a PEEK ferrule, 0.4 mm i.d. was attached to the outer (atmospheric) side of the tube, and a DELRIN cap with a center hole, 0.25 mm i.d., covered the inner (vacuum) side of the tube .
  • the probe was inserted into the source chamber via quick coupling, 9.53 mm i.d., in the center of the interface flange to the point that the end of the capillary was slightly bent while touching the wheel.
  • the outlet of the capillary was tapered, using fine sandpaper.
  • the diameter of the quartz wheel was 5.0 cm and the thickness 1.0 cm; the perimeter surface of the wheel was unpolished.
  • the wheel which was perfectly balanced on a stainless steel shaft, was propelled by a geared stepper motor 48 (Model ABS, Hurst, Princeton, IN) , with rotation speeds ranging from 0 to 12 rpm and 1800 steps for 1 full rotation.
  • the original repeller plate (R. M. Jordan Co.) with a center hole for the sample probe was used only for initial analysis of conventional MALDI samples; modification of the repeller plate was required to incorporate the wheel .
  • a rectangular hole of 12 x 30 mm was cut in the center of the plate and two pieces 52 of stainless steel foil (25 x 25 x 0.05 mm) were glued to the repeller plate 12 with a conductive glue 54 to create a slit (12 x 0.2 mm) 56 in the center of the plate.
  • the pieces of the foil were glued to the repeller plate at the ends opposite to the slit so that they remained flexible.
  • the distance of the wheel from the interface flange was adjusted such that the wheel was gently touching the stainless steel foil .
  • SEM Scanning Electron Microscopy
  • a digital delay generator (Model 9650A, EG&G, Princeton, NJ) triggered the desorption laser as well as a laboratory-built digital divider.
  • the output of the divider drove the stepper motor controller (Model EPC01, Hurst) for precise synchronization of the rotation of the wheel with the laser pulses.
  • the controller of the stepper motor was modified such that external pulses could be received to propel the motor and reset the controller counter.
  • a 500 MHz, 1-Gs/s digital oscilloscope (Model 9350AM, LeCroy, Chestnut Ridge, NY) allowed real time measurement and/or transfer of mass spectra to the computer.
  • a computer program running under DOS, transferred multiple files from the oscilloscope to a PC via a GPIB interface. Approximately 50 single-shot mass spectra (each consisting of 2000 data points) could be transferred to the computer memory in one second.
  • Capillary Electrophoresis Capillary Electrophoresis was performed using 75 ⁇ m i.d. and 375 ⁇ m o.d. fused silica capillaries (Polymicro Technologies) coated with polyvinylalcohol (47) to eliminate electroosmotic flow, with 10 mM solution of citric acid (electrophoresis grade, Schwarz/Mann Biotech, Cleveland, OH) as running buffer.
  • Electrophoresis was driven at 500 V/cm by a high voltage power supply (Model PS/EH30, Glassman, Whitehouse Station, NJ) .
  • the sample was injected from unbuffered solution either by electromigration at 50 V/cm or by pressure at 250 Pa.
  • the total length of the capillary was 15 cm and the effective length 10 cm.
  • Absorbance at 220 nm from a CE detector (Model Spectra 100, Spectra Physics) was recorded using Chrom Perfect (Justice Innovations, Mountain View, CA) . Referring to Fig.
  • the separation capillary (10 cm length) 32 was connected to a liquid junction (48, 49) 34 made of polycarbonate, which contained 10 mM of ⁇ CHCA matrix as cathodic buffer.
  • PEEK liners 62 (Model FS1L.15 PK and FS1L.4PK, Valco Instruments Co., Houston, TX) were inserted into holes in the polycarbonate liquid junction block 34 to center the separation and infusion capillaries precisely, with a gap of approximately 100 ⁇ m.
  • the sample was initially injected into the separation capillary, and the stepper motor was activated (0.33 rpm) within 5 seconds.
  • Methyl green (Sigma Chemical Co., St. Louis, MO) solutions in methanol and distilled water were initially used to explore deposition in a vacuum, ⁇ -cyano-4- hydroxycinnamic acid (o;CHCA) , 2 , 5-dihydroxybenzoic acid, 4- hydroxy-3-methoxycinnamic (ferulic) acid (all from Sigma Chemical Co.) , and 3-hydroxypicolinic acid (Aldrich Chemical, Milwaukee, WI) were used as matrices for MALDI, each consisting of 0.1 M stock solutions in 50% (v/v) methanol.
  • Angiotensins, angiotensinogens (see Table 1) , heptapeptide EDPFLRF and bovine insulin (BI) were purchased from Sigma Chemical Co and made as lmg/mL stock solutions in water.
  • One M stock solution of BI was prepared by dissolving in 0.1% trifluoroacetic acid (J.T. Baker Inc., Phillipsburg, NJ) .
