WO2003104800A1 - Identification de proteines presentes dans des melanges complexes - Google Patents

Identification de proteines presentes dans des melanges complexes Download PDF

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Publication number
WO2003104800A1
WO2003104800A1 PCT/US2001/023127 US0123127W WO03104800A1 WO 2003104800 A1 WO2003104800 A1 WO 2003104800A1 US 0123127 W US0123127 W US 0123127W WO 03104800 A1 WO03104800 A1 WO 03104800A1
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tagged protein
protein fragments
retrieved
masses
database search
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PCT/US2001/023127
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English (en)
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Randall Nelson
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Intrinsic Bioprobes Inc.
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Priority to AU2001280706A priority Critical patent/AU2001280706A1/en
Priority to PCT/US2001/023127 priority patent/WO2003104800A1/fr
Publication of WO2003104800A1 publication Critical patent/WO2003104800A1/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
    • 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
    • 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
    • G01N33/6818—Sequencing of polypeptides
    • 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
    • G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry

Definitions

  • the present invention relates to the field of protein identification. More specifically, the present invention relates to a novel method for the mass spectrometric detection and 5 identification of proteins present in complex mixtures at low to sub femtomole levels.
  • a fundamental and recurring goal in cell biology and biochemistry is the detection and identification of individual protein species against a complex background of numerous other molecular species present in the cell, tissue, or biological fluid.
  • the process requires the isolation/purification of a protein of interest from the 15 mixture, followed by appropriate characterization.
  • these approaches rely on either electrophoretic (2D-SDS-PAGE) or chromatographic (RP-HPLC) separation of proteins; methods that can be applied somewhat universally to proteins independent of their specific character.
  • More specific separation of a given protein from 20 a mixture generally relies on separation methods that target the function of the protein.
  • affinity chromatography is largely referred to as affinity chromatography.
  • affinity chromatography can be combined with mass
  • Three different affinity isolation approaches were taken in identifying tagged proteins.
  • the first approach is that of a mass spectrometric immunoassay (MSIA) in which tagged peptides are isolated from the complex mixture using affinity pipettor tips, and then eluted from the tip onto a target for matrix-assisted laser desorption/ionization - time of flight (MALDI-TOF) mass spectrometry.
  • MSIA mass spectrometric immunoassay
  • MALDI-TOF matrix-assisted laser desorption/ionization - time of flight
  • the second approach is that of using a beaded agarose/metal-chelate to selectively isolate the tagged peptides from the digested mixture. This approach is viewed beneficial in isolating metal binding motifs or posttransaltionally modified amino acids present in the peptides.
  • the third approach is that of performing the analysis using the technique of biomolecular interaction analysis mass spectrometry (BIA/MS), wherein the affinity isolation step is viewed in real-time using surface plasmon resonance (SPR). After isolation, tagged peptides are analyzed either directly from an SPR-active sensor using MALDI-TOF mass spectrometry, or eluted from the sensor and analyzed from the surface of a MALDI-TOF target.
  • affinity tag used in the examples given below strongly resembles metal binding domains present in naturally occurring proteins. Additionally because there are no trypsin cleavages sites intermixed within this tag, the metal binding tag is conserved throughout the treatment of the lysate with trypsin. Tryptic peptides containing the tag fused to native sequences of the protein under investigation can therefore be addressed using two different affinity recognition systems: a Ni 2+ chelate system or a monoclonal antibody system (using the tag as an epitope).
  • Still another object of the present invention is that of providing multiple means of data generation and analysis used in database searches.
  • masses are derived by subtracting the mass of the tag, and these values are used as input data for database search.
  • mass values for database search are derived by determining the differences in mass between adjacent signals in the mass spectra. These mass differences correspond to masses of untagged tryptic peptides of the target protein and are used directly to fuel database searches.
  • sequence data of the tagged protein is generated after affinity isolation by treatment of the tagged peptides with an exopeptidase while they are retained by an affinity receptor. This sequence data is then used as input for database searches.
