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EP1565752A2 - Methode permettant d'analyser des acides amines, des peptides et des proteines parspectrometrie de masse de leurs derives modifies par des charges fixees - Google Patents

Methode permettant d'analyser des acides amines, des peptides et des proteines parspectrometrie de masse de leurs derives modifies par des charges fixees

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Publication number
EP1565752A2
EP1565752A2 EP03781974A EP03781974A EP1565752A2 EP 1565752 A2 EP1565752 A2 EP 1565752A2 EP 03781974 A EP03781974 A EP 03781974A EP 03781974 A EP03781974 A EP 03781974A EP 1565752 A2 EP1565752 A2 EP 1565752A2
Authority
EP
European Patent Office
Prior art keywords
ion
amino acid
charge
mass
peptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03781974A
Other languages
German (de)
English (en)
Inventor
Gavin Ludwig Inst. for Cancer Research REID
Richard Ludwig Inst. for Cancer Research SIMPSON
Richard O'hair
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ludwig Institute for Cancer Research Ltd
Ludwig Cancer Research
Original Assignee
Ludwig Institute for Cancer Research Ltd
Ludwig Cancer Research
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Filing date
Publication date
Application filed by Ludwig Institute for Cancer Research Ltd, Ludwig Cancer Research filed Critical Ludwig Institute for Cancer Research Ltd
Publication of EP1565752A2 publication Critical patent/EP1565752A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle 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
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry

Definitions

  • the present invention is concerned with a method for the analysis of amino acids, peptides or proteins.
  • proteomics has become synonymous with (i) the identification and characterization of all proteins synthesized by a particular cell type or tissue at any given time, and quantitation of the global changes in protein expression levels observed between two different cell states (collectively known as expression proteomics) and, (ii) with the identification of components of functionally active protein complexes and characterization of the intricate protein-protein interactions involved in intracellular protein trafficking and signaling pathways (collectively known as cell-mapping proteomics).
  • MS mass spectrometry
  • MS approaches to proteomics generally involve one or two-dimensional electrophoretic (2DE) separation of protein mixtures, after which the protein spot or gel slice is cut out and subjected to in situ proteolysis using tiypsin.
  • Peptides are then extracted and subjected to mass spectrometric analysis.
  • the masses of these peptides are characteristic of the protein, and provide a peptide "mass fingerprint" which can be used in database searches to identify the protem [Henzel, WJ. Billeci T.M., Stults J.T., Wong S.C., Grimley C. and Watanabe C. Proc. Natl. Acad. Sci. USA. 1993, 90, 5011-5015.].
  • a more comprehensive approach, particularly for the identification and quantitation of individual components present in complex protein mixtures, is to subject each of the proteolytically derived peptides to tandem mass spectrometry.
  • Subsequent identification of each peptide may be performed by either database analysis of the uninte reted product ion spectrum [Eng, J.K. McCormack, A.L. and Yates, J.R. J. Am. Soc. Mass Spectrom. 1994, 5, 976-989.], through database searching of a partially derived amino acid "sequence tag" [Mann, M. and Wilm, M. Anal. Chem. 1994, 66, 4390-4399.], or by "de- novo" sequence analysis [Hunt, D. F., Yates, J.R., Shabanowitz J., Winston S. and Hauer, C.R. Proc. Natl. Acad. Sci. USA 1986, 83, 6233-6237.].
  • MS/MS spectra obtained during MS based peptide sequencing are unassignable (i.e., product ion spectra are too complex, too low in abundance, or lack sufficient ions to enable their interpretation) [Simpson, R.J., Connelly, L.M., Eddes, J.S., Pereira, J.J., Moritz, R.L. and Reid, G.E. Electrophoresis. 2000, 21, 1707-1732].
  • proteomics A fundamental aspect of proteomics research is the determination of protein expression levels between two different states of a biological system (i.e., relative quantification of protein levels), such as that encountered between a normal and diseased cell or tissue.
  • mR ⁇ A expression levels transcriptionomics
  • proteomics array-based gene expression monitoring or other gene expression methods for measuring mR ⁇ A abundances, alone, are insufficient for analyzing the cell's protein complement [Gygi, S.P., Rochon, Y., Franza, B.R. and Aebersold, R. Mol. Cell. Biol. 1999, 19, 1720-1730.].
  • the site specific labels allow efficient identification of those peptides containing the enriched amino acid mass "tags", via comparison with their unlabelled forms. Additionally, by comparing the relative abundances of the peptides from the isotopically enriched sample with those from a sample prepared using naturally abundant isotopes, quantitation of changes in the level of protein expression between the two samples may be obtained. This in vivo labelling approach is limited, however, to those systems where cells can be cultured under conditions suitable for incorporation of the isotopic label.
  • the second approach involves in vitro chemical derivatization with isotopically enriched labels following isolation of the proteins from the cellular matrix.
  • ICAT isotope coded affinity tag
  • Proteins from two different cell/tissue states are reduced and S-alkylated with either naturally abundant (light) or isotopically enriched (heavy) ICAT reagents, respectively, each containing a biotin moiety for subsequent affinity selection of cysteine-containing peptides by streptavidin affinity purification, leading to simplification of the mixture prior to MS analysis.
  • the abundance ratios of the proteolytically derived peptides containing the heavy isotope "diseased" sample compared to those originating from the light isotope "normal” sample (or between those containing structural labels) for each of these methods are indicative of changes in the level of protein expression between the two samples, thereby allowing their differential quantitation.
  • Proteins resolved by 2D gels or ID SDS-PAGE can be detected by autoradiography or storage phosphorimaging using in vivo or in vitro 32 P labelling [Ji H, Baldwin GS, Burgess AW, Moritz RL, Ward LD, and Simpson RJ. JBiol Chem 1993, 268, 13396-13405.; Boyle W.J., Geer Van der P. and Hunter T. Methods Enzymol. 1991, 201, 110-149.; Yan J.X., Packer N.H., Gooley A.A. and Williams K.L. J. Chromatogr. 1998, 808, 23-41.], or by western blotting using antibodies to detect phosphorylated proteins.
  • mass spectrometry has proven to be particularly useful for the analysis of protein phosphorylation [Neubauer, G. and Mann, M. Anal Chem 1999, 71, 235-242.].
  • mass spectrometric analysis of phosphopeptides is more complicated compared to that for unmodified peptides, due to their lower ionization efficiencies in positive ion MS analysis mode [Liao P.C., Leykam J., Andrews P.C., Cage D.A. and Allison J. Anal. Biochem.
  • phosphoserine- and phosphothreonine-containing peptides can readily undergo the facile loss of phosphoric acid (H 3 PO ) upon ESI and MALDI ionization, and upon low energy CID.
  • H 3 PO phosphoric acid
  • the sample may be enriched for phosphopeptides, as discussed above, in order to reduce the excess of unmodified peptides that suppress ionisation.
  • limitations still exist when attempting to identify the site of phosphorylation by tandem mass spectrometry, due to the lability of the phosphate side chain.
  • Phosphopeptide identification may also be performed by precursor ion scan mode monitoring of the characteristic phosphate specific product ions at m/z 79 (PO " ), and m/z 89 (H PO 4 " ), following collision induced dissociation (CID) of the phosphopeptide ions in negative ion mode, or by monitoring for the loss of H PO (98 Da) or HPO 3 (80 Da) in positive ion neutral loss scan mode [Carr S.A., Huddleston M.J., and Annan R.S. Anal. Biochem. 1996, 239, 180-192.; Schlosser A., Pipkorn R., Bossemeyer D., and Lehmann W.D. Anal Chem. 2001, 73, 170-176.].
  • CID collision induced dissociation
  • Product ion scan mode MS/MS of the intact phosphopeptide can sometimes be used to characterize the precise site of phosphate attachment within the peptide, as the b- and y- type fragment ions formed from peptide backbone cleavage may contain information on the specific location of the phosphorylated amino acid residue.
  • this technique is often hampered by extensive gas-phase dephosphorylation and elimination of phosphoric acid (H 3 PO 4 , 98 Da) from phosphoserine- and phosphothreonine, making it difficult to unambiguously locate the site of modification.
