WO1996036986A1 - Procedes et appareil pour sequencer des polymeres avec une certitude statistique en utilisant un spectrometre de masse - Google Patents

Procedes et appareil pour sequencer des polymeres avec une certitude statistique en utilisant un spectrometre de masse Download PDF

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Publication number
WO1996036986A1
WO1996036986A1 PCT/US1996/007146 US9607146W WO9636986A1 WO 1996036986 A1 WO1996036986 A1 WO 1996036986A1 US 9607146 W US9607146 W US 9607146W WO 9636986 A1 WO9636986 A1 WO 9636986A1
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Prior art keywords
polymer
mass
fragments
agent
charge ratio
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PCT/US1996/007146
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English (en)
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WO1996036986B1 (fr
Inventor
Dale H. Patterson
George E. Tarr
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Perseptive Biosystems, Inc.
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Priority claimed from US08/447,175 external-priority patent/US5869240A/en
Application filed by Perseptive Biosystems, Inc. filed Critical Perseptive Biosystems, Inc.
Priority to JP08535084A priority Critical patent/JP2001500606A/ja
Priority to EP96916490A priority patent/EP0827628A1/fr
Publication of WO1996036986A1 publication Critical patent/WO1996036986A1/fr
Publication of WO1996036986B1 publication Critical patent/WO1996036986B1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
    • C07K1/128—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/0027—Methods for using particle spectrometers
    • H01J49/0036—Step by step routines describing the handling of the data generated during a measurement

Definitions

  • the present invention relates generally to methods and apparatus for sequencing polymers, especially biopolymers, using mass spectrometry.
  • Biochemists frequently depend on reliable and fast determinations of the sequences of biological polymers. For example, sequence information is crucial in the research and development of peptide screens, genetic probes, gene mapping, and drug modeling, as well as for quality control of biological polymers when manufactured for diagnostic and/or therapeutic applications.
  • Various methods are known for sequencing polymers composed of amino acids, carbohydrates and nucleotides.
  • existing methods for peptide sequence determination include the N-terminal chemistry of the Edman degradation, N- and C-terminal enzymatic methods, and C-terminal chemical methods.
  • Existing methods for sequencing oligonucleotides include the Maxam-Gilbert base-specific chemical cleavage method and the enzymatic ladder synthesis with dideoxy base-specific termination method. Each method possesses inherent limitations that preclude it being used exclusively for complete primary structure identification.
  • Edman sequencing and adaptations thereof are the most widely used tools for sequencing certain protein and peptides residue by residue, while the enzymatic synthesis method is preferred for sequencing oligonucleotides.
  • oligonucleotides In the case of both peptides and oligonucleotides, an alternate approach to chemical sequencing is enzymatic cleavage sequencing. In the case of oligonucleotides, over 150 different enzymes have been isolated and found suitable for preparing oligonucleotide fragments. In the case of peptides, serine carboxypeptidases have proven popular over the last two decades because they offer a simple approach by which amino acids can be sequentially cleaved residue by residue from the C-terminus of a protein or a peptide. Carboxypeptidase Y (CPY), in particular, is an attractive enzyme because it non-specifically cleaves all residues from the C-terminus, including proline. (See, e.g., Breddam et al. (1987) Carlsburg Res. Commun. 52:55-63.)
  • Sequencing of peptides by carboxypeptidase digestion has traditionally been performed by a laborious, direct analysis of the released amino acids, residue by residue. Not only is this approach labor-intensive, but it is complicated by amino acid contaminants in the enzyme and protein/peptide solutions, as well as by enzyme autolysis. A further hindrance to any sequencing effort of this type is the absolute requirement for good kinetic information concerning the hydrolysis and liberation of each individual residue by the particular enzyme used.
  • MALDI-TOF matrix-assisted laser desorption ionization time-of-flight
  • carboxypeptidase digestion of peptides can be combined with MALDI-TOF to analyze the resulting mixture of truncated peptide. For example, eight consecutive amino acids have been sequenced from the C-terminus of human parathyroid hormone 1-34 fragment (Schar et al. (1991) Chimia 45: 123-126). Additionally, carboxypeptidase digestion of peptides has been combined with other mass spectrometry methods such as plasma desorption (Wang et al. (1992) Techniques Protein Chemistry III (ed., R.H. Angeletti; Academic Press, N.Y.) pp. 503-515).
  • one aspect of the present invention is directed to an integrated method for sequencing polymers using information gathered by mass spectrometry, which substantially overcomes the problems encountered in the related art.
  • the invention provides a method for obtaining sequence information about a polymer comprising a plurality of monomers of known mass.
  • One skilled in the art first provides a set of fragments, created by the hydrolysis of the polymer, each set differing by one or more monomers. The difference between the mass-to-charge ratio of at least one pair of fragments is determined.
  • One then asserts a mean mass-to-charge ratio which corresponds to the known mass-to-charge ratio of one or more different monomers.
  • the asserted mean is compared with the measured mean to determine if the two values are statistically different with a desired confidence level. If there is a statistical difference, then the asserted mean difference is not assignable to the actual measured difference.
  • additional measurements of the difference between a pair of fragments are taken, to increase the accuracy of the measured mean difference. The steps of such a method are repeated until one has asserted all desired ⁇ s for a single difference between one pair of fragments. The method is repeated for additional pairs of fragments until the desired sequence information is obtained.
  • the claimed methods are applicable to any polymer, including biopolymers such as DNAs, RNAs, PNAs, proteins, peptides and carbohydrates and modified froms of these polymers.
  • the set of polymer fragments may be created by hydrolysis of the intermonomer bonds of the polymers.
  • the instant invention contemplates both naturally-occurring and synthetic moieties characterized by a series of different monomers.
  • the polymer also can be modified.
  • the invention also contemplates the inclusion of a hydrolyzing agent to cause the hydrolysis. Hydrolyzing agents may be enzymatic or an agent other than an enzyme, and any combinations thereof.
  • the method of obtaining sequence information about a polymer includes providing a set of polymer fragments created by hydrolyzing said polymer, each fragment differing by one or more monomers of known mass; measuring the mass- to-charge ratio difference x between a pair of fragments.
  • a mean difference ⁇ which is related to a known mass-to-charge ratio of one or more monomers, and selects a desired confidence level for ⁇ .
  • the step of measuring the mass-to-charge ratio difference x between a pair of fragments is repeated to obtain a number of measurements n, thereby to determine the statistical mean mass-to-charge ratio difference x between the pair of fragments measured.
  • the measured mean x one can then determine the standard deviation 5 of the measured mean mass-to-charge ratio difference x previously determined and calculate a test statistic t ca ⁇ Cu i ated with the following algorithm:
  • Sequence information for the polymer is obtained by repeating the steps of the method for additional pairs of fragments.
  • the present invention further provides a method of obtaining sequence information about a polymer comprising a series of different monomers which involves: on a reaction surface, providing at least one amount of a hydrolyzing agent which hydrolyzes said polymer and breaks inter-monomer bonds, and a sample of polymer to form differing ratios of agent to polymer; incubating the same for a time sufficient to obtain a plurality of series of hydrolyzed polymer fragments; performing mass spectrometry on a plurality of the series to obtain mass-to-charge ratio data for hydrolyzed polymer fragments contained in the series; and, as described above, integrating data from a plurality of the series to obtain sequence information characteristic of the polymer sample.
  • the instant invention contemplates certain embodiments involving hydrolyzing agents capable of hydrolyzing a polymer to form sequence-defining ladders, as well as certain other embodiments having hydrolyzing agents capable of forming polymer maps.
  • the instant invention provides for hydrolyzing the polymer with combinations of such agents, as well as enzymatic and non-enzymatic hydrolyzing agents.
  • the hydrolyzing agent is disposed on a reaction surface in an array of discrete separate zones.
  • sets of polymer fragments are sequenced by hydrolyzing the polymer on a reaction surface having one or more different amounts of a hydrolyzing agent.
  • a hydrolyzing agent is provided in spatially separate differing amounts on the reaction surface such that parallel concentration dependent hydrolysis occurs.
  • the hydrolyzing agent is disposed as a gradient.
  • the agent is disposed on the reaction surface in a constant amount.
  • polymer is similarly disposed on the reaction surface.
  • differing agent to polymer ratios are disposed upon the reaction surface and incubated to obtain a plurality of series of hydrolyzed polymer fragments. The various manners in which such differing ratios can be accomplished will be obvious to the skilled practioner.