  • Methanol, ethanol and acetonitrile all HPLC grade
  • EXAMPLE I On-line MALDI Performance The performance of the new interface was tested using several oligopeptides with ⁇ CHCA as matrix.
  • 1 ⁇ M bovine insulin with 10 mM oCHCA aqueous solution was deposited for about 30 seconds on the wheel rotating at 0.33 rpm.
  • the averaged spectrum was obtained from 100 single shot spectra from 50 segments of the trace, i.e., 2 shots were applied to each segment (Fig. 6, trace B) .
  • MALDI spectra of samples of bovine insulin with ⁇ CHCA matrix prepared conventionally and by vacuum deposition appeared very similar. Mass resolution of the insulin peak in the case of the vacuum deposited sample was comparable with the value obtained with the original repeller. Improved manufacturing of the repeller plate and incorporation of time-lag focusing should further enhance the resolution. With the use of time-lag focusing, the ions could be accelerated in a more homogeneous electrical field after they travel away from the slit. On- line MALDI spectra of angiotensins and other small peptides with OCHCA matrix were also obtained.
  • the variations in the signal were ⁇ 18%
  • the ion signal of the peptide was much more constant than the same signal obtained from a conventional MALDI sample.
  • the peptide peak was present in every single shot spectrum.
  • a variety of typical matrices were examined with peptide samples.
  • An important advantage of the on-line interface of the invention is that it can use the same matrices that have been already developed for MALDI and it is not restricted to the liquid matrices.
  • Conventional matrices such as a-cyano-4 -hydroxycinnamic acid, 2,5- dihydroxybenzoic acid, 4-hydroxy-3-methoxycinnamic (ferulic) acid and 3-hydroxypicolinic acid were successfully tested with peptide samples (results not shown) .
  • the first laser shot was found to produce a lower ion signal from the analyte than the next nine laser shots. This result, together with a difference in mass spectra in the low-mass region (not shown) , suggests some chemical and physical changes (melting & solidifying) occurring on the sample surface. This behavior of the first shot was variable however, since for some samples, the intensity of the ion signal of the analyte produced by the first shot was similar to that of the consecutive shots. Similar phenomena can also be observed in conventional MALDI (67) .
  • the ion signal of the analyte produced by the consecutive shots gradually decayed as the sample was removed, with virtually all the sample depleted within 40 laser shots.
  • the results suggest that ⁇ 20 single shot spectra should be averaged to obtain the optimum signal-to-noise ratio in this case. Omitting of the first single shot spectrum is generally suggested. It should be pointed out that the number of spectra to be averaged depends on many factors, such as the analyte and matrix concentration and the desorption laser power density. Less spectra may be necessary when the amount of deposited sample is low or the desorption power density is higher.
  • MALDI -MS has already been shown to be a very sensitive method for determination of peptides (7,68).
  • On-line deposition of sample offers additional advantages in sample handling over conventional techniques. Virtually all of the sample solution is transferred to the source chamber and accumulated on the wheel . The segment of the sample trace (given by the width of the trace and a single step of the wheel) is slightly smaller than the spot size of the desorption laser meaning all sample can be used. Several tens of laser shots should utilize all deposited sample because the sample layer is very thin. The interaction of matrix with analyte in solid and gaseous phase during the desorption and ionization should be promoted because the trace consists of well-mixed analyte and matrix.
  • CE-MALDI-MS of a mixture of 300-500 pg of each of 12 angiotensins listed in Table 2, was carried out.
  • a liquid junction was used to connect the separation capillary to the infusion capillary of the interface.
  • Analytes eluting from the separation capillary were mixed with solution of the
  • MALDI matrix in the reservoir of the liquid junction and then drawn into the infusion capillary for deposition on the wheel .
  • CE-UV absorbance detection at 220 nm
  • a 10 mM citric acid solution was selected as a running buffer for the separation of the angiotensins because the pK a and ion mobility of citric acid are similar to that of ⁇ CHCA, as estimated from the literature (69) (A 10 mM ⁇ CHCA solution in 50% (v/v) methanol will be used in the reservoir of the liquid junction for CE-MALDI-MS.)