  • the phosphotyrosine side chain is relatively stable under MS and MS/MS conditions due to the relatively high stability of the arylphosphate modification, therefore the location of phosphotyrosine residues can be readily determined by the mass difference between two successive fragment ions of 243 Da. Indeed, the characteristic 'reporter' immonium ion of phosphotyrosine at 216.043 Da has been used for precursor ion experiments in positive ion mode MS/MS for selective identification of phosphotyrosine containing peptides [Steen H., Kuster B., Fernandez M., Pandey A., and Mann M. 2001. Anal. Chem. 73: 1440-1448].
  • a common way to overcome the lability of the phosphoserine and phospho threonine side chains during MS/MS is to replace the phosphate group with a more stable, less acidic
  • the method can also be used to incorporate an affinity "tag", thereby allowing enrichment of the derivatized peptides prior to their analysis
  • Mass spectrometry combined with cross-linking [Rappsilber, J., Siniossoglou, S., Hurt, E. C, and Mann, M. Anal. Chem. 2000, 72, 267-275.], or hydrogen/deuterium exchange [Yamada, ⁇ ., Suzuki, E., and Hirayama, K. Rapid Commun. Mass Spectrom. 2002, 16, 293-299] can be used for the rapid low-resolution evaluation of the three dimensional structures of proteins and protein complexes.
  • Cross-linking generally involves chemical [Uy, R., and Wold, F., 1977, In: “Protein Cross-linking” (Friedman, M., ed.), Plenum, New York.; Fancy, D.A. Current Opin. Chem. Biol. 2000, 4, 28-33.] or photochemical [Chowdhry, V., and Westheimer, F.H., Annu. Rev. Biochem. 1979, 48, 293-325.; Fancy, D.A. and Kodadek, T. Proc. Natl. Acad.
  • Tandem mass spectrometry (MS/MS) dissociation methods [McLuckey, S.A. and Goeringer, D.E. J. Mass Spectrom. 1997, 32, A61-A1A.] methods, whereby a precursor ion of interest is mass selected, subjected to fragmentation via collision-induced dissociation (CID) and then the resultant product ions mass analyzed to derive structural information relating to the amino acid sequence of the peptide or to indicate the presence and location of post- translational modifications, maybe used to address at least some of the limitations indicated above.
  • CID collision-induced dissociation
  • MS/MS scan modes whereby the mass spectrometer is set to detect diagnostic low mass product ions, or product ions offset by a given mass from the precursor ion, respectively, has been demonstrated to increase sensitivity by 1-2 orders of magnitude over conventional MS based detection methods [Wilm M; Neubauer G; Mann M. Anal. Chem. 1996, 68, 527-533.].
  • precursor ion or neutral loss scan modes can be used to indicate the presence of peptides containing certain structural features via formation of diagnostic "signature ions" formed upon CID MS/MS, giving greater specificity compared to that of MS based methods, and overcoming the problem of low abundance peptides overlapping with other higher abundance components present in the mixture.
  • MS/MS based approaches One of the limitations of MS/MS based approaches however, is that fragmentation giving rise to the product ion or neutral loss of interest is usually only one of many dissociation channels, thereby "diluting" the spectrum, limiting sensitivity and potentially complicating subsequent interpretation of the spectra. Also, MS/MS methods for quantitation of differential protein expression have not been described.
  • the present invention is based on a fixed-charge derivatization approach, which is designed to direct the dissociation of the peptide toward a single predictable fragmentation channel, resulting in the formation of a single product, thereby allowing its selective identification from a complex mixture by precursor ion or neutral loss scan mode MS/MS, then subjecting it to further structural interrogation, using MS/MS or multistage
  • MS/MS MS n
  • MS/MS MS/MS (MS n ) or by determination of an "accurate mass tag" of the precursor or product ion
  • Goodlet, D.R., Bruce, J.E. Anderson, G.A., Rist, B., Pasa-Tolic, L., Fiehn, O., Smith, R.D. and Aebersold, R. Anal. Chem. 2000, 72, 1112-1118.
  • the present invention provides methods of analysis of amino acids, peptides or proteins, the methods comprising:
  • the product ion having the second characteristic mass-to-charge ratio may be either a charged amino acid, peptide or protein containing product ion formed by neutral loss of the fixed charge from the precursor ion, or a product ion formed by charged loss of the fixed charge from the precursor ion.
  • the method of the invention comprises the further step of:
  • Step (6) maybe perfonned by first repeating steps (1), (2), (3) and (4) and then subjecting the product ion having the second characteristic mass-to-charge ratio formed by (i) neutral loss from the precursor, which will have a charge state the same as that of the precursor, or (ii) the complementary product ion to the charged product ion formed by charged loss from the precursor ion, which corresponds to a protein or peptide containing product ion having a charge state one lower than the precursor, to a further stage of dissociation to form a series of product ions having a range of mass to charge ratios, for the purpose of determining the amino acid sequence of the peptide and subsequently, the identity of its protein of origin.
  • step (6) may be carried out by use of high resolution mass analyzers to obtain mass accuracies of approximately 1-5 ppm on the product ion detected in step (5), or its complementary product ion (i.e., to derive an "accurate product ion mass tag").
  • This coupled with database searching, maybe employed for subsequent identification of those peptides found to contain a fixed charge derivative.
  • database searching algorithms can be improved such that unambiguous identification of the protein from which the peptide is derived has been achieved from this information alone.
  • the amino acid, peptide or protein ion may be dissociated by any suitable dissociation method including, but not limited to, collisions with an inert gas (known as collision-induced dissociation (CID or collisionally-activated dissociation (CAD); (ii) collisions with a surface (known as surface-induced dissociation or SID); (iii) interaction with photons (e.g. via a laser) resulting in photodissociation; (iv) thermal/black body infrared radiative dissociation (BIRD), and (v) interaction with an electron beam, resulting in electron-induced dissociation for singly charged cations (EID), electron-capture dissociation (ECD) for multiply charged cations, or combinations thereof.
  • CID collision-induced dissociation
  • SID surface-induced dissociation
  • BIRD thermal/black body infrared radiative dissociation
  • EID electron-induced dissociation
  • ECD electron-capture dissociation
  • the methods of analysis of the present invention may be used for amino acid, peptide or protein identification, differential quantitation, analysis of post translational modification status, analysis of cross-linking status or interaction of proteins.
  • the present invention provides methods of analysis of amino acids, peptides or proteins containing a fixed charge derivative, at a site other than at the C-terminal or N- terminal end thereof.
  • the fixed-charge derivative may be contained on the side-chain of a selected amino acid, or a side-chain of a selected amino acid residue contained within a protein or peptide.
  • the selected amino acid residue is that of a "rare" amino acid, as described in more detail below.
  • the fixed-charge derivative may be contained on a side-chain of a post translational modified amino acid residue, as described in more detail below.
  • the fixed- charge derivative may be on a cross-link contained between two proteins or peptides, as described in more detail below.
  • the selected amino acid residue may be one containing a S atom in the side chain thereof.
  • Preferred amino acid residues are methionine, cysteine, homocysteine or selenocysteine.
  • the selected amino acid residue may also be tryptophan or tyrosine.
  • the side chain may also contain an S-alkyl group.
  • Preferred amino acid residues are methionine, S- alkyl cysteine, S-alkyl homocysteine, S-alkyl tryptophan or S-alkyl tyrosine.
  • Derivatization of the side chain of a selected amino acid residue to introduce a fixed-charge may be achieved by strategies known in the art.
  • the fixed-charge may also be contained on an O-linked post-translationally modified amino acid residue (for example, a dehydroalanine residue formed by ⁇ -elimination from an O-linked post-translationally modified serine amino acid residue, or a dehydroamino-2- butyric acid residue formed by ⁇ -elimination from an O-linked post-translationally modified threonine amino acid residue) contained within a protein or peptide. Derivatization of a formerly post-translationally modified amino acid residue to introduce a fixed-charge may be achieved by strategies known in the art.
  • the fixed-charge may also be contained within a cross-linking reagent, or a cross-link contained between two amino acids, peptides or proteins. Derivatization of a cross-linking reagent or a cross-link contained between two amino acids, peptides or proteins to introduce a fixed-charge may be achieved by strategies known in the art.
  • Non-limiting examples of the fixed-charge include a sulfonium ion, a quaternary alkylammonium or a quaternary alkylphosphonium ion.
  • tandem mass spectrometry Analysis of the amino acid, peptide or protein ion may be performed by tandem mass spectrometry.