  • a series of concentrations of hydrolyzing agent can be dispersed across a row of the ⁇ L wells of the sample plate of the VoyagerTM MALDI-TOF Biospectrometry Workstation, available from PerSeptive Biosystems, Inc. Following passive evaporation, matrix may be added to each well and the sample plate "read" with a MALDI-TOF mass spectrometer.
  • time-dependent and concentration-dependent digestions should yield analogous sequence information, it is preferred to use a concentration-dependent approach because it is easily automated, all samples are ready at the same time, and less sample material is lost due to transfer from reaction vessels to the analysis plate.
  • concentration-dependent on plate hydrolysis with subsequent analysis on a MALDI mass spec, because it requires only a few pmol of total peptide as a combined result of the sensitivity of MALDI and no sample loss upon moving from digestion to analysis.
  • a suitable light-absorbent matrix may be added to the polymer fragments at any time prior to measuring the mass-to-charge ratios.
  • matrix may be preloaded onto the reaction surface, or, alternatively, added to the hydrolyzing mixture, prior to, during, or after hydrolysis.
  • the method provides also combining the agent and polymer with other useful moieties.
  • moieties which selectively shift the mass of hydrolyzed fragments prior to mass spectrometry analysis are included.
  • moieties capable of improving ionization of hydrolyzed fragments are included.
  • the method provides for including a light-absorbent matrix.
  • the instant method also contemplates embodiments in which any one or more of the above-described moieties are combined with the agent and polymer prior to mass spectrometry analysis.
  • Other aspects of the instant invention are related to apparatus and kits for sequencing polymers.
  • the apparatus and kits of the invention in various embodiments include either a mass spectrometer associated with a computer responsive thereto, or a computer associated with a mass spectrometer.
  • the apparatus of the invention includes a mass spectrometer having a means for generating ions, a means for accelerating ions, and a means for determining ions.
  • the mass spectrometer is associated with a computer which is responsive to the mass spectrometer, wherein the computer has the means for performing the methods of the invention.
  • the apparatus of the invention in yet other embodiments includes a computer readable disc having thereon the information necessary to, in combination with a mass spectrometer, perform the methods of the invention.
  • the apparatus includes the computer itself, having means for performing the methods of the invention.
  • one embodiment of the apparatus of the instant invention involves a novel form of sample plate or sample holder for a mass spectrometer.
  • the sample plate or sample holder comprises a reaction surface with spacially separate areas having differing ratios of polymer and hydrolyzing agent. After a suitable incubation period during which the hydrolyzing agent hydrolyzes inter-monomer bonds within the polymer in each area, a plurality, typically all, of the areas containing hydrolyzed polymer fragments are ionized, typically serially, in the mass spectrometer and data representative of the mass to charge ratios of these fragments are obtained. One or more of the areas will have ratios of hydrolyzing agent to polymer suitable for more or less optimal generation of useful ladder elements or other polymer fragments.
  • Some areas on the sample holder may have overly hydrolyzed polymer fragments useless for deriving sequence information. Other areas may contain substantially unhydrolyzed polymer.
  • mass spectrometry analysis of all areas at least some mass to charge ratio data can be obtained from fragments generated in one or more areas.
  • the method of the invention obviates the necessity to empirically prepare samples to ascertain the appropriate ratio of hydrolyzing agent to polymer, as well as optimal reaction time and carefully controlled reaction temperature, heretofore required.
  • different hydrolyzing agents can be used in different series of areas on the sample holder so as to further generate useful hydrolyzed fragments, and the data from these may also be integrated to improve the sequencing process.
  • the mass spectrometer sample plate or sample holder has a planar solid surface with at least one amount of a hydrolyzing agent capable of hydrolyzing a polymer disposed thereon.
  • the hydrolyzing agent is disposed on the reaction surface in a dehydrated form.
  • the hydrolyzing agent is immobilized on the reaction surface.
  • the hydrolyzing agent is disposed on the reaction surface in the form of a liquid or gel which is resistant to physical dislocation.
  • a light-absorbent matrix is disposed on the surface of the sample holder. Additionally, any one or more of such embodiments of the sample holder may further have microreaction vessels on their surface.
  • sample holders are disposable. It is further contemplated that the reaction surface is fabricated from a variety of substrates and assumes a variety of configurations suitable for use with a mass spectrometer. As disclosed herein, all embodiments of the sample plate or sample holder are useful to adapt a mass spectrometry apparatus for sequencing a polymer.
  • peptide ladders created using the traditional solution-phase digestion approach i.e., aliquots of samples are removed at selected time intervals from enzymatic digests, suffer from a number of disadvantages. For example, large amounts of development time, enzyme and peptide are required to obtain significant digestion in a short amount of time while preserving all possible sequence information.
  • an alternative strategy is to perform the digestion on the MALDI sample surface.
  • the overall polymer sequencing effort is superior to the prior art time-dependent digestions in terms of: inherent simplicity of the method and elimination of laborious optimization requirements; reduced loss of sample due to transfer from reaction vessel to reaction surface; reduced amounts of enzyme and peptide used; and, particularly important for large-scale application, ease of use/automation.
  • the mass spectrometry sample plate or sample holder of the instant invention provides advantages heretofore unavailable to the skilled practitioner. For example, certain embodiments minimize reagent handling and greatly facilitate sample processing. The skilled practitioner need only provide a sample of polymer. Virtually all other experimental parameters are pre-optimized.
  • FIGURE 1 is an exemplary sample plate or sample holder for MALDI analysis.
  • the wells serve as micro-reaction vessels in which on-plate digestions may be performed.
  • the physical dimensions of the plate are 57 x 57 mm and the wells are 2.54 mm in diameter.
  • FIGURES 2A, 2B and 2C depict several MALDI spectra from a time-dependent CPY digestion of ACTH 7-38 fragment [FRWGKPVGKKRRPVKVYPNGAEDESAEAFPLE] (SEQ. ID. No. 22) at 1 min (2A), 5 min (2B) and 25 min (2C).
  • the nomenclature of the peak labels denotes the peptide populations resulting from the loss of the indicated amino acids. Peaks representing the loss of 19 amino acids from the C-terminus are observed.
  • FIGURE 3 is a MALDI mass spectrum representing pooled 15 s, 105 s, 6 min and 25 min quenched aliquots from a time-dependent CPY digestion of ACTH 7-38 fragment. All amino acid losses are observed except for those of Glu(28), Asn(25), and Pro(24) which were present as small peaks in the 6 min aliquot and subsequently diluted to undetectable concentrations in this pooled fraction. All conditions are stated in the text
  • FIGURES 4A and 4B depict various MALDI spectra from on-plate digestions of ACTH 7-38 fragment at various concentrations of Carboxypeptidase Y (CPY): 6.10 x 10 "4 U/ ⁇ L (4A); 1.53 x 10 ⁇ U/ ⁇ L (4B).
  • Panels A and B show the spectra obtained from digests using CPY concentrations of 6.10 x 10 "4 and 1.53 x 10 '3 Units/ ⁇ L, respectively.
  • Laser powers significantly above threshold were used to improve the signal-to-noise ratio of the smaller peaks in the spectrum at the expense of peak resolution.
  • FIGURES 5 A, 5B, and 5C depict various MALDI spectra of the following three selected peptides: osteocalcin 7-19 fragment [GAPVPYPDPLEPR] (SEQ. ID. No. 13) (5A), angiotensin 1 [DRVYLHPFHL] (SEQ. ID. No. 8) (5B), and bradykinin [RPPGFSPFR] (SEQ. ID. No. 5) (5C) resulting from on-plate digestions using CPY concentrations of 3.05 x 10 "3 , 3.05 x 10 "4 , and 6.10 x 10 "4 Units/ ⁇ L, respectively.
  • FIGURES 6A-6E depict various MALDI spectra of exonuclease hydrolysis of a nucleic acid polymer (SEQ. ID. No. 23) at various concentrations of Phosphodiesterase I (Phos I): 0.002 ⁇ U/ ⁇ L (6A); 0.005 ⁇ U/ ⁇ L (6B); 0.01 ⁇ U/ ⁇ L (6C); 0.02 ⁇ U/ ⁇ L (6D); 0.05 ⁇ U/ ⁇ L (6E).
  • Phos I Phosphodiesterase I
  • FIGURE 7 depicts a MALDI spectrum of a hydrolyzed nucleic acid polymer (SEQ. ID. No. 23) combined with a light-absorbent matrix.