  • Angiotensins were injected by electromigration at 50V/cm within 5 s from a 8.3 ⁇ g/mL (each) aqueous peptide mixed solution. The final amounts injected ranged from 300 to 500 pg.
  • ⁇ CHCA was used not only as the MALDI matrix but also as the electrolyte in the CE buffer.
  • indirect detection at 335 nm was used for CE-UV of the angiotensin mixture because of the large ratio between absorption coefficients of matrix and peptides at this wavelength.
  • citric acid was used instead in CE to show that the conditions of CE separation are not restricted by requirements of MS detection.
  • the infusion flow rate must be high enough to collect all analyte ions migrating out of the capillary (49) .
  • the velocity of the infused solution has to be higher than the electromigration velocity of analyte migrating towards the cathode located in the liquid junction.
  • the infusion flow of 50% (v/v) methanol was about 300 nL/min and most peptide ions were estimated to enter the infusion capillary.
  • the capillary and the anodic reservoir were filled with 10 mM citric acid solution and the cathodic reservoir in the liquid junction with 10 mM ⁇ CHCA solution in 50% (v/v) methanol.
  • Angiotensins were injected by electromigration at 50V/cm within 5 s from a 8.3 ⁇ g/mL (each) aqueous peptide mixed solution in the same quantities as in CE-UV.
  • the stepper motor ran at 0.33 rpm and the laser repetition rate was adjusted to 20 Hz, i.e., 2 shots per segment. As shown in Fig. 12, all twelve peptides were resolved and identified on the 2D MS-electropherogram.
  • CE-MALDI-MS migration time in CE-MALDI-MS was normalized to the peak 5 in CE-UV (migration time of 52.5 s) . This normalized time does not include the period needed for sample transport from the liquid junction to the desorption region (approximately 100 s) . Temporal halfwidth of CE-MALDI-MS peaks was lower than those detected by UV, which means that broadening caused by the liquid junction, laminar flow, adsorption in the infusion capillary and deposition process is lower than the broadening caused by finite size of light beamwaist in the commercial UV detector.
  • a sampling device such as a capillary array or microchip having several sample channels can be used for simultaneous introduction and high throughput analysis of multiple samples.
  • a capillary array or microchip having several sample channels can be used for simultaneous introduction and high throughput analysis of multiple samples.
  • FIG. 13B an array of capillaries
  • the beam of the desorption laser is used for simultaneous deposition of multiple samples on rotating wheel 22.
  • the beam of the desorption laser is used for simultaneous deposition of multiple samples on rotating wheel 22.
  • a cleaning beam 29 can be provided, at a different orientation from that of the desorption laser, to accomplish this purpose.
  • the cleaning beam can be an entirely separate laser from the desorption laser.
  • a single laser beam can be split to perform both functions. As only about 5% of the energy of the nitrogen laser beam used in these examples is needed for desorption, the remaining energy of the beam can be used for the cleaning function. Cleaning can also be accomplished mechanically or through the use of heat.
  • the mass spectrometer described herein was a time-of- flight mass spectrometer. This configuration is simple, very fast and particularly efficient in that it uses most of the desorbed ions to record the entire mass spectrum.
  • Other mass spectrometers such as a Fourier transform ion cyclotron resonance mass spectrometer, a quadruple mass spectrometer or an ion trap mass spectrometer can also be used.
  • tandem systems such as a quadrupole filter/TOF MS system, are particularly useful.
  • the method of the invention also enables the use of other mass spectrometry techniques, such as MS-MS, which is particularly important for analysis of proteins.
  • the particular advantages of the method of the invention are on-line coupling, very short analysis time, high throughput for a single sample, further increase of throughput by multiplexing (the simultaneous analysis of several samples) , high sensitivity and compatibility with existing time-of-flight focusing techniques.
  • the potential of the technique can be usefully exploited, e.g., for sensitive analysis, trace analysis, analysis of both small and large molecules, DNA sequencing, mutation analysis, screening and on-stream analysis.