  • the tandem mass spectrometer may be equipped with electrospray ionization (ESI) or matrix assisted laser deso ⁇ tion ionization (MALDI) interfaces to transfer the protem or peptide ion from solution into the gas-phase.
  • ESI electrospray ionization
  • MALDI matrix assisted laser deso ⁇ tion ionization
  • Mass analysers that are applicable to tandem mass spectrometry fall into two basic categories: tandem-in-space and tandem-in-time. Combinations of these types also can be used.
  • Tandem-in-space mass spectrometers have discreet mass analysers for each stage of mass spectrometry; examples include sector (commonly double focusing sector and "hybrid” combinations of sector and quadrupole analyser instruments), time of flight and triple quadrupole instruments, as well as “hybrid” combinations of time of flight and quadrupole instruments. Tandem-in-time mass instruments have only one mass analyser, and each stage of mass spectrometry takes place in the same region, but is separated in time via a sequence of events. Examples of tandem in time mass analysers include both two- and three-dimensional quadrupole ion trap and Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometers.
  • FT-ICR Fourier-transform ion cyclotron resonance
  • the methods of the invention in certain embodiments also include one or more steps of protein extraction, protein separation, reduction and alkylation of cysteine disulfides 5 and/or digestion.
  • the fixed-charge derivatization approach in the method of the present invention may be applied to the quantitation of differential protein expression based on the inco ⁇ oration of suitable isotopic (e.g., C, N, H) or structural labels to the fixed charge.
  • suitable isotopic e.g., C, N, H
  • the fixed-charge derivatization approach in the method of the present invention may be also applied to the
  • the fixed-charge derivatization approach in the method of the present invention may also be applied to the identification and characterization of protein-protein interactions via inco ⁇ oration of the fixed-charge derivative into a suitable cross-linking reagent.
  • X preferably is any halogen, sulfonic ester, perchlorate ester or chlorosulfonate.
  • R ⁇ R 5 are H
  • Ri' - R 6 ' are 12 C.
  • the substituted acetophenone is an isotopically encoded substituted acetophenone, or a salt thereof, or a solvate thereof, preferably in which at least one of, more preferably two or more, and more preferably still at least three of R R 5 are 2 H, and Ri'-R ⁇ ' are 12 C, or in which R ⁇ -R 5 are H, and at least one of, preferably two or more, and more preferably at least three of Ri '-R ⁇ ' are 13 C.
  • At least one of Ri-R 5 is a functional group containing an atom other than hydrogen or carbon.
  • the substituted acetophenone is water soluble.
  • Certain water soluble molecules include the foregoing molecules in which Ri is SO 2 H, and R -R 5 are H, and Ri '- R 6 ' are 12 C; Ri is H, R 2 is SO 2 H, and R 3 -R 5 are H, and Ri'-R 6 ' are 12 C; R 1-2 are H, R 3 is SO 2 H, and R 4 -R 5 are H, and R -Re' are 12 C; Ri is SO 3 H, and R 2 -R 5 are H, and Ri'-Re' are 1 C; Ri is H, R 2 is S0 3 H, and R 3 -R 5 are H, and R '-R 6 ' are 12 C; or R ⁇ 2 are H, R 3 is SO 3 H, and R 4 -R 5 are H, and Ri'-R ⁇ ' are 12 C.
  • Isotopically encoded water soluble substituted acetophenone molecules include the foregoing wherein Ri is SO 2 H, and at least one of, and preferably at least three of R 2 -R 5 are 2 H, and Rt'- R ⁇ ' are 12 C; R 1 is SO 2 H, and R 2 -R 5 are H, and at least one of, and preferably at least three of R ⁇ - Rg' are U C; R is SO H, and at least one of, and preferably at least three of R] and R 3 -R 5 are H, and Ri'-R 6 ' are 12 C; R 2 is SO 2 H, and R !
  • R 3 -R 5 are H, and at least one of, and preferably at least three of R R 6 ' are 13 C;
  • R 3 is SO 2 H, and at least one of, and preferably at least three of R R 2 and R 4 -R 5 are 2 H, and R ⁇ - R 6 ' are 12 C;
  • R 3 is SO 2 H, and R t - R and j-Rs are H, and at least one of, and preferably at least three of Ri'- Rg' are 13 C;
  • Ri is SO 3 H, and at least one of, and preferably at least three of R 2 -R 5 are 2 H, and Ri'- R 6 ' are 12 C;
  • Ri is SO 3 H, and R 2 -R 5 are H, and at least one of, and preferably at least three of Ri'- R 6 ' are 13 C;
  • R 2 is SO H, and at least one of, and preferably at least three of Ri and R 3 - R 5 are 2 H, and
  • Ri '- R 6 ' are C;
  • R 2 is SO 3 H, and Ri and R 3 -R 5 are H, and at least one of, and preferably at least three of Ri '- R 6 ' are C;
  • R 3 is SO 3 H, and at least one of, and preferably at least three of Ri-R 2 and R R 5 are 2 H, and Rj'- R 6 ' are 12 C; or
  • R 3 is SO 3 H, and R R 2 and R 4 -R 5 are H, and at least one of, and preferably at least three of Ri'-are 13 C. hi these molecules, it will be understood that other combinations and amounts of the listed substituents are possible and the invention embraces such combinations.
  • substituted acetophenones (or salts thereof, or solvates thereof) are provided that have the following formula:
  • X is a sulfonic ester, perchlorate ester or chlorosulfonate.
  • Preferred substituted acetophenones are those in which R 1 -R 5 are H, and Ri'-Re' are 12 C.
  • the invention also includes substituted acetophenones that are isotopically encoded substituted acetophenones, salts thereof, and solvates thereof.
  • Certain preferred substituted isotopically encoded acetophenones are those in which at least one of, and preferably at least three of R R 5 are 2 H, and Ri'-R ⁇ are 12 C, or those in which R 1 -R 5 are H, and at least one of, and preferably at least three of Ri'-R 6 ' are 13 C.
  • the invention further includes water soluble substituted acetophenones, salts thereof, and solvates thereof, having the following formula:
  • X is any halogen, sulfonic ester, perchlorate ester or chlorosulfonate.
  • X is Br or I. 5
  • the water soluble substituted acetophenones are those in which Ri is SO 2 H, R 2 -R 5 are H, and Ri'- R 6 ' are 12 C; Ri is H, R 2 is SO 2 H, R 3 -R 5 are H, and Ri'- R 6 ' are 12 C; Ri- 2 are H, R 3 is SO 2 H, R ⁇ Rs are H, and Ri'- Re' are 12 C; Ri is SO 3 H,
  • the water soluble substituted acetophenone also can be isotopically encoded forms of those described herein, preferably wherein X is any halogen, sulfonic ester, perchlorate ester or chlorosulfonate.
  • the isotopically encoded water soluble substituted acetophenones are those in which Ri is SO H, at least one of, and preferably at least three of R 2 - R 5 are 2 H, and Ri'-R ⁇ ' are 12 C; Ri is SO 2 H, R -R 5 are H, and at least one of, and 5 preferably at least three of R ⁇ '-R 6 ' are 13 C; R is SO 2 H, at least one of, and preferably at least three of Ri and R 3 -R 5 are 2 H, and Ri'-Re' are 12 C; R 2 is SO 2 H, Ri and R 3 -R 5 are H, and at
  • Ri '-R 6 ' 1 ⁇ least one of, and preferably at least three of Ri '-R 6 ' are C;
  • R is SO 2 H, at least one of, and preferably at least three of R ⁇ -R 2 and r R 5 are 2 H, and Ri'-R ⁇ ' are 12 C;
  • R 3 is SO 2 H, Ri-R 2 and R 4 - R 5 are H, and at least one of, and preferably at least three of Ri'-R 6 ' are 13 C;
  • Ri is 0 SO 3 H, at least one of, and preferably at least three of R 2 - R 5 are 2 H, and R ⁇ '-R 6 ' are 12 C;
  • Ri is SO 3 H, R2-R 5 are H, and at least one of, and preferably at least three of Ri'-R 6 ' are 13 C;
  • R 2 is SO 3 H, at least one of, and preferably at least three of Ri and R 3 -R 5 are 2 H, and R ⁇ '-R 6 ' are
  • kits for analysis of amino acids, peptides or proteins by mass spectrometry include one or more containers containing the substituted acetophenones described herein.