  • the instant invention relates to methods, kits and apparatus for sequencing polymers using mass spectrometry.
  • the present invention provides an integrated strategy for obtaining sequence information about a polymer comprising a plurality of monomers of known mass. Specifically, using sets of polymer fragments and mass spectrometry, the invention provides a method of interpretation of sequence data obtained by mass spectrometry which allows the rapid, automated and cost effective sequencing of polymers with a statistical certainty.
  • the present invention further provides methods wliich utilize polymers and hydrolyzing agents disposed upon a reaction surface. The hydrolyzing agents are enzymatic or non-enzymatic. The hydrolyzing agents react with the polymer to produce sequence-defining polymer ladders or polymer maps.
  • the methods of this invention further involve the step of obtaining mass spectrometry data relating to hydrolyzed polymer series and integrating the data from a plurality of polymer series to determine the polymer sequence.
  • the mass spectrometry method of this invention is applicable to all manner of ion formation and all modes of mass analysis.
  • the kits and apparatus of this invention relate, in part, to a mass spectrometer sample plate or sample holder for adapting a mass spectrometer to obtain sequence information about a polymer in accordance with the method of the instant invention.
  • the sample plate has disposed thereon hydrolyzing agent, in dehydrated, immobilized, liquid and/or gel form, and/or a light-absorbent matrix.
  • certain of the sample plates of the instant invention are disposable.
  • Other embodiments of the apparatus of the instant invention relate to mass spectrometers, computers and computer discs suitable for use with the aforementioned methods of sequencing polymers.
  • a "polymer” is intended to mean any moiety comprising a series of different monomers suitable for use in the method of the instant invention. That is, any moiety comprising a series of different monomers whose intermonomer bonds are susceptible to hydrolysis are suitable for use in the method disclosed herein.
  • a peptide is a polymer made up of particular monomers, i.e., amino acids, which can be hydrolyzed by either enzymatic or chemical agents.
  • a DNA is a polymer made up of other monomers, i.e., bases nucleotides, which can be hydrolyzed by a variety of agents.
  • a polymer can be a naturally-occurring moiety as well as a synthetically-produced moiety.
  • the polymer is a biopolymer selected from, but not limited to, the following group: proteins, peptides, DNAs, RNAs, PNAs (peptide nucleic acids), carbohydrates, and modified versions thereof
  • Sequence information as used herein is intended to mean any information relating to the primary arrangement of the series of different monomers within the polymer, or within portions thereof. Sequence information includes information relating to the chemical identity of the different monomers, as well as their particular position within the polymer. Polymers with known primary sequences, as well as polymers with unknown primary sequences, are suitable for use in the methods of the instant invention. It is contemplated that sequence information relating to terminal monomers as well as internal monomers can be obtained using the methods disclosed herein. In certain applications, sequence information can be obtained using a sample of an intact, complete polymer. In other applications, sequence information can be obtained using a sample containing less than the intact complete polymer, for example, polymer fragments.
  • polymer fragments can be naturally-occurring, artifacts of isolation and purification, and/or generated in vitro by the skilled artisan. Additionally, polymer fragments can be initially derived from and prepared by a variety of fractionation and separation methods, such as high performance liquid chromatography, prior to use with the methods of the instant invention.
  • reaction surface of the instant method includes any surface suitable for hydrolyzing the subject polymer with the subject agent.
  • the reaction surface can be fabricated from a variety of substrates, such as but not limited to: metals, foils, plastics, ceramics, and waxes. All reaction surfaces must be suitable for use with a mass spectrometer apparatus.
  • the reaction surface of the instant invention can assume any configuration suitable for use with a particular mass spectrometer apparatus.
  • the reaction surface can be a planar solid surface.
  • the surface may have microreaction vessels disposed thereon.
  • the reaction surface can assume the configuration of a probe suitable for use with certain mass spectrometer apparatus.
  • the reaction surface can be activated and/or derivatized to enhance or facilitate polymer sequencing in accordance with the instant invention.
  • the instant invention relates to a method of data analysis of the mass-to-charge ratios obtained by mass spectrometry. As exemplified below in further detail, the method provides a set of fragments, created by hydrolysis of the polymer, each set differing by one or more monomers. The difference between the mass-to-charge ratio of at least one pair of fragments is determined. One then asserts a mean mass-to-charge ratio which corresponds to the known mass-to-charge ratio of one or more different monomers.
  • the asserted mean is compared with the measured mean to determine if the two values are statistically different with a desired confidence level. If there is a statistical difference, then the asserted mean difference is not assignable to the actual measured difference. In some embodiments, additional measurements of the difference between a pair of fragments are taken, to increase the accuracy of the measured mean difference. The steps of the method are repeated until one has asserted all desired mean differences for a single difference between one pair of fragments.
  • the claimed invention is an integrated method for generating sequence information about a polymer comprising a plurality of monomers of known mass.
  • the method involves the interpretation of mass-to-charge ratio data of a set of fragments obtained from the polymer, to statistically identify monomer differences between pairs of fragments.
  • known molecular masses have been compared to MALDI derived masses for a few mass measurements, and researchers have attempted to make general statements on the instrumental mass accuracy.
  • the methods of the claimed invention involve multiple integrated steps which may be automated according to the invention.
  • the difference, x between the mass-to-charge ratio of at least one pair of fragments is measured.
  • ⁇ corresponds to a known mass-to-charge ratio of one or more differing monomers.
  • analyses x analyses x to determine if it is statistically different from the ⁇ with a selected confidence level.
  • the asserted ⁇ is not assignable to the mass difference x with the selected confidence level. The steps described above are repeated until all desired ⁇ s have been asserted, and then can be repeated for additional pairs of fragments.
  • the analysis to determine if x is statistically different from ⁇ comprises taking repeated measurements of x, a number of times n, to determine a measured mean mass-to-charge ratio difference x between at least one pair of fragments. A standard deviation s of the measured mean x can then be determined, and the measured mean x compared to the asserted mean ⁇ to determine if they are statistically different with the desired confidence level.
  • a set of polymer fragments are obtained, either by on plate digestion, or from an external source, and one or more measurements of the mass-to-charge ratio of a pair of the fragments are taken. Peaks representing the loss of one or more monomers can be analyzed using t-statistics to allow assignments to be made with a desired confidence interval. The two-tailed t-test for one experimental mean,
  • x is the experimental mean mass difference
  • ⁇ is the asserted mass difference
  • N is the number of replicates performed
  • 5 is the experimental standard deviation of the mean
  • this technique is to be used for the sequence determination of peptides of unknown sequence.
  • researchers have attempted to make general statements of instrumental mass accuracy (e.g. better than 0.1%). Ascribing this mass accuracy to any individual mass measurement for the purpose of residue assignment holds no statistical validity, therefore making true residue assignment and direct application to unknowns difficult.
  • statistical levels of confidence must be placed on residue assignments.
  • the above-described method of integrating data can further comprise the steps of: providing, on a reaction surface, at least one amount of hydrolyzing agent which hydrolyzes a polymer to break intermonomer bonds aud produce a set of polymer fragments, and a sample of the polymer such that differing ratios of agent to polymer are formed on the reaction surface; incubating the combined polymer and agent for a time sufficient to obtain a plurality of series of hydrolyzed polymer fragments; and, performing mass spectrometry on a plurality of the series to obtain mass-to-change ratio data.
  • a set of polymer fragments created by the endohydrolysis of a polymer can be used to practice the instant invention.
  • the use of an endohydrolase creates a set of fragments defining a map of said polymer.
  • the mass-to-charge ratio of the fragments is measured, and a hypothetical identity is asserted for the fragment measured.
  • the hypothetical identity corresponds to a known identity of a fragment of a reference polymer.
  • Information on reference polymers is easily included in a database to be used with this method. After selecting a desired confidence level, one determines whether the mass-to-charge ratio of the asserted hypothetical fragment is statistically different from the mass-to-charge ratio of the asserted hypothetical fragment.
  • the steps are repeated for different additional hypothetical fragments. This method is repeated until sufficient information is obtained about the fragments that one can identify the polymer with a desired confidence level.
  • one essentially determines whether the fragments of the polymer corresponds to fragments of a known polymer with enough certainty to identify the polymer. It is preferable that the hypothetical identities which are asserted correspond to a known identity derived from a computer database of known sequences.
  • the methods of the invention also contemplate providing multiple different sets of fragments of the same polymer, i.e. maps and ladders, to obtain the maximum amount of sequence information possible.
  • the sets of polymer fragments can be created by any method.