  • Mass spectrometric data obtained using the process described herein will originate only from a select region within a protein - the region containing the tag.
  • the masses used in the database search must therefore form a nested set of data that overlaps only one region of the target protein.
  • the data used in the identification process can be used in a second verification analysis by overlapping a region of the identified protein.
  • FIG. 1 is a schematic representation of a MSIA strategy for identification of affinity tagged proteins.
  • An antibody recognizing the affinity tag is used to selectively retrieve tagged peptides created by the chemical or enzymatic digestion of a complex biological mixture containing a tagged protein. After retrieval, the tagged peptides are eluted from the affinity media and analyzed by mass spectrometry. Mass data from the tagged peptides is then used in the identification of the tagged protein.
  • FIG. 2 illustrates the MSIA approach using a monoclonal antibody recognizing an epitope engineered into a protein. The top trace is a MALDI-TOF mass spectrum of unfractioned, trypsinized E.
  • Coli lysate containing all proteins of the expression system The bottom trace is a MALDI-TOF mass spectrum of components affinity retained during the MSIA approach. Tryptic peptides containing the affinity tag (epitope) are selectively retrieved from the digest mixture and mass analyzed. From the resulting masses, the tagged protein is identified as glutathione-s-transferase using database searches fueled by either the mass differences between the ion signals or the determined masses minus the mass of the tag.
  • FIG.3. is a schematic representation of protein identification via the MSIA approach and an additional step involving exopeptidase digestion. Using the approach shown/demonstrated in Figs. 1 and 2, tagged peptides are selectively retrieved from the digested mixture.
  • FIG. 4 shows the results of applying the process described in Fig. 3 to tagged peptides retrieved from trypsinized ⁇ . coli lysate containing a tagged protein.
  • FIG. 5 shows results obtained when an agarose/NTA/Ni 2+ affinity media was applied to ⁇ . coli expressing a protein in which the tag was a metal-binding motif.
  • the upper MALDI-TOF spectrum shows proteins obtained from undigested ⁇ . coli lysate through interaction with the affinity reagent. Due to its complexity, the spectrum offers no useful information on the identity of the tagged protein.
  • the lower trace was obtained from the same lysate that had been digested for 30-minutes with trypsin. Mass data from the simplified spectrum is used in the identification of the tagged protein as glutathione-s-transferase.
  • FIG. 6 is a schematic representation of BIA/MS analysis of tagged proteins present in complex biological mixtures.
  • Tags present in proteins either naturally or engineered into proteins at the gene or protein level, are used to retrieved polypeptides from complex mixtures (after digestion with specific chemicals or proteases - the digestion of the entire biological system results in tagged peptides) for the purpose of identifying the tagged proteins.
  • SPR-BIA is used to selectively isolate, detect and quantitate the peptides from the digested media.
  • MALDI-TOF mass spectrometry is used to unambiguously identify and/or structurally characterize the tagged peptides.
  • FIG 7 shows serial activation and immobilization of mAb 7G8 to the two flow cells (FC1 and FC2) present on the surface of the biosensor chip.
  • the flow cells were first activated using EDC-mediated binding of NHS to the carboxylated dextran of the chip.
  • Monoclonal 7G8 was immobilized through coupling of primary amines to the NHS-activated surfaces.
  • Antibody on FC1 was deactivated after immobilization by exposure of the flow cell to trypsin.
  • FIG.8 shows sensorgrams showing the SPR response observed during the serial routing of unfractioned, trypsinized E. coli lysate across the surfaces of the active (FC2) and blank (FC1) flow cells.
  • a 30-uL of trypsinized lysate was flowed over the flow cells at a rate of 10 ⁇ L/min.