  • Certain reagent kits for analysis of amino acids, peptides or proteins by mass spectrometry of the invention include a container containing a substituted acetophenone, or a salt thereof, or a solvate thereof, having the following formula:
  • X is a halide, preferably Br or I.
  • Certain preferred substituted acetophenones in the kits are those in which Ri-R 5 are H, and R ⁇ '-R 6 ' are 12 C.
  • Other preferred substituted acetophenones in the kits are isotopically encoded substituted acetophenones, or salts thereof, or solvates thereof, preferably those in which at least one of, and preferably at least three of R ⁇ -R 5 are H, and Ri'-Re' are C, or in which R ⁇ -R 5 are H, and at least one of, and preferably at least three of Ri '-R 6 ' are C.
  • the water soluble derivatives of the substituted acetophenones listed above also can be included in the kits. Instructions for use of the substituted acetophenones in the analysis of amino acids, peptides or proteins by mass spectrometry also may be included.
  • the reagent kit further can contain one or more containers containing: cysteine disulfide reducing agents, cysteine alkylating reagents, proteases or chemical cleavage agents, and/or solvents.
  • the cysteine disulfide reducing agents preferably include dithiothreitol (DTT), 3-mercaptoethanol, tris-carboxyethyl phosphine (TCEP), and/or tributylphosphine (TBP).
  • the cysteine alkylating reagents preferably include alkylhalides (e.g. iodoacetic acid, iodoacetamide), vinylpyridine and/or acrylamide.
  • the proteases or chemical cleavage agents preferably include trypsin, Endoproteinase Lys-C, Endoproteinase Asp-N, Endoproteinase Glu-C, pepsin, papain, thermolysin, cyanogen bromide ⁇ hydroxylamine hydrochloride, 2-[2'-nitrophenylsulfenyl]-3-methyl-3'-bromoindole (BNPS- skatole), iodosobenzoic acid, pentafluoropropionic acid and/or dilute hydrochloric acid.
  • the solvents preferably include urea, guanidine hydrochloride, acetonitrile, methanol and/or water.
  • the invention also includes amino acids, or peptides comprising an amino acid, derivatized to include a side chain fixed-charge sulfonium ion, quaternary alkylammonium ion or quaternary alkylphosphonium ion.
  • amino acid is derivatized using the substituted acetophenone described herein.
  • the amino acid is derivatized using a substituted acetophenone, or a salt thereof, or a solvate thereof, having the following formula:
  • X is a halide, preferably Br or I.
  • Certain preferred substituted acetophenones used to derivatize the amino acids or peptides are those in which Ri-R 5 are H, and Ri'-Re' are 12 C.
  • Other preferred substituted acetophenones used to derivatize the amino acids or peptides are isotopically encoded substituted acetophenones, or salts thereof, or solvates thereof, preferably those in which at least one of, and preferably at least three of Ri-R 5 are 2 H, and
  • Ri '-Re' are C, or in which Ri-R 5 are H, and at least one of, and preferably at least three of Rj'-R ⁇ ' are C.
  • the water soluble derivatives of the substituted acetophenones listed above also can be used to derivatize the amino acids or peptides.
  • the amino acid derivative is isotopically encoded.
  • the invention in another aspect provides methods for providing an internal standard in a mass spectrometer method comprising adding to a sample a predetermined quantity of the fixed charge derivatized amino acid or peptide described above.
  • GAILMGAILA SEQ ID NO:l
  • GAILMGAILA SEQ ID NO:l
  • methionine side chain fixed-charge derivative (A) CID MS/MS of the [M+H] + ion.
  • B CID MS/MS of the methionine side chain fixed-charge acetophenone (AP) sulfonium [M(AP)] + ion.
  • AP methionine side chain fixed-charge acetophenone
  • AP methionine side chain fixed-charge acetophenone
  • AP methionine side chain fixed-charge acetophenone
  • AP methionine side chain fixed-charge acetophenone
  • AP methionine side chain fixed-charge acetophenone
  • FIG. 1 Quadrupole ion trap tandem mass spectrometry of doubly charged GAILMGAILA (SEQ ID NO: 1) and its methionine side chain fixed-charge derivative.
  • A CID MS/MS of the [M+2H] 2+ ion.
  • B CID MS/MS of the methionine side chain fixed-charge acetophenone (AP) sulfonium [M(AP)+H] 2+ ion.
  • AP methionine side chain fixed-charge acetophenone
  • GAILMGAILK (SEQ ID NO:2) and its methionine side chain fixed-charge derivative.
  • A CID MS/MS of the [M+H] + ion.
  • B CTD MS/MS of the methionine side chain fixed-charge acetophenone (AP) sulfonium [M(AP)] + ion.
  • AP methionine side chain fixed-charge acetophenone
  • AP methionine side chain fixed-charge acetophenone
  • GAILMGAILK SEQ ID NO:2
  • methionine side chain fixed-charge derivative (A) CID MS/MS of the [M+2H] 2+ ion.
  • GAILMGAILR (SEQ ID NO:3) and its methionine side chain fixed-charge derivative.
  • A CID MS/MS of the [M+H] + ion.
  • B CTD MS/MS of the methionine side chain fixed-charge acetophenone (AP) sulfonium [M(AP)] + ion.
  • AP methionine side chain fixed-charge acetophenone
  • AP methionine side chain fixed-charge acetophenone
  • AP methionine side chain fixed-charge acetophenone
  • AP methionine side chain fixed-charge acetophenone
  • AP methionine side chain fixed-charge acetophenone
  • AP methionine side chain fixed-charge acetophenone
  • AP methionine side chain fixed-charge acetophenone
  • AP methionine side chain fixed-charge acetophenone
  • AP methionine side chain fixed-charge acetophenone
  • GAILMGAILR SEQ ID NO:3 and its methionine side chain fixed-charge derivative.
  • A CTD MS/MS of the [M+2H] 2+ ion.
  • B CID MS/MS of the methionine side chain fixed-charge acetophenone (AP) sulfonium
  • GAILMGAILK (SEQ ID NO:2).
  • B CID MS/MS product ion spectra of the [M(AP)+H] 2+ ion obtained at a collision energy (laboratory frame) of 12V.
  • C CID MS/MS product ion spectra of the [M(AP)+H] 2+ ion obtained at a collision energy (laboratory frame) of 22V.
  • FIG 11. (A) Total ion current trace from a 200 ⁇ m I.D. capillary RP-HPLC - MS analysis of 10 pmol of a reduced and S-carboxyamidomethylated tryptic digest of bovine serum albumin, following derivatization using bromoacetophenone. Individual mass spectra for two regions of the total ion current chromatogram, indicated by arrows, are shown in Figures 1 IB and 1 lC. Figure 12. (A) Total ion current trace from a 200 ⁇ m ID.
  • the peptides are IETMR (SEQ JJD NO:9), ETYGDMADCCEK (SEQ ID NO:10), TVMENFVAFVDK (SEQ ID NO: 11), and MPCTEDYLSLILNR (SEQ ID NO: 12).
  • Individual neutral loss scan mode mass spectra for two regions of the total ion current chromatogram, indicated by arrows, are shown in Figures 12B and 12C.
  • GAILMGAILR (SEQ ID NO:3). Expanded regions of the m/z range for the triply and doubly charged precursors are shown in panels (B) and (C) respectively.
  • FIG. 15 Summed neutral loss scan mode CID MS/MS spectrum of a 1.0 : 0.1 pmol/ ⁇ L mixture of do and d 5 containing methionine side chain fixed-charge acetophenone sulfonium ion derivative of GAILMGAILR (SEQ ID NO:3). Individual neutral loss mass spectra for the triply and doubly charged precursors are shown in panels (B) and (C), respectively.
  • FIG. 16 (A) Total ion current trace from a 75 ⁇ m ID. capillary RP-HPLC • MS analysis of a 1.0 : 1.0 pmol mixture of d 0 and d 5 containing methionine side chain fixed-charge acetophenone (AP) sulfonium ion derivative of GAILMGAILR (SEQ ID NO:3). (B) Summed mass spectra from the region indicated in panel (A). (C) Total ion current trace from a 75 ⁇ m ID.
  • Fixed-charge includes any charge localised to a specific heteroatom contained within the protein or peptide, or to a specific heteroatom contained within the derivatization reagent (e.g., in solution or in the gas-phase), by the attachment of any moiety.
  • Fixed charge derivatization means the introduction of a fixed charge as defined above.