  • Certain of the claimed methods contemplate the step of hydrolyzing the polymer with a hydrolyzing agent to obtain the fragments, or synthesizing fragments, as well as merely providing a set of fragments which have been obtained previously.
  • the term "hydrolyzing agent” is intended to mean any agent capable of disrupting inter-monomer bonds within a particular polymer. That is, any agent which can interrupt the primary sequence of a polymer is suitable for use in the methods disclosed herein.
  • Hydrolyzing agents can act by liberating monomers at either termini of the polymer, or by breaking internal bonds thereby generating fragments or portions of the subject polymer.
  • a preferred hydrolyzing agent interrupts the primary sequence by cleaving before or after a specific monomer(s); that is, the agent specifically interacts with the polymer at a particular monomer or particular sequence of monomers recognized by the agent as the preferred hydrolysis site within the polymer. All of the currently preferred hydrolyzing agents described herein are commercially available from reagent suppliers such as Sigma Chemicals (St. Louis, MO).
  • an excipient is added to, and used in conjunction with, the hydrolyzing agent.
  • the excipients contemplated herein facilitate lyophilization and/or dissolution of the hydrolyzing agent.
  • fucose and other sugars suitable for use with the instant invention are contemplated. Suitable for use is intended to mean that no interference with mass spectrometry is encountered by the use thereof.
  • Other excipients useful in the instant invention are pH modifiers, such as ammonium acetate.
  • Still other excipients suitable for use in the methods and apparatus disclosed herein are those which act as stabilizers of the integrity of the hydrolyzing agent. With respect to excipients, the identity of those suitable will be obvious to the skilled artisan using only routine experimentation. While certain preferred excipients are described above, identification of suitable equivalents is within the skill of the ordinary artisan.
  • the hydrolyzing agent is a hydrolase enzyme.
  • Some hydrolases are endohydrolases, others are exohydrolases.
  • the particular hydrolase used is determined by the nature of the polymer and/or the type of sequence information desired. Its identity can be readily determined by the skilled artisan using no more than routine experimentation.
  • currently preferred endohydrolases include but are not limited to: endonucleases, endopeptidases, endoglycosidases, trypsin, chymotrypisin, endoproteinase Lys-C , endoproteinase Arg-C , and thermolysin.
  • exohydrolases include but are not limited to: exonucleaes, exoglycosidases, and exopeptidases.
  • the currently preferred exonucleases include, but are not limited to: phosphodiesterase types I and II, exonuclease VII, ⁇ -exonuclease, T7 gene 1 exonuclease, exonuclease III, BAL-31, exonuclease I, exonuclease V, exonuclease II, and DNA polymerase III.
  • exoglycosidases include, but are not limited to: ⁇ -mannosidase I, ⁇ -mannosidase, ⁇ -hexosaminidase, ⁇ -galactosidase, ⁇ - fucosidase I, ⁇ -fiicosidase II, ⁇ -galactosidase, ⁇ -neuraminidase, ⁇ -glucosidase I and ⁇ - glucosidase II.
  • exopeptidases include, but are not limited to: carboxypeptidase Y, carboxypeptidase A, carboxypepetidase B, carboxypeptidase P, a inopeptidase 1, LAP, proline aminodipeptidase, leucine amino peptidase, and cathepsin C.
  • the hydrolyzing agent is an agent other than an enzyme.
  • an agent can be a chemical, such as an acid.
  • preferred agents other than an enzyme include but are not limited to: cyanogen bromide, hydrochloric acid, sulfuric acid, and pentafluoroproprionic fluorohydride.
  • hydrolysis can be accomplished using partial acid hydrolysis in accordance with the methods disclosed herein. Again, the identity of a hydrolyzing agent other than an enzyme will be determined by the nature of the polymer and the type of sequence information desired. It is within the skilled practitioner's ability to identify a suitable agent, as well as the circumstances under which such an agent is preferred.
  • the instant method further provides for use of combinations of the above-described individual hydrolyzing agents.
  • combinations of enzymes can be used in the claimed invention.
  • Combinations of hydrolyzing agents other than enzymes can also be used.
  • combinations of enzymes with agents other than enzymes can also be used in the instant method. Again, the exact combination and the circumstances under which such a combination is appropriate will depend upon the nature of the polymer and the sequence information desired. The skilled practitioner will know when combinations of hydrolyzing agents are suitable for use in the methods disclosed herein.
  • hydrolyzing agent/polymer sequence-specific interactions are well known in the art.
  • polymers such as proteins and DNAs specifically interact with proteinases and nucleases, respectively.
  • Certain of the preferred proteinases specifically recognize the C-terminus (carboxypeptidase Y) or the N- terminus (amino peptidase 1) of a protein's amino acid sequence.
  • Certain of the preferred nucleases specifically recognize the 5' or the 3' terminus of a polynucleotide' s base sequence.
  • the claimed invention can be applied to the sequencing of any natural biopolymer such as proteins, peptides, nucleic acids, carbohydrates, etc., as well as synthetic biopolymers such as PNA and phosphotiolated nucleic acids.
  • the ladders could conceivably be created enzymatically using exohydrolases, endohydrolases or the Sanger method and/or chemically by truncation synthesis or failure sequencing. It is preferable to use on-plate digestion and interpretation of peptide ladders created from carboxypeptidase Y, carboxypeptidase P and aminopeptidase I digestions of numerous peptides.
  • exohydrolases generate a series of hydrolyzed fragments comprising a sequence-defining "ladder" of the polymer. That is, these agents generate a series of hydrolyzed fragments, each hydrolyzed fragment within the series being a "ladder element,” which collectively comprise a sequence-defining "ladder” of the polymer.
  • Ladder elements represent hydrolyzed fragments from which monomers have been consecutively and/or progressively liberated by the exohydrolase acting at one or the other of the polymer's termini. Accordingly, ladder elements are truncated hydrolyzed polymer fragments, and ladders per se are concatenations of these collective truncated hydrolyzed polymer fragments.
  • sequence information relating to the amino acid sequence of a protein can be obtained using carboxypeptidase Y, an agent which acts at the carboxy terminus.
  • hydrolyzing agents other than exohydrolases which also act at one or the other of a polymer's termini generate ladder elements which collectively comprise a series of sequence- defining ladders.
  • the well-known Edman degradation technique and associated reagents can be adapted for use with the methods of the instant invention for this purpose.
  • the above-described subtractive-type sequencing method through which repetitive removal of successive amino-terminal residues from a protein polymer can occur, can also be accomplished with hydrolyzing agents other than enzymes as disclosed herein.
  • sequence information can also be obtained using hydrolyzing agents which act to disrupt internal inter-monomer bonds.
  • an endohydrolase can generate a series of hydrolyzed fragments useful ultimately in constructing a "map" of the polymer. That is, this agent generates a series of related hydrolyzed fragments which collectively contribute information to a sequence-defining "map" of the polymer.
  • peptide maps can be generated by using trypsin endohydrolysis in tandem with cyanogen bromide endohydrolysis to obtain hydrolyzed fragments with overlapping amino acid sequences. Such overlapping fragments are useful for reconstructing ultimately the entire amino acid sequence of the intact polymer.
  • this combination of hydrolyzing agents generates a useful plurality of series of hydrolyzed fragments because trypsin specifically catalyzes hydrolysis of only those peptide bonds in which the carboxyl group is contributed by either a lysine or an arginine monomer, while cyanogen bromide cleaves only those peptide bonds in which the carbonyl group is contributed by methionine monomers.
  • trypsin and cyangogen bromide hydrolysis in tandem, one can obtain two different series of hydrolyzed "mapping" fragments.
  • mapping fragments are then examined by mass spectrometry to identify specific hydrolysates from the second cyanogen bromide hydrolysis whose amino acid sequences establish continuity with and/or overlaps between the specific hydrolysates from the first hydrolysis with trypsin. Overlapping sequences from the second hydrolysis provide information about the correct order of the hydrolyzed fragments produced by the first trypsin hydrolysis. While these general principles of peptide mapping are well-known in the prior art, utilizing these principles to obtain sequence information by mass spectrometry as disclosed herein has heretofore been unknown in the art.
  • a sample of polymer includes biological fluids containing (or suspected to contain) the polymer of interest.
  • a sample of polymer is also intended to include isolated and purified polymer. Additionally, a sample of polymer can be aqueous or non-aqueous.
  • Adding a sample of polymer to the reaction surface can be accomplished in a variety of ways.