  • Responses of 30 and 10 RU indicate the retention of 30 and 10 picograms of material on the FC2 and FC1, respectively
  • FIG. 9 shows MALDI-TOF mass spectra obtained by targeting the active (FC2) and blank (FC1) flow cells. Ion signals are observed unique to the active flow cell
  • FIG 11 shows sensorgrams resulting from SPR-BIA of the unfractionated, trypsinized E. coli lysate using an NTA (nitrilotiracetic acid) sensor chip in the presence (Ni 2+ - active) or absence (Ni 2+ -free) of Ni 2+ .
  • NTA nitrilotiracetic acid
  • FIG 12 shows MALDI-TOF mass spectra of material eluted from the NTA sensor chip using a MALDI matrix solution.
  • Several peptide species are observed in the spectrum resulting from Ni 2+ -active SPR-BIA, whereas fewer are observed to result from Ni 2+ -free SPR-BIA.
  • the subtracted spectrum shows four signals resulting from specific interaction with the Ni 2+ -chelate surface.
  • FIG. 14 shows a coverage map of a identified protein (GST) where the mass analyzed tagged peptides are observed to form a nested set of fragments within the protein.
  • the nested set approach is used as an additional restriction to database searching and is able to reconfirm or validate the protein identification.
  • the present invention is a novel method that is useful for the detection and identification of tagged proteins present in complex biological systems.
  • Protein identification using mass spectrometry has evolved over the past decade to a routine process where the mass values determined from endopeptidase-generated fragments of a given protein are used to fuel searches of protein or genomic databases. These high-accuracy mass values, in combination with the known specificity of the enzyme are correlated with in-silico (virtual) maps of proteins present within an organism and are able to identify proteins with high degrees of confidence.
  • contiguous lengths of amino acid sequences within a protein, determined using tandem mass spectrometry or through treating peptides with exopeptidases can be used in the database searches with equally successful results. In either case, the endopeptidase treatment used to generate the initial proteolytic fragments is performed after the isolation of the target protein.
  • tags - fragments of the protein containing sites recognized by an affinity receptor - referred to as tags - can be targeted for identification analysis.
  • tagged proteins present in complex mixtures can first be digested using specific chemicals or enzymes and the resulting tagged peptides affinity- isolated from the digested mixture in preparation for mass analysis, database search and identification.
  • Chemicals and enzymes that can be used in the digestion of the mixture include but are not limited to: HC1, o-iodobenzoic acid, cyanogen bromide, trypsin, endoprotemases Glu-C; Lys-C; Arg-C, Staph N8, Asp- ⁇ , chymotrypsin, pepsin, papain, subtilisin, thermolysin, thrombin, and Factor X.
  • Affinity tags present in the proteins can be either natural in origin, genetically engineered into the target protein or can be introduced into the protein post-translationally through chemical or enzymatic modification of specific sites within the protein, e.g., biotinylation of sites, or phosphorylation of sites using kinases.
  • Affinity tags used in the process include but are not limited to: epitopes, binding domains or motif:-, (e.g. 6xHis tags), posttranslationally modified amino acid residues (e.g.
  • Affinity receptors can be organic, inorganic or biological in character, and include, but are not limited to: monoclonal or polyclonal antibodies, antibsera, avidin, streptavidin, nucleotides, metal-chelates, and ion exchange functionalities.
  • the affinity receptors can be immobilized, either covalently or non- covalently, to a variety of solid supports to ease the separation of the affinity-isolated tagged peptides from the digest mixture
  • solid supports include, but are not limited to; beaded affinity media (e.g., agarose, glass or polyacrylamide), paramagnetic affinity beads, affinity-derivatized pipettor tips, or planar supports capable of being introduced into a mass spectrometer.
  • Nolatilization ionization for mass spectrometry can be in the form of matrix-assisted laser desorption/ionization (MALDI), electrospray ionization (ESI), liquid secondary ion mass spectrometry (LSIMS), plasma desorption mass spectrometry (PDMS) or massive cluster impact (MCI) coupled to a variety of mass analyzers such as time-of-flight (TOF) mass spectrometers, quadrupole ion (QI) mass spectrometers, quadrupole ion-trap (QIT) mass spectrometers, Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, electrostatic/magnetic sector (EB) mass spectrometers used singularly or in combination with each other.