  • the fixed charge may be introduced either by introducing a neutral reagent to subsequently form the fixed charge at a specific site within the protein or peptide, or by introduction of a reagent containing the fixed charge to a specific site within the protein or peptide.
  • the fixed charge derivative thus formed preferably has a structure such that it allows the exclusive formation of a product ion upon dissociation that is characteristic of the fixed charge derivative.
  • “Protem”, as used herein, means any protein, including, but not limited to peptides, enzymes, glycoproteins, hormones, receptors, antigens, antibodies, growth factors, etc., without limitation. Proteins may be endogenous, or produced from other proteins by chemical or proteolytic cleavage. Preferred proteins include those comprised of at least 15-20 amino acid residues.
  • m cludes cross-linked proteins
  • Peptide as used herein includes any substance comprising two or more amino acids and mcludes di-, Lri-, oligo and polypeptides etc according to the number of amino acids linked by amide(s) bonds. Peptides may be endogenous, or produced from other peptides or proteins by chemical or proteolytic cleavage. Preferred peptides include those comprised of up to 15-20 amino acid residues. The term includes cross-linked peptides.
  • the amino acids are ⁇ -ammo acids
  • either the L-optical isomer or the D-optical isomer can be used.
  • the L -isomers are generally preferred.
  • alkyl is used herein to refer to a branched or unbranched, saturated or unsaturated, monovalent hydrocarbon. Suitable alkyl groups include, for example, structures containing one or more methylene, methine and/or methyne groups. Branched structures have a branching motif similar to i-propyl, t-butyl, i-butyl, 2-ethylpropyl, etc. As used herein, the term encompasses "substituted alkyls," and "cyclic alkyl.”
  • Substituted alkyl refers to alkyl as just described including one or more substituents such as, for example, alkyl, aryl, acyl, halogen (i.e., alkylhalos, e.g., CF 3 ), hydroxy, amino, amide, alkoxy, alkylamino, acylamino, thioamido, acyloxy, aryloxy, aryloxyalkyl, ether, ester, disulfide, mercapto, thia, aza, oxo, both saturated and unsaturated cyclic hydrocarbons, heterocycles and the like. These groups may be attached to any carbon or substituent of the alkyl moiety.
  • these groups may be pendent from, or integral to, the alkyl chain.
  • the substituted alkyl group may be covalently attached to an insoluble bead or polymer, and contain a chemical or photochemical cleavage site between the insoluble bead or polymer and the alkyl group.
  • aryl is used herein to refer to an aromatic substituent, which may be a single aromatic ring or multiple aromatic rings which are fused together, linked covalently, or linked to a common group such as a methylene or ethylene moiety.
  • the common linking group may also be a carbonyl as in acetophenone.
  • the aromatic ring(s) may include phenyl, naphthyl, biphenyl, diphenylmethyl and benzophenone among others.
  • aryl encompasses "arylalkyl” and "substituted aryl.”
  • Substituted aryl refers to aryl as just described including one or more functional groups such as lower alkyl, acyl, halogen, alkylhalos (e.g. CF 3 ), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy, phenoxy, mercapto and both saturated and unsaturated cyclic hydrocarbons which are fused to the aromatic ring(s), linked covalently or linked to a common group such as a methylene or ethylene moiety
  • the linking group ma.y also be a carbonyl such as in cyclohexyl phenyl ketone.
  • substituted aryl encompasses "substituted arylalkyl.”
  • arylalkyl is used herein to refer to a subset of “aryl” in which the aryl group is attached to another group by an alkyl group as defined herein.
  • substituted arylalkyl defines a subset of "substituted aryl” wherein the substituted aryl group is attached to another group by an alkyl group as defined herein.
  • acyl is used to describe a ketone substituent, ⁇ (O)R, where R is alkyl or substituted alkyl, aryl or substituted aryl as defined herein.
  • GAILMGAILA SEQ ID NO:l
  • GAILMGAILK SEQ ID NO:2
  • GAILMGAILR SEQ ID NO:3
  • GAILAGAILA SEQ ID NO:4
  • GAILAGAILK SEQ ID NO:5
  • GAILAGAILR SEQ ID NO:6
  • Polymer supported pyridyl bromide perbromide, iodoacetic acid and iodoacetamide were from Sigma (St. Louis, MI, USA). Methanol, and acetonirrile (Chromar grade) were purchased from Mallinkrodt (Paris, KY, USA). Formic acid and acetic acid were obtained from BDH
  • d 5 -bromoacetophenone was synthesized according to the method of Frechet et al [Frechet, J.M.J., Farrall, M.J. and Nuyens, L.J. J. Macromol Sci-Chem. 1977, All, 507- 514.] Briefly, 1.25 mL of ds-acetophenone was added to 4.8 g Polymer supported pyridyl bromide perbromide ( ⁇ 3 meq Br 3 " /g resin) in 30 mL methanol then allowed to react with stirring at room temperature for 4 hours. The resultant ⁇ -bromo ketone was obtained in pure form by filtration of the reaction mixture and evaporation of the solvent, followed by recrystallization prior to use.
  • Mass spectrometric analysis was performed using either a (i) quadrupole ion trap (Finnigan-MAT model LCQ-DECA, San Jose, CA), (ii) quadrupole-time-of-flight (Micromass model Q-TOF2, Manchester, UK), or (ii) triple quadrupole (Finnigan model TSQ, San Jose, CA) mass spectrometer, all equipped with electrospray ionization interfaces.
  • Electrospray interface conditions were optimized to maximize the intensity of the ion of interest. Typical conditions were: spray voltage, -4.5 kV; nitrogen source gas, 1 psi; cone gas, 100 (arbitrary units); source temperature, 50°C; desolvation temperature, 150°C; cone voltage, 50V (singly charged ions), 30V (doubly charged ions) and
  • Samples (1 pmol/ ⁇ L in 50:50:1 H O:CH 3 CN:acetic acid) were introduced to the mass 20 spectrometer by a home built nano-electrospray ionization source at a flow rate of 200 nL/min.
  • the spray voltage was maintained at -1.8 kV.
  • the heated capillary temperature was 150°C.
  • the argon collision gas pressure was maintained at 1.5 mtorr.
  • the instrument was operated under unit resolution conditions.
  • Neutral loss mode MS/MS scans (neutral losses of 83 and 85.5 Da for do- and d 5 - containing doubly charged ions, and 55.3 and 57 Da for do- 25 and d 5 - containing triply charged ions) were performed at collision energies of 18V and 13V, respectively.
  • Product ion CID MS/MS spectra of peptide ions selectively identified by neutral loss scans were then acquired at 18V and 3 IV, and 13V and 18V for doubly and triply charged ions, respectively. All spectra shown are the average of 20 scans.
  • Capillary RP-HPLC was performed using (i) a column (200 ⁇ m ID. x 150 mm O.D 30 fused silica), packed with Brownlee RP-300, 7 ⁇ m dimethyloctyl silica, developed at a flow rate of 3.6 ⁇ L min "1 using a linear 60 minute gradient from 0-100%B, where solvent A was 0.1M aqueous acetic acid, and solvent B was 0.1M aqueous acetic acid containing 60% acetonitrile, or (ii) a column (75 ⁇ m I.D.
  • cysteine the amino acid targeted in the popular ICAT and related methods for selective mass spectrometric detection and quantitation
  • 96.9% contain cysteine
  • 98.35% coverage could be attained if proteins containing both amino acids were targeted for analysis, as only 1.65% of entries do not contain either methionine or cysteine. Therefore, approaches directed toward the identification of proteins containing methionine (or both methionine and cysteine) should cover a significantly greater fraction of the proteins represented in the database than approaches targeting for cysteine alone.
  • Methionine is a rare amino acid (see above).
  • the chemistry and biological applications of sulfonium ions are well documented, giving precedent for developing reagents to form these ions in applications involving specific
  • 25 chain is virtually independent of pH (even down to pHl) whereas the reactivity of all other nucleophilic functional groups (e.g. cysteine, lysine and histidine residues) decreases at low pH (due to protonation).