  • the sample can be introduced as individual aliquots, or the sample can be introduced in a continuous mode such as sample eluting from a preparative or qualitative column. In both cases, the sample can be introduced manually or by automated means.
  • the instant method Upon adding a sample of polymer and hydrolyzing agent to the reaction surface, the instant method provides that differing concentrations of agent or ratios of agent to polymer are formed on said reaction surface. For example, if the polymer sample contains a uniform amount of polymer, then the method contemplates that differing amounts of agent be disposed on the reaction surface. This would produce differing agent to polymer ratios.
  • the differing amounts of agent can be in the form of discrete separate zones to which a constant amount of polymer is added. Alternatively, the differing amounts of agent can be in the form of a non-discrete gradient of agent ranging from low to high amounts of agent, perhaps in the form of strip of appropriate length and width.
  • differing agent to polymer ratios are produced.
  • the agent and polymer can assume any configuration and be present in any amount(s); all that is required is that the combination of agent and polymer results in differing ratios of the same disposed on the reaction surface.
  • differing ratios of agent to polymer can also be accomplished by disposing a constant amount of agent on the reaction surface and adding varying amounts of polymer, e.g., a polymer gradient or discrete separate zones of differing amounts of polymer or polymer solution.
  • polymer gradient polymer eluted from a column in the form of a gaussian-distributed gradient is currently preferred.
  • the instant method further provides for incubating the above-described agent to polymer ratios for a time required to obtain the requisite plurality of series of hydrolyzed polymer fragments.
  • Incubating can proceed under any conditions suitable for hydrolyzing the polymer and for any amount of time required to obtain a plurality of series of hydrolyzed fragments.
  • the disclosed methods permit sequencing information to be obtained in relatively short time periods, for example, in less than 1 hour.
  • the incubation time can be shortened or lengthened depending upon the nature of the polymer and/or hydrolyzing agent(s). It will be obvious to one skilled in the art how to identify appropriate incubation times and optimize the same. Incubation reactions can be terminated by evaporation.
  • a "plurality of series" of hydrolyzed polymer fragments is intended to mean that hydrolyzed fragments are produced by at least two different agent:polymer ratios, and that each agen polymer ratio generates a series of hydrolyzed fragments. For example, if a constant amount of polymer is added to two separate zones of agent containing different amounts of agent, each zone represents one agen polymer ratio and each zone produces one series of hydrolyzed fragments. When taken together, the two zones are a plurality which collectively contain a plurality of series of hydrolyzed polymer fragments.
  • the instant methods teach obtaining sequence information by performing mass spectrometry on a plurality of series of hydrolyzed fragments to obtain mass-to-charge ratio data for hydrolyzed polymer fragments contained therein. This contemplates that at least two different agent.polymer ratios be provided and analyzed by mass spectrometry.
  • the claimed invention may be practiced using any type of mass spectrometry known in the art.
  • any manner of ion formation can be adapted for obtaining mass-to-charge ratio data, including but not limited to : matrix-assisted laser desorption ionization, plasma desorption ionization, electrospray ionization, thermospray ionization, and fast atom bombardment ionization.
  • any mode of mass analysis is suitable for use with the instant invention including but not limited to: time-of-flight, quadrapole, ion trap, and sector analysis.
  • a currently preferred mass spectrometer instrument is an improved time-of-flight instrument which allows independent control of potential on sample and extraction elements, as described in copending U.S.S.N.
  • the mass spectrometers used to practice the instant invention include a means to generate ions, a means to accelerate ions, and, a means to detect ions.
  • Any ionization method may be used, for example, desorption, negative ion fast atom bombardment, matrix- assisted laser desorption and electrospray ionization. It is preferable to use matrix-assisted laser desorption mass spectrometry.
  • any of the methods of the instant invention as described herein can further comprise the step of eluting from a liquid chromatography column a sample comprising a polymer or polymer fragments for which sequence information is to be obtained.
  • the sample eluted from the column is rendered compatible with a mass spectrometer by contact with a suitable buffer prior to the step of determining mass to charge ratio.
  • the method of the instant invention also provides for including moieties useful in mass spectrometry.
  • a light-absorbent matrix can be introduced at any point prior to performing mass spectrometry analysis by laser desorption.
  • Light-absorbent matrices are particularly useful for analysis of biopolymers.
  • Matrix-assisted laser desorption ionization techniques, as well as various matrices suitable therefor, are well known in the art and have been described, for example, in U.S. 5,288,644 (issued February 22, 1994) and U.S.S.N. 08/156,316 (Atty. Docket No. Vestec-14-2, allowed April 18, 1995), the disclosures of which are herein incorporated by reference.
  • moieties useful in the instant method include those capable of selectively shifting the mass of certain hydrolyzed fragments. These, too, can be added at any point prior to mass spectrometry analysis.
  • mass-shifting moieties include, but are not limited to, those moieties which produce reaction products such as: alkyl, aryl, alkenyl, acyl, thioacyl, oxycarbonyl, carbamyl, thiocarbamyl, sulfonyl, imino, guanyl, ureido, and silyl reaction products. Attachment of such moieties to hydrolyzed polymers is achieved using art-recognized attachment chemistries. The particular moiety best suited to a particular sequence determination will depend upon the nature of the polymer and the hydrolyzed fragments. The skilled artisan will be able to determine which moiety to use, if any.
  • moieties suitable for use with the instant method are those which can improve ionization of hydrolyzed fragments. Such moieties can be introduced at any time prior to mass spectrometry analysis.
  • ionization-improving moieties include, but are not limited to, those moieties which produce reaction products such as: amino, quarternary amino, pyridino, imidino, guanidino, oxonium, and sulfonium reaction products. Preparation and/or use of such moieties are well known in the art.
  • the instant invention provides a mass spectrometer sample plate or sample holder.
  • sample plate and “sample holder” are used synonymously.
  • the instant sample plate is useful for adapting any mass spectrometer apparatus for obtaining sequence information in accordance with the disclosed methods.
  • the sample holder has a planar solid surface on which is disposed hydrolyzing agent.
  • the sample holder has the form of a probe useful in certain mass spectrometer apparatus.
  • the agent can be in dehydrated, immobilized, liquid and/or gel form.
  • the agent is resistant to physical dislocation and is chemically stable for at least about one to two months, thereby facilitating both transport and storage. These considerations are particularly useful for commercial applications involving the sample plate of the present invention.
  • the agent can be disposed in separate discrete zones of differing amounts, or in a non-discrete gradient. Alternatively, the agent can be disposed in a constant amount on the surface of the sample plate.
  • the sample plate has a light- absorbent matrix disposed on its surface; this can be with or without hydrolyzing agent.
  • At least one amount of a dehydrated agent capable of hydrolyzing a polymer is disposed on the planar solid surface of the sample plate.
  • at least one amount of an immobilized agent capable of hydrolyzing a polymer can be disposed thereon.
  • the sample plate has disposed thereon at least one amount of a hydrolyzing agent in liquid or gel form, said liquid or gel form being resistant to physical dislocation.
  • the sample plate can also have microreaction vessels arranged on its surface. In one embodiment, these vessels can be depressions on the plate's surface resulting from chemical- etching or similar techniques.
  • the sample plate can be fabricated from a variety of substrates including but not limited to: metals, foils, plastics, ceramics, and waxes.
  • the sample plate is disposable.
  • the sample plate disclosed herein is a component of a kit useful for sequencing polymers by mass spectrometry.
  • the surface can comprise an array of discrete separate zones of differing amounts of said agent.
  • the surface comprises a non-discrete gradient of said agent or a constant amount of said agent.
  • any embodiment can further comprise a light-absorbent matrix, and/or microreaction vessels, and/or be fabricated of a disposable material.
  • the instant invention provides a kit having a sample plate or holder comprising a reaction surface, said surface providing differing amounts of a hydrolyzing agent to hydrolyze said polymer into said fragments.
  • the kit contains a sample plate or holder further comprising a matrix suitable for matrix-assisted laser desorption mass spectrometry.
  • the claimed invention also relates to other mass spectrometer apparatus and kits for performing the methods above.
  • the apparatus of the invention for obtaining sequence information about a polymer comprises a mass spectrometer having a means for generation ions from a sample, a means for acceleration of ions generated, and a detection means.