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Electron Tubes For Measurement (AREA)

Abstract

L'invention concerne une interface universelle permettant d'introduire en ligne et en continu des échantillons liquides directement dans la chambre source (5) d'un spectromètre (20) de masse à temps de vol en vue d'une analyse à ionisation-désorption laser assistée par matrice (MALDI). L'échantillon liquide renferme une matrice solide ou liquide que l'on utilise lors des analyses MALDI classiques et il est déposé directement sur un porte-échantillons (22) mobile dans la chambre source (5). Le porte-échantillons (22) est à pression subatmosphérique, d'où un séchage rapide de l'échantillon. L'échantillon séché est ensuite désorbé du porte-échantillons (22) au moyen d'un laser à azote (28) dans une zone de désorption (26). Le procédé de l'invention permet un travail sur plusieurs échantillons, à savoir le dépôt parallèle de divers échantillons à partir d'un ensemble de capillaires ou de canaux micropuce (24), la désorption séquentielle qui s'ensuit étant assurée par un laser de lecture. Cet arrangement est particulièrement utile pour des analyses de spectrométrie de masse à haut rendement.
EP98924853A 1997-05-23 1998-05-22 Interface de depot en ligne d'echantillons liquides pour spectroscopie de masse a temps de vol a ionisation-desorption laser assistee par matrice (maldi-tof) Withdrawn EP0986746A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US4748997P 1997-05-23 1997-05-23
US47489P 1997-05-23
PCT/US1998/010537 WO1998053308A1 (fr) 1997-05-23 1998-05-22 Interface de depot en ligne d'echantillons liquides pour spectroscopie de masse a temps de vol a ionisation-desorption laser assistee par matrice (maldi-tof)

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EP0986746A1 true EP0986746A1 (fr) 2000-03-22

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EP (1) EP0986746A1 (fr)
JP (1) JP2002502543A (fr)
WO (1) WO1998053308A1 (fr)

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CA2386832C (fr) * 1999-10-29 2009-09-29 Mds Inc. Photoionisation sous pression atmospherique, nouveau procede d'ionisiation pour spectrometrie de masse avec chromatographie en phase liquide
WO2001095367A2 (fr) * 2000-06-05 2001-12-13 Pharmacia & Upjohn Company Ionisation a electronebulisation a sources multiples pour la spectrometrie de masse
GB2367685B (en) * 2000-07-26 2004-06-16 Masslab Ltd Ion source for a mass spectrometer
JP3413491B2 (ja) * 2000-08-10 2003-06-03 岡崎国立共同研究機構長 質量分析用インターフェイス、質量分析計、及び質量分析方法
CA2429633A1 (fr) * 2000-11-16 2002-05-30 Ciphergen Biosystems, Inc. Procede d'analyse de spectres de masse
DE10112386B4 (de) 2001-03-15 2007-08-02 Bruker Daltonik Gmbh Flugzeitmassenspektrometer mit Multiplex-Betrieb
WO2006084354A1 (fr) * 2005-02-08 2006-08-17 Mds Inc., Doing Business Through It's Mds Sciex Division Procede et dispositif pour depot d'echantillons
BRPI0806471A2 (pt) * 2007-01-12 2011-09-27 Univ Texas técnicas de separação de baixo fluxo de interface
JP5837273B2 (ja) * 2009-03-03 2015-12-24 公益財団法人野口研究所 質量分析法用測定試料及びその調製方法
US10090144B2 (en) 2014-03-18 2018-10-02 Micromass Uk Limited Liquid extraction matrix assisted laser desorption ionisation ion source

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JPH01137551A (ja) * 1987-11-25 1989-05-30 Jeol Ltd 質量分析装置用イオン源
US5382793A (en) * 1992-03-06 1995-01-17 Hewlett-Packard Company Laser desorption ionization mass monitor (LDIM)
US6068749A (en) * 1996-01-19 2000-05-30 Northeastern University Subatmospheric, variable pressure sample delivery chamber for electrospray ionization/mass spectrometry and other applications

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JP2002502543A (ja) 2002-01-22

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