  • nucleophilic functional groups e.g. cysteine, lysine and histidine residues
  • sulfonium ions have been exploited to: (a) tag methionine sites in peptides and proteins [Lundblad. Techniques in Protem Modification, Chapter 8 "The Modification of Methionine” CRC Press. Florida, 1995.; Liu T.-H. "The role
  • the reagent used in these preliminary studies reacts specifically with the side chain of methionine in solution under acidic pH conditions to form a stable side chain sulfonium ion derivative (- CH(CH ) 2 S(CH 3 )R ⁇ + ) (step (i) of Scheme 1). Under these conditions, the reagent does not react with the free thiol of cysteine containing peptides.
  • the reagent may however, be used for derivatization of cysteine-containing peptides after initial S-alkylation of the cysteine thiol side chain under basic pH conditions to yield the derivative (-CHCH 2 SR ) (step (i) of Scheme 2).
  • step (ii) of Scheme 2 provided that the R 2 substituent used for the initial cysteine alkylation enhances sulfonium ion stability (i.e., the charge is stable) (step (ii) of Scheme 2).
  • conditions could be chosen so that the initial S-alkyl cysteine derivative is not stable to sulfonium ion formation, allowing specific derivatization of methionine containing peptides in cases where cysteines have been previously reduced and S-alkylated to enable efficient proteolysis during sample preparation.
  • Neutral loss mode scan mode CID tandem mass spectrometry (MS/MS) scans, or post data acquisition neutral loss product ion detection software following conventional CID MS/MS, can be used to selectively identify methionine and cysteine containing peptides via the characteristic loss of CH 3 SRi from methionine and RiSR 2 from cysteine, with formation of [M+nH-CH 3 SRi] (n+1)+ and [M+nH-RiSR 2 ] (n+1)+ product ions, respectively.
  • Methionine and cysteine containing peptides can therefore be identified individually, through the use of an R 2 group that does not correspond to -CH 3 , (i.e., the side chain alkyl group of methionine) or simultaneously, via initial alkylation of cysteine where the R 2 group corresponds to -CH 3 .
  • Reactions should be easy to perform using readily available reagents. Many of the potentially useful reagents are commercially available. Other potentially useful reagents may be synthesized by simple methods described in the literature. Importantly, the ability to inco ⁇ orate a suitable isotopic label into the selected derivative is an important consideration in allowing subsequent quantitation of differential protein expression using the same approach. Thus, isotopically labelled derivatives of these reagents should be commercially available or readily synthesized.
  • isotopically labeled d 5 -bromoacetophenone has been readily prepared here from d 5 -acteophenone using the brominating reagent, poly(4- vinylpyridinium tribromide), in a one-step process (see Materials and Methods). Reactions should be fast, proceed to completion and the resultant sulfonium ions must be chemically and thermally stable in solution [Stirling, M.J. and Patai, S. The Chemistry of the Sulfonium Group, Wiley: New York, 1981.; Stirling C.J.M. Sulfonium Salts in Organic Chemistry of Sulfur Oae, S. Ed.
  • the peptide was reacted for 4, 8, 16, and 32 hours with a 100 fold molar excess of each alkylating reagent then analysed by mass spectrometry to determine the extent of reaction. After 16 hours, only the acetophenone sulfonium ion derivative had reacted to completion [March J. "Advanced Organic Chemistry", 4th Ed., Wiley, New York, 1992, pp 343.; Halvorsen, S. J. Chem. Soc. Chem Comm, 1978, 327.; Yoh, L. Tetrahedron Lett, 1988, 29, 4431]. The ethyl derivative was found to have the least reactivity and stability in solution (only 1% reaction after 16 hours).
  • Scheme 3 (pathway 3) is highly unlikely given that the least stable carbocation is formed (CH 3 + ). Problems associated with Scheme 3 (pathways 4 and 5) can be avoided by choosing Ri groups which do not yield stable ions (e.g. avoid benzyl groups etc) and which do not form an alkene (e.g. avoid ethyl or substituted ethyl groups and higher homologues), respectively. Differentiation between the fragmentation reactions of
  • the ⁇ -carbonyl containing alkylating reagents, iodoacetic acid, iodoacetamide and bromoacetophenone are those that exhibit the favourable solution and gas phase reaction properties discussed above.
  • acetophenone allows ready incorporation of a suitable isotopic label (for example, via synthesis of d 5 - bromoacetophenone or C 6 -bromoacetophenone), to enable subsequent quantitation, and has therefore been the reagent used from here on.
  • any reagent having the general structure XR where X is any suitable leaving group consisting of, for example, halides, sulfonic esters, perchlorate esters or chlorosi fonates [March J. "Advanced Organic Chemistry", 4th Ed., Wiley, New York, 1992, pp 352-357], and R is any substrate with a carbon adjacent to the leaving group, such as alkyl, allyl or ⁇ -carbonyl groups, with the exception of those indicated above, and where an isotopic or structural label can be inco ⁇ orated to allow its use for differential quantitation, would be of interest.
  • X is any suitable leaving group consisting of, for example, halides, sulfonic esters, perchlorate esters or chlorosi fonates
  • R is any substrate with a carbon adjacent to the leaving group, such as alkyl, allyl or ⁇ -carbonyl groups, with the exception of those indicated above, and where an isotopic or structural label can be inco
  • triply charged ion examples were the ,only partial exceptions, where some charged side chain loss of protonated S-methyl acetophenone (CH 3 SCH 2 COC 6 H 5 +H + ), labeled as (-CH 3 S(AP)+H + ) in Figures 5A and 8A, (with the corresponding protonated S-methyl acetophenone
  • Pathway 1 of Scheme 4 involves direct proton transfer by an intramolecular nucleophile (Nu), such as the basic side chains of arginine or lysine, the N-terminal amino group or backbone amide carbonyl groups, with elimination of the neutral to yield a novel amino acid derivative, 3-amino-l-butenoic acid (vinyl glycine), with a residue mass of 83 Da.
  • Nu intramolecular nucleophile
  • Nu intramolecular nucleophile
  • arginine or lysine the basic side chains of arginine or lysine, the N-terminal amino group or backbone amide carbonyl groups
  • the fragmentation may be directed by a nucleophilic attack mechanism, whereby an adjacent amide carbonyl attacks the side chain to facilitate the neutral loss, yielding a cyclic product (shown in Pathway 2 of Scheme 4 as the 6 membered cyclic product formed from nucleophilic attack by the amide carbonyl on the N-terminal side of the modified methionine residue).
  • a nucleophilic attack mechanism whereby an adjacent amide carbonyl attacks the side chain to facilitate the neutral loss, yielding a cyclic product (shown in Pathway 2 of Scheme 4 as the 6 membered cyclic product formed from nucleophilic attack by the amide carbonyl on the N-terminal side of the modified methionine residue).
  • This mechanism is consistent with the growing recognition that nucleophilic attack processes are the major processes involved in the fragmentation of protonated peptide ions [O'Hair, R.A.J. J. Mass Spectrom. 2000, 35, 1377-1381. Schlosser, A. and Lehmann,
  • Intramolecular transfer of the acidic proton present in either the precursor ion (Pathway 1), or the product ion formed following methionine side chain cleavage (Pathway 2A or 2B) would result in "mobilization" of this proton, thereby allowing it to be involved in subsequent fragmentation reactions according to the mobile proton model developed for rationalizing the fragmentation of protonated peptide ions [Wysocki, V.H., Tsaprailis, G., Smith, L.L. and Breci, L.A. J. Mass Spectrom. 2000, 35, 1399-1406.].
  • the acidic proton formed upon loss of the side chain can be readily mobilized onto the peptide backbone by the direct proton transfer mechanism for cleavage in pathway 1 of Scheme A, or by ring opening of the cyclic product ion shown in pathway 2A of Scheme A, allowing subsequent fragmentation to take place at sites along the backbone, whereas, in the case of the singly protonated ions formed directly by electrospray, where the ionising proton is initially located on a basic side chain such as arginine or lysine, significant energy to overcome the barriers to proton transfer must be initially supplied in order for subsequent fragmentation of the backbone to occur.
  • a basic side chain such as arginine or lysine
  • MS of the doubly protonated product ion formed from dissociation of the acetophenone modified GAILMGAILA (SEQ ID NO:l) peptide yielded less extensive products than those formed by MS/MS of the unmodified peptide, with b 9 and b 2+ ions dominating.
  • the proton initially present in the precursor and the acidic proton formed by loss of the side chain from the doubly charged sulfonium ion derivative are not as "mobile” as those of the doubly protonated peptide where one proton is expected to reside on the amino terminal and one along the backbone; again consistent with an initial cyclic product formed from the fixed-charge derivative.