  • the apparatus additionally comprises a computer responsive to the mass spectrometer comprising a means for determining the mass to charge ratio difference x between a pair of polymer fragments; a means for asserting a mean difference ⁇ between the mass-to-charge ratio of the pair of fragments, wherein ⁇ corresponds to a known mass-to-charge ratio of one or more monomers; and a means for analyzing x to determine if it is statistically different from ⁇ with the desired confidence level, and a means for determining when the desired number of possible ⁇ s have been asserted.
  • a computer responsive to the mass spectrometer comprising a means for determining the mass to charge ratio difference x between a pair of polymer fragments; a means for asserting a mean difference ⁇ between the mass-to-charge ratio of the pair of fragments, wherein ⁇ corresponds to a known mass-to-charge ratio of one or more monomers; and a means for analyzing x to determine if it is statistically different from ⁇ with the desired confidence
  • the information necessary for the claimed methods can be incorporated onto a computer-readable disc, which can render a computer responsive to a mass spectrometer for performing the analysis.
  • Claimed software will automate the process of acquiring and interpreting the data in an intelligent fashion using software feedback control.
  • the data interpretation software would control the number of acquisitions (minimum of 2) that are required to statistically differentiate multiple candidates for an amino acid assignment. The operator would have control of specifying to what minimum statistical level of confidence the assignment(s) must meet.
  • Aliquots of 1 ⁇ L were taken from the reaction vial at reaction times of 15 s, 60 s, 75 s, 105 s, 2 min, 135 s, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15 min and 25 min. At 25 min, 15 ⁇ L of 5 x 10 "3 units/ ⁇ L CPY was added to the reaction vial.
  • a pooled peptide solution was prepared by combining 2 ⁇ L of the 15 s, 105 s, 6 min and 25 min aliquots. Into individual ⁇ L wells on the MALDI sample plate, 1 ⁇ L of each aliquot solution was placed and allowed to evaporate to dryness before insertion into the mass spectrometer.
  • All on-plate digestions were performed by pipetting 0.5 ⁇ L of the peptide at a concentration of 1 pmol/ ⁇ L into each often 1 ⁇ L wells across one row of a sample plate configured similarly to the sample plate manufactured and supplied by PerSeptive Bio Systems, Inc. of Framingham, MA and adapted for use with their trademarked mass spectrometry apparatus known as VoyagerTM. All peptides listed in Table 1 were purchased from Sigma and were of the highest purity offered. To initiate the reaction in the first well, 0.5 ⁇ L of 0.0122 units/ ⁇ L CPY was added.
  • MALDI-TOF mass analysis was performed using the VoyagerTM BiospectrometryTM Workstation (PerSeptive Biosystems, Cambridge, MA). A 28.125 KV potential gradient was applied across the source containing the sample plate and an ion optic accelerator plate in order to introduce the positively charged ions to the 1.2 m linear flight tube for mass analysis.
  • a low mass gate was used to prevent the matrix ions from striking the detector plate.
  • the guide wire was pulsed for a brief period deflecting the low mass ions (approximately ⁇ 1000 daltons). All other spectra were recorded with the low mass gate off.
  • Figure 2 illustrates the MALDI spectra of the 1 min, 5 min and 25 min time aliquots that were removed from a solution-phase time-dependent CPY digestion of ACTH 7-38 fragment.
  • the nomenclature of the peak labels denotes the peptide populations resulting from the loss of the indicated amino acids. Peaks representing the loss of 19 amino acids from the C-terminus are observed.
  • the prior-art time-dependent method presented herein is the result of extensive method optimization and is optimized for obtaining the maximum sequence information in the shortest amount of time. For this particular optimized case, detectable amounts of all populations were observed over 25 min in the three selected time aliquots. This was not the case for numerous preliminary solution-phase digestions that were performed during the method optimization that led to the choice of these optimized conditions. At higher concentrations of CPY the peaks representing the loss of Glu(28) and Pro(24) were often not observed, indicating that CPY cleaves these residues very readily when alanine and tyrosine are at the penultimate positions, respectively.
  • solution-phase digestion suffers from a number cf disadvantages.
  • a large amount of time, enzyme and peptide is required for method optimization in order to obtain significant digestion in a short amount of time while preserving all possible sequence information.
  • For each peptide from which sequence information is to be derived some time-consuming method development must be performed since a set of optimum conditions for one peptide is not likely to be useful for another peptide given the composition-dependent hydrolysis rates of CPY.
  • An alternative strategy is to perform the concentration-dependent hydrolysis on the MALDI sample surface as described below.
  • Figure 1 depicts a VoyagerTM sample plate for MALDI analysis comprised of a 10 x 10 grid of 1 ⁇ L wells etched into the stainless steel base. These wells serve as micro-reaction vessels in which on-plate digestions may be performed. The physical dimensions of the plate are 57 x 57 mm and the wells are 2.54 mm in diameter.
  • MALDI spectra corresponding to the on-plate concentration dependent digestions of the ACTH 7-38 fragment for CPY concentrations of 6.10 x 10 "4 , and 1.53 x 10 "3 units/ ⁇ L, respectively, are illustrated in panels A and B of Figure 4.
  • Panel A and B show the spectra obtained from digests using CPY concentrations of 6.10 x 10 "4 and 1.53 x 10 "3 units/ ⁇ L, respectively.
  • Laser powers significantly above threshold were used to improve the signal-to-noise ratio of the smaller peaks in the spectrum at the expense of peak resolution.
  • T e symbol * indicates doubly charged ions and # indicates an unidentified peak at m/z - 2517.6 daltons.
  • the lower concentration digestion yielded 12 significant peaks representing the loss of 11 amino acids from the C-terminus.
  • the digestion from the higher concentration of CPY showed some overlap of the peptide populations present at the lower concentration as well as peptide populations representing the loss of amino acids through the Val(20).
  • the concentration of the peptides representing the loss of the first few amino acids have decreased to undetectable levels (approximately ⁇ 10 fmol) with the exception of the Leu(37) peak.
  • the ACTH 7-38 fragment sequence can be read 19 amino acids from the C- terminus without gaps, stopping at the same amino acid run of peptide-RRKKP as the time- dependent digestion.
  • Figure 4 represents 2 of the 9 CPY concentrations that were performed simultaneously.
  • Angiogenin 20 ENGLPYHLDQSI(FR)R 1781.0 +0.5 mid
  • the bolded amino acids indicate that a peak representing the loss of that residue was observed in one or more of the MALDI spectra taken across the row of digestions. In order to be able to identify a residue, the peak representing the loss of that amino acid and the preceding amino acid must be present.
  • the residues that are enclosed in parenthesis are those for which the sequence order could not be deduced.
  • CPY offered some sequence information from the C-terminus for most of the peptides digested, lending no sequence information in only three of the 22 cases. In two of these three cases, the C-terminus was a lysine followed by an acidic residue at the penultimate position. CPY has been reported to possess reduced activity towards basic residues at the C-terminus, and the presence of the neighboring acidic residue seems to further reduce its activity. In the case of the lutenizing hormone releasing hormone (LH-RH), the C-terminal amidated glycine followed by proline at the penultimate position inhibited CPY activity which agrees with reports of CPY slowing at both proline and glycine residues (Hayashi et al.
  • CPY is known to hydrolyze amidated C- terminal residues of dipeptides and is shown here to cleave those of physalaemin, kassinin, subtance P, bomesin, and ⁇ -MSH.
  • CPY was able to derive sequence information from all of the peptides, except LH-RH, that possess blocked N-terminal residues (physalaemin, bombesin and ⁇ -MSH). This is significant as these peptides would lend no information to the Edman approach. A number of the peptides were sequenced until the detection of the truncated peptide peaks were impaired by the presence of CHCA matrix ions ( ⁇ 600 daltons). The sequencing of the other peptides did not go as far as a combination of residues at the C-terminus and penultimate position that inhibited CPY activity were encountered.
  • Figure 5 shows selected on-plate digestions of osteocalin 7-19 fragment, angiotensin 1 and bradykinin resulting from on-plate digestions using CPY concentrations of 3.05 x 10 "3 , 3.05 x 10 " , and 6.10 x 10 "4 units/ ⁇ L, respectively.
  • Na denotes a sodium adduct peak
  • # denotes a matrix peak at m/z - 568.5 daltons.
  • Bradykinin is shown to sequence until the matrix begins to interfere with peak detection. For all three of the selected peptides, the total sequence information obtained for the overall 9 well digestion is represented in the single digestion shown. For many other peptides this was not the case. The total sequence information is often derived from 2 or more of the wells as is the case with ACTH 7-38 fragment given in Figure 4.