  • MALDI matrix-assisted laser desorption/ionization
  • ESI electrospray ionization
  • LIMS liquid secondary ion mass spectrometry
  • PDMS plasma desorption mass
  • this improved approach to the identification of proteins present in complex biological mixture is comprised of the ordered steps of: 1) digesting or treating of the complex mixture with a specific protease to create tagged protein fragments; 2) isolating the tagged proteolytic fragments from the digested mixture using an affinity receptor; 3) analyzing by mass spectrometry the affinity isolated fragments to accurately determine their masses; 4) processing the mass data in manners favorable to database searching, and, 5) using the processed mass data in combination with the specificity of the isolating enzyme to determine the identity of the tagged protein via database search.
  • the process offers several advantages in terms of speed and analytical accuracy over current methodologies used to identify proteins.
  • the total sample workup of the analyte entails the two simple steps of digestion and affinity isolation. In that these steps can be performed rapidly (in less than one-hour) and on many samples in parallel, the process is conducive to high-throughput protein identification.
  • the process is less prone to analytical artifacts because the initial digest of the complex mixture results in digestion of potentially interfering proteins, which in their native state may bind to the affinity media via their higher-order structure.
  • protein identification using masses of the tagged peptides may proceed through more than one process, i.e., the same data can be treated in multiple routines to validate the identification.
  • mass spectrometric data obtained from tagged peptides will form nested sets corresponding to linear sequences within the tagged protein, again enhancing the database searching routines.
  • An additional advantage is that mass spectrometric sensitivity is generally much higher for small molecules than it is for large proteins. Mass measurement accuracy dramatically improves in the peptide region, where monoisotopic mass resolution is achievable, resulting in high-precision database searches that are less prone to misidentification.
  • tandem mass spectrometric techniques are more ideally suited for tag-containing peptides than for proteins. These approaches are used to actively (i.e., during mass spectrometric analysis) derive partial sequence information from the peptide fragments.
  • ESI techniques micro or nanospray coupled to a variety of tandem MS instruments may be used to partially sequence the tagged peptides.
  • PSD post-source decay
  • ISD in-source decay
  • the partial sequence data may be used to augment database searches.
  • An alternative for deriving sequence is to digest the peptides while retained by the affinity receptor using select exopeptidases. The exopeptidase treatment results in ragged end peptides, still retained on the affinity media, that can be rinsed free of reagents and then analyzed by protein ladder sequencing.
  • FIG. 1 shows a general mass spectrometric immunoassay (MSIA) strategy for identification of affinity tagged proteins.
  • MSIA mass spectrometric immunoassay
  • the tagged peptides are retrieved from the digested biological mixtures using affinity-pipette tips derivatized with a monoclonal antibody with high affinity towards the tag. Once retrieved and washed free of non-specific components, the tagged peptides are eluted from the affinity tip in preparation for mass spectrometry.
  • the digest/retrieval/identification process was applied to trypsinized E. coli whole lysate containing tagged glutathione S-transferase (GST) protein.
  • GST glutathione S-transferase
  • the peptide contained the 16 amino acid sequence HTTPHHTTPHHTTPHH that contains three copies (two overlapping) of the epitope HTTPHH recognized by monoclonal antibody 7G8 (mAb7G8, see below).
  • the inserted amino acids are shown in italics, with the tag-containing sequence underlined.