  • the energy resolved CID breakdown and appearance curves for the doubly charged sulfonium ion derivative of GAILMGAILK (SEQ D NO:2) is shown in Figure 9A.
  • the breakdown curve of the precursor [M(AP)+H] 2+ ion, the appearance/breakdown curve of the initial neutral loss product ion (-CH 3 S(AP)), as well as the appearance curves of the individual product ions and that of the summed product ion abundances (sum of product ions) are indicated. It can be seen that there is essentially no overlap between the breakdown of the [M(AP)+H] 2+ ion and the appearance of the individual product ions, indicating that these ions are not formed directly from the initial precursor.
  • the directed fragmentation of the fixed-charge derivatives gives the ability to use low energy CID conditions for formation of the initial neutral loss product ion and then to obtain a "pseudo MS 3 " spectrum under "high” energy CID conditions to obtain further structural information.
  • the appearance curves for the formation of the summed product ion abundances from the doubly protonated unmodified methionine and alanine containing peptides are shown in Figure 10.
  • GAILAGAILA (SEQ ID NO:4) 21.7 8.1 NA
  • GAILMGAILA (SEQ ID NO:l) 22.7 (+1.0) a 8.6 (+0.5) a NA
  • GAILM(AP)GAILA b (SEQ ID NO:l) 34.5 (+12.8) a 14.8 (+6.7) a NA
  • GAILAGAILK (SEQ ID NO:5) 36.8 10.8 NA
  • GAILMGAILK (SEQ ID NO:2) 38.4 (+1.6) a 11.4 (+0.6) a 11.4
  • GAILM(AP)GAILK b (SEQ ID NO:2) 43.0 (+6.2) a 21.5 (+10.7) a 11.6
  • GAILAGAILR (SEQ ID NO:6) 47.1 10.8 NA
  • GAILMGAILR (SEQ ID NO:3) 48.6 (+1.5) a 11.6 (+0.8) a NA
  • GAILM(AP)GAILR (SEQ ID NO:3) 53.7 (+6.6) a 22.1 (+11.3) a 11.7
  • sulfenylhalide would also react with cysteine containing peptides to yield an unsymmetrical disulfide (Scheme 5B), however, specificity of the reaction to tryptophan may be imparted by initial S- alkylation of cysteine, or reduction of the unsymmetrical disulfide prior to sulfonium ion formation.
  • the solution phase and gas phase criteria for sulfonium ion formation from 2- indole S-alkyl tryptophan modified residues are the same as those discussed above for methionine and for S-alkyl cysteine sulfonium ion formation.
  • methionine and tryptophan containing peptides could be identified individually, through the use of an Ri group that does not correspond to -CH 3 , (i.e., the side chain alkyl group of methionine) or simultaneously, via initial alkylation of tryptophan where the Ri group corresponds to -CH 3 .
  • 2-indole S-alkyl tryptophan and S-alkyl cysteine containing peptides could be identified individually, through the introduction of an Ri group to the S-alkyl tryptophan derivative that does not match that of the Ri group used for initial alkylation of cysteine, or simultaneously, via initial alkylation of tryptophan using the same Ri group as that used for initial cysteine alkylation.
  • similar electrophilic aromatic substitution reactions could also be used for the introduction of an S-alkyl group to the side chain of tyrosine residues, followed by subsequent formation of a sulfonium ion derivative.
  • RiSXi any reagent having the general structure RiSXi, where X ⁇ is any suitable leaving group consisting of, for example, halides, sulfonic esters, perchlorate esters or chlorosulfonates
  • X ⁇ is any suitable leaving group consisting of, for example, halides, sulfonic esters, perchlorate esters or chlorosulfonates
  • Ri is any substrate with a carbon adjacent to the leaving group, such as alkyl, allyl or c.-carbonyl groups, with the exception of those indicated above, and where an isotopic or structural label can be inco ⁇ orated to allow its use for differential quantitation
  • R 2 X 2 where X is any suitable leaving group consisting of, for example, halides, sulfonic esters, perchlorate esters or chlorosulfonates
  • R 2 is any substrate with a carbon adjacent to the leaving group, such as alkyl, allyl or ocarbonyl groups, with the exception of those indicated above, and where an isotopic or structural label can be inco ⁇ orated to allow its use for differential quantitation.
  • Sulfonium ions of tryptophan and cysteine containing peptides may also be formed directly, by initial reaction with a sulfenylhalide (X 2 RSX ⁇ ) containing a suitable leaying group X 2 , to subsequently yield a cyclic sulfonium ion derivative of tryptophan (Scheme 6 A) or cysteine (Scheme 6B).
  • reagent having the general structure X RSX l5 where Xi is any suitable leaving group consisting of, for example, halides, sulfonic esters, perchlorate esters or chlorosulfonates [March J. "Advanced Organic Chemistry", 4th Ed., Wiley, New York, 1992, pp 352-357]
  • R is any substrate with a carbon adjacent to the leaving group, such as alkyl, allyl or ⁇ -carbonyl groups, with the exception of those indicated above, and where an isotopic or structural label can be inco ⁇ orated to allow its use for differential quantitation
  • X 2 is any suitable leaving group consisting of, for example, halides, sulfonic esters, perchlorate esters or chlorosulfonates [March J. "Advanced Organic Chemistry", 4th Ed., Wiley, New York, 1992, pp 352-357]. Cysteine.
  • Cyclic sulfonium ions may also be formed specifically from cysteine containing peptides, by initial alkylation with a reagent (X 2 RXi) (Scheme 7A) or by initial disulfide exchange with a reagent (X 2 RSSX ⁇ ) (Scheme 7B), each containing a suitable leaving group X 2 , with subsequent formation of a cyclic sulfonium ion derivative.
  • R is any substrate with a carbon adjacent to the leaving group, such as alkyl, allyl or ⁇ -carbonyl groups, with the exception of those indicated above, and where an isotopic or structural label can be inco ⁇ orated to allow its use for differential quantitation
  • X 2 is any suitable leaving group consisting of, for example, halides, sulfonic esters, perchlorate esters or chlorosulfonates under neutral or basic pH conditions, or protonated amines or alcohols under acidic pH conditions
  • Xi is, for example, nitrobenzoic acid
  • R is any substrate with a carbon adjacent to the leaving group, such as alkyl, allyl or ⁇ -carbonyl groups, with the exception of those indicated above, and where an isotopic or structural label can be inco ⁇ orated to allow its use for differential quantitation
  • X 2 is any suitable leaving group consisting of, for example, halides, sulfonic esters, perchlorate esters or chlorosulfonates [March J. "Advanced Organic Chemistry", 4th Ed., Wiley, New York, 1992, pp 352-357].
  • fixed-charge sulfonium, quaternary alkylammonium or quaternary alkylphosphonium ion reagents should result in charged loss of the side chain via cis 1,2 elimination, with formation of a dominant low mass product ion with formation of its complementary neutral peptide species (if the initial charge state of the precursor was one), or a charged peptide ion species with a charge state one lower than the precursor (if the initial charge state of the precursor was greater than one).
  • These characteristic low mass product ion(s) can be selectively detected using a parent ion scan mode MS/MS experiment.
  • This side chain cleavage may be further enhanced by oxidation of the thioether [Steen, H. and Mann, M. J.
  • R X any reagent having the general structure R X, where X is any suitable leaving group consisting of, for example, halides, sulfonic esters, perchlorate esters or chlososulfonates [March J. "Advanced Organic Chemistry", 4th Ed., Wiley, New York, 1992, pp 352-357], and R + is any substrate with a carbon adjacent to the leaving group, such as alkyl, allyl or - carbonyl groups, and with a terminal sulfonium, quaternary alkylammonium or quaternary alkylphosphonium ion, and where an isotopic or structural label can be inco ⁇ orated to allow its use for differential quantitation.
  • X is any suitable leaving group consisting of, for example, halides, sulfonic esters, perchlorate esters or chlososulfonates
  • R + is any substrate with a carbon adjacent to the leaving group, such as alkyl, ally
  • a first sample is derivatized with either sulfonium or tertiary alkyl fixed-charge reagents containing naturally abundant (light) isotopes, then mixed with a second sample derivatized with the same reagent containing isotopically emiched (heavy) isotopes, prior to their introduction to the mass spectrometer.