  • the interpretation of data utilized an automated process of acquiring and interpreting the data using software feedback control.
  • the data interpretation software controls the number of acquisitions (minimum of 2) that are required to statistically differentiate multiple candidates for an amino acid assignment.
  • the operator has control of specifying to what minimum statistical level of confidence the assignment(s) should meet.
  • Table 2 represents a comparison of the actual average masses of the sequenced residues of the ACTH 7-38 fragment and the experimental mass differences with associated standard deviations and 95% confidence intervals calculated for the time-dependent digestion.
  • the number of replicates indicate the number of spectra that possessed the detectable adjacent peaks required for the mass difference measurement of that particular residue.
  • the need for a significant number of measurements in order to estimate the mean is obvious from the table as the 95% confidence level decreases as the square root of the number of measurements.
  • the actual mass fell within ⁇ 3 ⁇ the experimental mass distribution. Calculated t- values for each case were less than the tabulated t-value for the 95% confidence interval signifying that the experimental mass is not significantly different than the actual known mass.
  • a calculated t-value for each possible amino acid must be compared with the tabulated value.
  • Table 3 represents calculated t- values for 19 sequenced amino acid experimental means in the ACTH 7-38 fragment given the asserted means of 20 common unmodified amino acids.
  • the abie value is given at the end of each column.
  • a t ca ic i ted ⁇ t ta bie indicates that the experimental mean is not significantly different that the mean of the asserted amino acid at 95%> confidence interval.
  • Each t ca ⁇ cu ⁇ at ed for which this is the case is indicated in bold.
  • Table 4 summarizes the results of the statistical amino acid assignments for the 19 amino acids sequenced from the C-terminus of ACTH 7-38 fragment using the prior art time-dependent strategy.
  • the masses of the listed amino acids could not be statistically differentiated from the experimentally derived mass difference at the given confidence levels.
  • the amino acids indicated in bold are the known residues existing at the given positions.
  • the confidence intervals indicated are the highest levels at which all amino acid masses other than those indicated are statistically different from the experimental mean.
  • Gin and Lys for the amino acid assignment of residue 21 could not be made as the experimental mean (128.15 daltons) exactly bisected the asserted means of Gin (128.13 daltons) and Lys (128.17 daltons). The same phenomenon occurred in the assignment of residue 37.
  • the experimental mean 113.63 daltons bisected the asserted means of Leu(Ile) (113.16 daltons) and Asn (114.10 daltons).
  • the assignments of the amino acids at positions 28 and 38 were difficult due to the small number of replicates taken (2 and 3, respectively). Residue 28 was assigned Gln/Lys/GIu/Met at a confidence interval greater than 95%) but less than 98%.
  • Table 3 shows that, for this residue, the asserted amino acid mass that resulted in the smallest calc late d was that of methionine. Using a confidence interval of 80%, the correct assignment of Glu is deemed statistically improbable. Likewise, the assignment of residue 38 was made as Gln/Lys/GIu at a confidence level of 95%, but the correct assignment (Glu) is again statistically improbable at an 80%> level. Since the errors are randomly distributed, all amino acids can be differentiated (except Leu and He) by sufficient population sampling.
  • mass shift reagents used to move peptide populations out of the interfering matrix are a possible chemical means for improving experimental error relating to peptides appearing in the low mass ( ⁇ 600 daltons) region.
  • the use of reflectron and/or extended flight tube geometries are also expected to be instrumental methods suitable for reducing this error.
  • the protocol disclosed herein for statistical assignment of residues using the on-plate strategy involves multiple sampling from each well in which digestion is performed.
  • the number of replicates required depends on the amino acid(s) that is(are) being sequenced at any one CPY concentration. For example, more replicates are required for mass differences around 113-115 daltons (Ile/Leu, Asn and Asp) and 128-129 daltons (Gln/Lys/GIu) than for mass differences around 163 (Tyr) or 57 (Gly) in order to be able to assure that all but one assignment are statistically unlikely.
  • the experimental errors for this method appear to be as random (multiple replicates per sample) as for the time-dependent digestion (one replicate per sample).
  • the method disclosed herein has also been used to obtain sequence information about a nucleic acid polymer containing 40 bases. Hydrolysis using an exonuclease specific for the 3' terminus was conducted using different concentrations of Phos I (phosphodiesterase I) ranging from 0.002 ⁇ U/ ⁇ L to 0.05 ⁇ U/ ⁇ L. Hydrolysis was allowed to proceed for 3 minutes. Spectra of hydrolyzed sequences using MALDI-TOF are depicted in Figures 6A-6E. Data integration as disclosed herein confirmed the sequence to be:
  • this strategy can be applied to the sequencing of any natural biopolymer such as proteins, peptides, nucleic acids, carbohydrates, and modified versions thereof as well as synthetic biopolymers such as PNA and phosphothiolated nucleic acids.
  • the ladders can be created enzymatically using exohydrolases, endohydrolases or the Sanger method and/or chemically by truncation synthesis or failure sequencing.

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Abstract

Le procédé et l'appareil décrits ici sont utiles pour séquencer des polymères en utilisant un spectromètre de masse. Ce procédé fait appel à différents rapports d'agent d'hydrolyse sur polymère, cet agent étant disposé sur une surface de réaction destinée à être utilisée avec le spectromètre de masse, ainsi qu'à l'intégration de données obtenues par analyse avec un spectromètre de masse sur une pluralité de séries de fragments d'un polymère hydrolysé. Des logiciels d'ordinateurs permettent de mettre en ÷uvre les paradigmes d'interprétation statistique. L'appareil comprend un support pour l'échantillon destiné au spectromètre de masse, qui porte l'agent d'hydrolyse et qui peut être adapté à n'importe quel spectromètre de masse pour le séquençage de polymères.
PCT/US1996/007146 1995-05-19 1996-05-17 Procedes et appareil pour sequencer des polymeres avec une certitude statistique en utilisant un spectrometre de masse WO1996036986A1 (fr)

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EP96916490A EP0827628A1 (fr) 1995-05-19 1996-05-17 Procedes et appareil pour sequencer des polymeres avec une certitude statistique en utilisant un spectrometre de masse

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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998045700A2 (fr) * 1997-04-09 1998-10-15 Engels Joachim W Procede de sequençage de biopolymeres par spectrometrie de masse
WO1998054571A1 (fr) * 1997-05-28 1998-12-03 The Walter And Eliza Hall Institute Of Medical Research Diagnostics d'acide nucleique par spectrometrie de masse ou separation de masse et clivage specifique de la base
WO1999012040A2 (fr) * 1997-09-02 1999-03-11 Sequenom, Inc. Detection de polypeptides par spectroscopie de masse
US6043031A (en) * 1995-03-17 2000-03-28 Sequenom, Inc. DNA diagnostics based on mass spectrometry
WO2000020870A1 (fr) * 1998-10-01 2000-04-13 Brax Group Limited Caracterisation de polypeptides par clivage et spectrometrie de masse
US6146854A (en) * 1995-08-31 2000-11-14 Sequenom, Inc. Filtration processes, kits and devices for isolating plasmids
US6225450B1 (en) 1993-01-07 2001-05-01 Sequenom, Inc. DNA sequencing by mass spectrometry
US6225047B1 (en) 1997-06-20 2001-05-01 Ciphergen Biosystems, Inc. Use of retentate chromatography to generate difference maps
US6423966B2 (en) 1996-09-19 2002-07-23 Sequenom, Inc. Method and apparatus for maldi analysis