  • E. coli cells containing the tagged GST expression vector were grown at 37 °C in
  • a 10 ⁇ L aliquot of the lysate was mixed with 90 ⁇ L of either ⁇ BS buffer (0.01 M ⁇ EPES, 0.15 M ⁇ aCl, 3 mM EDTA, 0.005% v/v Surfactant P20, p ⁇ 7.4) or ⁇ TA buffer (0.01 M ⁇ EPES, 0.15 M ⁇ aCl, 50 ⁇ M EDTA, 0.005% v/v Surfactant P20, p ⁇ 7.4 ) containing 0.0005 mg/mL trypsin. Digestion was allowed to proceed for 30-minutes at 40°C prior to quenching by the addition of bovine pancreatic trypsin inhibitor. Final concentration of lysate is ⁇ 0.001 mg/mL.
  • Mouse monoclonal antibody 7G8 was generated against MAP-conjugated peptide P ⁇ TTP ⁇ TTP ⁇ TT using standard methods. Hybridoma supematants were screened by ELISA against the immunogen and by Western blot against the pGEX-5X-l construct described above. An analysis of antibody binding to individual synthesized peptides contained within PHHTTPHHTTPHHTT indicated that the smallest continuous sequence sufficient for efficient antibody binding is HTTPHH. mAb7G8 was isolated from ascites fluid using a protein A/agarose microcolumn.
  • Fluid containing ⁇ 20 mg/mL protein was diluted by a factor of ten with HBS buffer and drawn through the column, after which the column was rinsed three times (500 ⁇ L) with HBS buffer and once (500 ⁇ L) with doubly-distilled water.
  • mAb7G8 was eluted from the microcolumn using two- 25 ⁇ L aliquots of 10 mM sodium acetate (pH 2.0). The eluted antibody was then mixed with 100 ⁇ L of sodium phosphate buffer (pH 8.2) and coupled to n-hydroxysuccinimide activated pipettor tips via primary amine linkage.
  • Mass spectrometry was performed on tagged peptides eluted from the affinity pipettor tips using a MALDI-TOF mass spectrometer and experimental methods that are known in the art.
  • FIG. 2 shows the results of retrieving tagged peptides using mAb7G8 derivatized affinity pipettor tips.
  • the top trace is a MALDI-TOF spectrum of the trypsinized E. coli whole lysate. Because of the complexity of the analytical solution, the spectrum does not yield any particularly useful information on the analyte.
  • the first of the methods for protein identification using this data is to subtract the mass of the tag from the masses determined in the mass spectra. The resulting numerical values represent masses of tryptic fragments of the original, non-tagged protein.
  • mass values may be used to fuel database searches capable of identifying the protein.
  • a few precautions must be taken to insure an accurate database search.
  • the exact mechanism of tag-insertion, and more importantly, variations in gene sequence due to e.g., restriction enzyme specificity must be known. For instance, if the insertion mechanism of the tag results in a perturbation of gene sequence (e.g., dropping of a codon or frame shift), then the protein sequence in the region of the tag is disrupted and the determined mass values will not accurately depict the original gene sequence.
  • two codons coding for Pro229 and Arg230 are removed from the original sequence of the GST when treated with BamH I and ⁇ coR I.
  • Mass Difference Method A second method of database search using the data shown in FIG 2 is to simply use the mass differences between the observed ion signals to fuel the database search. This method requires the addition of 18 Da to each mass difference in order to correspond to the in-silico generated numerical values of the tryptic peptide fragments. Three search values are calculated from the masses determined during MALDI-TOF; 446.1, 619.0, and 1,047.1 Da. Database search using these values (PeptideSearch, http//www.mann.embl- heildelberg.de) with a 0.5 Da error window results in three versions of GST, out of which one (accession number U13858) is the correct version.
  • Possible solution to the terminal heterogeneity problem are to insert tags or construct cloning vectors so that the tag is a terminus of the expressed protein, or to include a specific proteoiytic cleavage site (e.g., thrombin or Factor Xa) into the tag that can be cleaved (prior to trypsin treatment) to create tag-containing peptides with a common terminus.
  • a specific proteoiytic cleavage site e.g., thrombin or Factor Xa
  • FIG. 3 shows an alternative strategy to protein identification where tagged peptides are partially sequenced using an additional step of exopeptidase digestion. The approach begins as in Example 1 with digestion of unfractionated biological mixture and the retrieval of tagged peptides from the digested mixture. Once retrieved, the tagged peptides are exposed briefly (while still retained in the affinity pipette) to high-levels of enzymes or chemicals in order to produce ragged-end peptides that vary in mass by the masses of the amino acids present in their sequence.
  • Tagged peptides retrieved using mAb7G8-derivatized pipettor tips were treated with various concentrations of carboxypeptidase Y (CPY) while still retained in the affinity pipettors. Eight different concentrations of CPY were used (ranging from 0 - 5 mg/mL) during the parallel processing of the affinity-retrieved peptides.
  • the digest involved drawing the CPY solutions ( ⁇ 5 uL) into the affinity pipettes and allowing 5 minutes for digestion.
  • FIG. 4 shows the results of the post-capture processing. Because of redundancies in the spectra, results from only four of the digests are shown.
  • the CPY digestion has resulted in the C-terminal sequencing of 12 residues of the sequence corresponding to that expected from the parent protein (see the sequence of the GST fusion protein above).
  • a search of the OWL database using the sequence tag - S, G, R, I/L, N, T, D - resulted in three matches: glutathione s-transferase or glutathione s- transferase variants.
  • FIG. 5 shows a MALDI-TOF spectrum of peptides retrieved using the same procedure described in Example 1, with the exception of using ⁇ i 2+ /nitrilotriacetic acid (NTA)/agarose affinity receptor.
  • Example 5 Isolation and Identification of Tagged GST using Monoclonal Antibody /Epitope
  • FIG. 6 shows the general approach to protein identification using surface plasmon resonance biomolecular interaction analysis mass spectrometry (SPR-BIA/MS).
  • SPR-BIA/MS surface plasmon resonance biomolecular interaction analysis mass spectrometry
  • Both flow cells (FC 1 and FC 2) on the sensor chip were prepared using a serial derivatization procedure in which the flow cells were activated through exposure to N-hydroxysuccinimide (0.1 M prepared in HBS buffer) containing 0.1 M N'-ethyl-N'-(dimethylaminoproply) carbodiimide, derivatized by exposure to mAb7G8, and then blocked by exposure to 1 M ethanolamine hydrochloride (in HBS buffer, adjusted to pH 8.5).
  • FC 1 was deactivated (for use as a blank) by exposing the surface of the flow cell to 0.005 mg/mL trypsin dissolved in HBS buffer (5 minutes at 10 ⁇ L/min). The flow cells were then allowed to equilibrate by continuously flowing HBS buffer (10 ⁇ L/minute) through the biosensor for a period of approximately 12 hours.
  • BIA proceeded by routing the trypsinized E. coli lysate, in HBS buffer, serially across the flow cells. Both flow cells were monitored in real-time using SPR, and the signal from the blank flow cell (FC1) subtracted from that of the active flow cell (FC2). 30 ⁇ L of the analytical solution was driven across the flow cells at 10 ⁇ L/minute before the injection was terminated. The chip was undocked from the biosensor, rinsed with two successive 200 ⁇ L aliquots of distilled water, and allowed to dry.
  • the chip was prepared for mass spectrometry by applying ⁇ 100 nL of ⁇ -cyano-4-hydroxycinnamic acid ( ⁇ 50 mM dissolved in 1:2 acetonitrile:1.5 % trifluoroacetic acid (ACCA)) to each of the flow cells using a thin-gauge wire. Mass spectrometry was performed using a MALDI-TOF mass spectrometer and experimental methods that are known in the art.
  • FIG. 7 shows the activation and derivatization of the two flow cells (FCl and FC2) of a CM5 sensor chip with mAb7G8.
  • An antibody density of ⁇ 8 nanograms/flow cell was determined by SPR-BIA, indicating approximately 50 femtomole of IgG present on the surface of each of the flow cells. After the serial derivatization, FCl was addressed with trypsin in order to deactivate the antibody.