WO2002093166A1 (fr) * 2001-05-15 2002-11-21 Wolfgang Altmeyer Procede de determination qualitative et/ou quantitative du genre, de l'espece, de la race et/ou de l'origine geographique de materiaux biologiques
WO2004097369A2 (fr) * 2003-04-25 2004-11-11 Sequenom, Inc. Procedes et systemes de fragmentation et systemes de sequençage de novo
US6994969B1 (en) 1999-04-30 2006-02-07 Methexis Genomics, N.V. Diagnostic sequencing by a combination of specific cleavage and mass spectrometry
US7125726B2 (en) 2001-04-30 2006-10-24 Artemis Proteomics Ltd Method and kit for diagnosing myocardial infarction
EP2289090A2 (fr) * 2008-04-28 2011-03-02 Thermo Fisher Scientific (Bremen) GmbH Procédé et dispositif de commande de systèmes de mesure, programme d'ordinateur correspondant, et support d'enregistrement lisible par ordinateur correspondant
US9153424B2 (en) 2011-02-23 2015-10-06 Leco Corporation Correcting time-of-flight drifts in time-of-flight mass spectrometers
US9249456B2 (en) 2004-03-26 2016-02-02 Agena Bioscience, Inc. Base specific cleavage of methylation-specific amplification products in combination with mass analysis
US9394565B2 (en) 2003-09-05 2016-07-19 Agena Bioscience, Inc. Allele-specific sequence variation analysis

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4032538A3 (fr) 2009-03-02 2022-10-26 Massachusetts Institute of Technology Procédés et produits pour établir un profil enzymatique in vivo
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US11428689B2 (en) 2016-05-05 2022-08-30 Massachusetts Institute Of Technology Methods and uses for remotely triggered protease activity measurements
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU1514782A1 (ru) * 1987-07-16 1989-10-15 Tikhookeanskij I Bioorg Khim D Способ обнаружения бактериального эндотоксина
US5288644A (en) * 1990-04-04 1994-02-22 The Rockefeller University Instrument and method for the sequencing of genome

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU1514782A1 (ru) * 1987-07-16 1989-10-15 Tikhookeanskij I Bioorg Khim D Способ обнаружения бактериального эндотоксина
US5288644A (en) * 1990-04-04 1994-02-22 The Rockefeller University Instrument and method for the sequencing of genome

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE WPI Section Ch Week 9018, Derwent World Patents Index; Class B04, AN 90-137766, XP002014080 *
J. G. VAN RAAPHORST: "THE EVALUATION OF MEASUREMENT DATA IN THERMAL IONISATION MASS SPECTROMETRY", INTERNATIONAL JOURNAL OF MASS SPECTROMETRY AND ION PHYSICS, vol. 31, 1979, AMSTERDAM NL, pages 65 - 69, XP002014079 *
R. J. COLTON: "SECONDARY ION MASS SPECTROMETRY : HIGH-MASS MOLECULAR AND CLUSTER IONS", NUCLEAR INSTRUMENTS & METHODS IN PHYSICS RESEARCH, vol. 218, 1983, AMSTERDAM NL, pages 276 - 286, XP002014078 *
ROEPSTORFF P: "MASS SPECTROMETRY OF PROTEINS", TRAC, TRENDS IN ANALYTICAL CHEMISTRY, vol. 12, no. 10, 1 November 1993 (1993-11-01), pages 413 - 421, XP000403883 *

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US6235478B1 (en) 1995-03-17 2001-05-22 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6043031A (en) * 1995-03-17 2000-03-28 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6197498B1 (en) 1995-03-17 2001-03-06 Sequenom, Inc DNA diagnostics based on mass spectrometry
US6277573B1 (en) 1995-03-17 2001-08-21 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6146854A (en) * 1995-08-31 2000-11-14 Sequenom, Inc. Filtration processes, kits and devices for isolating plasmids
US6812455B2 (en) 1996-09-19 2004-11-02 Sequenom, Inc. Method and apparatus for MALDI analysis
US6423966B2 (en) 1996-09-19 2002-07-23 Sequenom, Inc. Method and apparatus for maldi analysis
WO1998045700A2 (fr) * 1997-04-09 1998-10-15 Engels Joachim W Procede de sequençage de biopolymeres par spectrometrie de masse
WO1998045700A3 (fr) * 1997-04-09 1999-03-11 Joachim W Engels Procede de sequençage de biopolymeres par spectrometrie de masse
WO1998054571A1 (fr) * 1997-05-28 1998-12-03 The Walter And Eliza Hall Institute Of Medical Research Diagnostics d'acide nucleique par spectrometrie de masse ou separation de masse et clivage specifique de la base
US6811969B1 (en) 1997-06-20 2004-11-02 Ciphergen Biosystems, Inc. Retentate chromatography—profiling with biospecific interaction adsorbents
US6818411B2 (en) 1997-06-20 2004-11-16 Ciphergen Biosystems, Inc. Retentate chromatography and protein chip arrays with applications in biology and medicine
CN100351990C (zh) * 1997-06-20 2007-11-28 生物辐射实验室股份有限公司 滞留层析和蛋白质芯片排列在生物学和医药上的应用
US7112453B2 (en) 1997-06-20 2006-09-26 Ciphergen Biosystems, Inc. Retentate chromatography and protein chip arrays with applications in biology and medicine
US7105339B2 (en) 1997-06-20 2006-09-12 Ciphergen Biosystems, Inc. Retentate chromatography and protein chip arrays with applications in biology and medicine
US6225047B1 (en) 1997-06-20 2001-05-01 Ciphergen Biosystems, Inc. Use of retentate chromatography to generate difference maps
US6579719B1 (en) 1997-06-20 2003-06-17 Ciphergen Biosystems, Inc. Retentate chromatography and protein chip arrays with applications in biology and medicine
US6881586B2 (en) 1997-06-20 2005-04-19 Ciphergen Biosystems, Inc. Retentate chromatography and protein chip arrays with applications in biology and medicine
US6844165B2 (en) 1997-06-20 2005-01-18 Ciphergen Biosystems, Inc. Retentate chromatography and protein chip arrays with applications in biology and medicine
US7329484B2 (en) 1997-06-20 2008-02-12 Bio-Rad Laboratories, Inc. Retentate chromatography and protein chip arrays with applications in biology and medicine
US7575935B2 (en) 1997-06-20 2009-08-18 Bio-Rad Laboratories, Inc. Retentate chromatography and protein chip arrays with applications in biology and medicine
WO1999012040A2 (fr) * 1997-09-02 1999-03-11 Sequenom, Inc. Detection de polypeptides par spectroscopie de masse
WO1999012040A3 (fr) * 1997-09-02 1999-09-02 Sequenom Inc Detection de polypeptides par spectroscopie de masse
EP1296143A3 (fr) * 1997-09-02 2004-02-04 Sequenom, Inc. Détection de polypeptides par spectroscopie de masse
US6387628B1 (en) 1997-09-02 2002-05-14 Sequenom, Inc. Mass spectrometric detection of polypeptides
US6322970B1 (en) 1997-09-02 2001-11-27 Sequenom, Inc. Mass spectrometric detection of polypeptides
US6846679B1 (en) 1998-10-01 2005-01-25 Xzillion Gmbh & Co., Kg Characterizing polypeptides through cleavage and mass spectrometry
WO2000020870A1 (fr) * 1998-10-01 2000-04-13 Brax Group Limited Caracterisation de polypeptides par clivage et spectrometrie de masse
US6994969B1 (en) 1999-04-30 2006-02-07 Methexis Genomics, N.V. Diagnostic sequencing by a combination of specific cleavage and mass spectrometry
US7125726B2 (en) 2001-04-30 2006-10-24 Artemis Proteomics Ltd Method and kit for diagnosing myocardial infarction
WO2002093166A1 (fr) * 2001-05-15 2002-11-21 Wolfgang Altmeyer Procede de determination qualitative et/ou quantitative du genre, de l'espece, de la race et/ou de l'origine geographique de materiaux biologiques
WO2004097369A3 (fr) * 2003-04-25 2005-11-17 Sequenom Inc Procedes et systemes de fragmentation et systemes de sequençage de novo
AU2004235331B2 (en) * 2003-04-25 2008-12-18 Sequenom, Inc. Fragmentation-based methods and systems for De Novo sequencing
WO2004097369A2 (fr) * 2003-04-25 2004-11-11 Sequenom, Inc. Procedes et systemes de fragmentation et systemes de sequençage de novo
US9394565B2 (en) 2003-09-05 2016-07-19 Agena Bioscience, Inc. Allele-specific sequence variation analysis
US9249456B2 (en) 2004-03-26 2016-02-02 Agena Bioscience, Inc. Base specific cleavage of methylation-specific amplification products in combination with mass analysis
EP2289090A2 (fr) * 2008-04-28 2011-03-02 Thermo Fisher Scientific (Bremen) GmbH Procédé et dispositif de commande de systèmes de mesure, programme d'ordinateur correspondant, et support d'enregistrement lisible par ordinateur correspondant
EP2289090B1 (fr) * 2008-04-28 2021-07-28 Thermo Fisher Scientific (Bremen) GmbH Procédé et dispositif de commande de systèmes de mesure, programme d'ordinateur correspondant, et support d'enregistrement lisible par ordinateur correspondant
US9153424B2 (en) 2011-02-23 2015-10-06 Leco Corporation Correcting time-of-flight drifts in time-of-flight mass spectrometers

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