  • FIG. 8 shows the sensorgrams resulting from the serial routing of trypsinized E. coli lysate across the surface of the flow cells.
  • Three sensorgrams are shown, one for each of the two flow cells, and one of the real-time background subtraction of the signal from the blank flow cell from that of the active flow cell (FC2-FC1). Changes in response of 30 and 10 RU are observed for the active (FC2) and blank (FCl) flow cells, indicating the retention of 30 and 10 picograms of material, respectively.
  • the background subtracted sensorgram shows a response change of 20 RU, indicating the retention of —20 picograms of material unique to FC2.
  • FIG. 9 shows the mass spectra produced during the MALDI-TOF analysis of material directly from the surface of the flow cells (FC2 and FCl), and the spectrum resulting from the background subtraction routine (FC2-FC1). Minor signals at m/z ⁇ 2,800 Da and ⁇ 6,500 Da are observed to cancel out as a result of the subtraction, suggesting that the compounds responsible for these signals were retained through interactions with something other than the immobilized antibody.
  • the mole amount of peptides present on the chip is estimated to range between 5.5 femtomole (high end of range - if the 20 picograms of retained material was represented by only the 3.6 kDa ion species) to 800 attomole (low end of range - if the 20 picograms of material were distributed evenly between all six ion signals).
  • Identification of the tagged protein proceeded as described in Example 1, with the mass of the tag subtracted from the determined molecular weights, and these subtracted values used to fuel a database search as tryptic peptides. GST was identified as the leading candidate.
  • EXAMPLE 6 Isolation and Identification of Tagged GST using Ni -chelate/Histidine Recognition System and SPR-BIA/MS: Elution and Mass Difference Method A second SPR-BIA/MS approach was used for protein identification.
  • the experimental procedure follows that described in Example 4 with the exception of using a NTA chip.
  • flow cells were exposed to 20 ⁇ L of Ni 2+ -charged NTA buffer (0.01 M HEPES, 0.15 M NaCl, 50 ⁇ M EDTA, 0.005% v/v Surfactant P20, pH 7.4 containing 500 ⁇ M NiCl 2 ) resulting in a NTA-chelated nickel surface sufficient for binding histidine-rich polypeptides when brought in contact with the digested mixture.
  • FIG. 11 shows sensorgrams resulting from SPR-BIA of trypsinized E. coli lysate using a Biacore X biosensor equipped with an NTA biosensor chip.
  • Four sensorgrams are given: two from analyses using Ni + -active flow cells and two from Ni 2+ -free flow cells.
  • the Ni 2+ -active analyses exhibit behavior characteristic of the specific binding of ligands to the affinity surface of biosensor chip.
  • a plateau is observed in both sensorgrams representing the loading of the NTA surface with Ni 2+ , followed by a gradual increase in response, during exposure to digested lysate, indicating the selective retention of material on the surface of the flow cell.
  • solution flow was returned to NTA buffer in order to rinse the flow cell free of unbound digest components.
  • the SPR response shows a relatively slow rate of dissociation ( - 3.5 x 10 "4 sec "1 ), indicating a reasonable avidity between the tag and the Ni 2+ surface.
  • Response readings taken just prior to undocking of the sensor chip indicate — 1,000 RU (1,000 picograms) and 650 RU (650 picograms) of material selectively retained in flow cells 1 and 2, respectively.
  • the blank flow cells exhibit no, or a slightly negative response as a result of incubation with digested lysate indicating little or no retention of lysate compounds during the analysis (the negative response is likely due to an incubation anomaly occurring at - 690 seconds into the analysis).
  • the low response changes observed in these blank sensorgrams suggests that the -1,650 picograms of material bound in the active flow cells is due to ligands specific to the Ni 2+ -chelate.
  • FIG. 12 shows a MALDI-TOF mass spectra of material eluted from the sensor chip.
  • the third spectrum shown in Fig. 12 is the background subtracted spectrum resulting from subtraction of the Ni 2+ -free spectrum from the Ni 2+ -active spectrum.
  • the -1,650 picograms of material retained during Ni -active SPR-BIA is represented mass spectrometrically by the four ion signals in the - 4 kDa range.
  • SPR-BIA and MALDI-TOF data it is estimated that approximately 100 femtomole of each of the - 4 kDa species was isolated onto the Ni 2+ -active sensor chip. No estimates of the extraction efficiency from the chip can be made at this time.
  • a feature of the pentanucleotide CCACA coding sequence used for this tag is that after at least three repeats it yields the same repeating amino acid sequence (HTTPH) no matter in which of the three possible reading frames it is translated.
  • HTTPH repeating amino acid sequence
  • the gene/protein discovery process may be accomplished by insertion of cDNA or genomic DNA, from e.g., genomic libraries, into appropriately designed cloning vectors that contain the repeating pentanucleotide or other tag coding sequence.
  • BIA/MS may be used to detect and identify the resulting tag-containing polypeptides using the methods describe above.
  • the BIA/MS approaches presented above are quite sensitive.
  • the level of detection experienced using the elution method is equal to that experienced by other groups using a BIA/MS method in which retained species were eluted from the sensor chip and analyzed from a conventional mass spectrometer target.
  • the elution method described above is significantly simpler in that extensive washing of the biosensor unit is not required.
  • An advantage of the elution method is re-use of the sensor chips. Over twenty analyses have been performed using a single NTA chip without significant loss in activity of the sensor surface. A further advantage would be the possibility of analyzing the eluent using electrospray (ESI) mass spectrometry.
  • ESI electrospray

Abstract

La présente invention concerne un procédé général d'identification de niveaux à l'état de trace de protéines marquées présentes dans des systèmes biologiques complexes. Ledit procédé implique la digestion enzymatique ou chimique du système biologique, la récupération sélective de peptides marqués à partir du système digéré à l'aide d'un système de reconnaissance d'affinités, d'une spectrométrie de masse des espèces isolées par affinité et d'une identification de protéines via une recherche dans une base de données à l'aide des masses des espèces récupérées et de la spécificité de l'enzyme/agent chimique. L'invention concerne des procédés de préparation d'échantillons, de recherche dans une base de données et de validation de données.
PCT/US2001/023127 2001-07-19 2001-07-19 Identification de proteines presentes dans des melanges complexes WO2003104800A1 (fr)

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AU2001280706A AU2001280706A1 (en) 2001-07-19 2001-07-19 Identification of proteins present in complex mixtures
PCT/US2001/023127 WO2003104800A1 (fr) 2001-07-19 2001-07-19 Identification de proteines presentes dans des melanges complexes

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102012214202A1 (de) * 2012-08-09 2014-02-13 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Verfahren zum Charakterisieren oberflächenadhäsiver Eigenschaften von Peptiden und Proteinen

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DATABASE PUBMED [online] CAPRIOLI ET AL.: "Peptide sequence analysis using exopeptidases with molecular analysis of the truncated polypeptides by mass spectrometry", XP001055013, accession no. NCBI Database accession no. 2425658 *
GRIMM ET AL.: "A rapid and sensitive procedure for the micro-purification and subsequent characterization of peptides and protein samples by N-terminal sequencing and matrix assited laser desorption ionization time of flight mass spectrometry", JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS, vol. 18, 1998, pages 545 - 554, XP001055012 *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102012214202A1 (de) * 2012-08-09 2014-02-13 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Verfahren zum Charakterisieren oberflächenadhäsiver Eigenschaften von Peptiden und Proteinen
DE102012214202B4 (de) * 2012-08-09 2016-05-04 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Verfahren zum Charakterisieren oberflächenadhäsiver Eigenschaften von Peptiden und Proteinen

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