  • the abundance ratios of the product ions formed by neutral loss or parent ion scan mode MS/MS of the light isotope tag containing peptide ions, compared to those containing the heavy isotope tag can be determined, which is indicative of changes in the abundance levels of proteins between the two samples.
  • the product ion representing the peptide product can then be automatically selected for further structural interrogation by "high" energy MS/MS or MS 3 .
  • a 10 times expanded region of the spectrum around m/z 700-800 shows the level of chemical noise associated with the mass spectrum.
  • the neutral loss scan mode MS/MS spectra of the same mixture neutral losses of 83 Da and 85.5 Da, respectively for the doubly charged precursors, and neutral losses of 55.3 Da and 57 Da, respectively for the triply charged precursors
  • Figure 13B the neutral loss scan mode MS/MS spectra of the same mixture (neutral losses of 83 Da and 85.5 Da, respectively for the doubly charged precursors, and neutral losses of 55.3 Da and 57 Da, respectively for the triply charged precursors) are shown in Figure 13B.
  • the m/z 700-800 regions for each spectrum have been expanded 10 times to indicate the level of chemical noise.
  • a neutral side chain derivative could be introduced (e.g., an alkyl thiol), followed by subsequent derivatization to form a fixed charge sulfonium ion. Therefore, sulfonium, quaternary alkylammonium, or quaternary alkylphosphonium ion chemistries as outlined above, could also be used for selective detection of peptides containing the modified amino acid residue.
  • the fixed charge derivatization method described here would result in the exclusive loss of the modified side chain, allowing improved detection of these peptides at higher levels of sensitivity and with better control of the subsequent dissociation process for subsequent structural elucidation.
  • the fixed charge derivatization method of the present invention would also allow differential quantitation of the O-linked post translational modification status.
  • the fixed-charge derivatization approach could also be extended toward the improved characterization of protein-protein interactions by inco ⁇ oration of the fixed-charge derivative into a suitable cross-linking reagent prior to cross-linking reactions, or by deritivatization of a cross-link contained between two proteins or peptides after a cross- linking reaction which, upon CID MS/MS, fragments via the specific loss of a neutral or charged entity from the cross-link. While the inco ⁇ oration of a labile MS/MS "tag" on a cross linker has been used by Back et al.
  • the method of the present invention could be employed here to direct the fragmentation of cross linked peptides toward exclusive cleavage of the fixed-charge, to yield either a characteristic neutral loss or low mass product ion (Scheme 10).
  • the product ion containing the cross linked peptide may then be automatically selected for further structural interrogation by higher energy MS/MS or by MS 3 . If quantitative comparison of cross linking between two different samples was required, an isotopic label could be also inco ⁇ orated into the cross-link.
  • any of the examples discussed above containing fixed-charge derivatives could also be selectively pre-enriched prior to mass spectrometric analysis by known chromatographic methods [Tang, J.R. and Hartley, B.S. Biochem. J. 1967, 102, 593.; Degen, J. and Kyte, J. Anal. Biochem. 1978, 89, 529-539.; Kyte, J., Degen, J. and Harkins, R.N. Methods Enzymology 1983, 91, 367-377.; Gevaert, K., Van Damme, J., Goethals, M., Thomas, G.R., Hoorelbeke, B., Demol, H., Martens, L..
  • the fixed-charge derivatives could be selectively pre-enriched by solid phase capture methods, using fixed charge reagents covalently coupled to beads or insoluble polymers [Weinberger, S.R.; Viner, RI. and Ho, P. Electrophoresis. 2002, 23, 3182-3192.; Zhou, H.; Ranish, J. A.; Watts, J. D.; Aebersold, R. Nature Biotechnol. 2002, 20, 512-515.; Qiu, Y; Sousa, E. A.; Hewick, R. M.; Wang, J. H.
  • a preferred isotopically encoded reagent of this form is a 13 C 6 labelled version, whose structure is shown below. 13 C labelling is preferred over deuterium labelling in order to minimise any chromatographic separation of the isotopically labelled versus unlabelled reagents. This reagent would be expected by those of skill in the art to have the same properties as the unlabelled form.
  • a more preferred reagent is a water soluble form of the foregoing reagent, as this added property confers benefits such as allowing for more streamlined sample preparation workflows.
  • water soluble forms of the reagents are shown below, as well as their C 6 labelled versions. Note that the addition of a sulfinic or sulfonic acid functional group could be achieved at the ortho, meta- or para positions relative to the acetyl group. The synthetic strategy can be chosen to direct the synthesis toward the any one of these positions; the para substituted position is shown below.
  • a non-limiting method for the synthesis of these derivatives may be obtained using methods known to those skilled in the art [Buess, CM. and Kharasch, N. J. Am. Chem. Soc. 1950, 72, 3529-3532.; Kharasch, N. and Swidler, R. J. Org.Chem. 1954, 19, 1704-1707.; Nogels Textbook of Practical Organic Chemistry. 4th ed. 1978. p 773-778.; Szmant, H.H. and Irwin, D.A. J. Am. Chem. Soc.
  • the derivative may be (iii) cleaved by thiolysis to yield the p-acetylthiobenzene derivative (iv) oxidised to the sulfinic or sulfonic acids, then (v) brominated.
  • a bromine atom is shown as the leaving group (i.e., X, of RX) in the foregoing structures, an iodine also preferred.
  • Other leaving groups that could be used include: halides such as a chlorine atom, as well as sulfonic esters, perchlorate esters, chlorosulfonates, or protonated amines or alcohols [March J.
  • the invention also includes reagent kits for analysis of amino acids, peptides or proteins by mass spectrometry.
  • the reagent kits include one or more of the foregoing compounds and other reagents such as protein disulfide reducing agents (e.g., dithiothreitol (DTT), /3-mercaptoethanol, tris-carboxyethyl phosphine (TCEP), tributylphosphine (TBP)), cysteine alkylating reagents (e.g., alkylhalides (e.g., iodoacetic acid, iodoacetamide) as well as vinylpyridine or acrylamide), proteases or chemical cleavage agents (e.g., tiypsin, Lys-C, Asp-N, pepsin, thermolysin, cyanogen bromide, dilute acid).
  • protein disulfide reducing agents e.g., dithiothreitol (DTT), /3-mercaptoethanol, tris-carboxyethyl phosphine (
  • the reagent kits also can contain solvents. Typical solvents that are used include urea, guanidine hydrochloride, acetonitrile, methanol, water.
  • solvents that are used include urea, guanidine hydrochloride, acetonitrile, methanol, water.
  • the various reagents can be used to perform the foregoing methods steps in solution, or on a solid phase, (e.g., by having the reagents immobilized on a column).
  • Amino acids or peptides labelled using the method of the invention can be used, in addition to analysis of amino acids, peptides and proteins, for internal standards.
  • the derivatized peptides described herein can be used in a manner analogous to several recent reports that used known quantities of an isotopically encoded peptide derivative spiked into a sample as an internal standard in order to perform absolute quantitation of the selected peptide of interest (see Gygi, Steven P.; Peng, Junmin. PCT Int. Appl. (2003) WO 2003078962; Gerber, Scott A.; Rush, John; Stemman, Olaf; Kirschner, Marc W.; Gygi, Steven P. Proc. Natl. Acad. Sci. U.S.A. (2003), 100, 6940-6945.). Note that these reports do not employ the use of fixed charge derivatives amenable to tandem mass spectrometry detection as are describe herein.

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Abstract

L'invention concerne des méthodes, des réactifs et des trousses qui permettent d'identifier des acides aminés, des peptides et des protéines, d'effectuer une quantification différentielle et d'analyser la modification post-traductionnelle et l'état de réticulation. Ces méthodes consistent : à dériver un mélange de peptides ou de protéines d'acides aminés afin de former au moins un peptide ou une protéine d'acide aminé contenant un ion à charge fixe différent de celui qui se trouve à son extrémité C-terminale ou N-terminale ; à introduire le mélange de peptides ou de protéines d'acides aminés contenant le peptide ou la protéine d'acide aminé dérivé à charge fixe dans un spectromètre de masse ; à faire passer le mélange de peptides ou de protéines d'acides aminés contenant le peptide ou la protéine d'acide aminé dérivé à charge fixe à travers un premier spectromètre de résolution de masse afin de sélectionner les ions précurseurs de protéines ou de peptides qui présentent un premier rapport masse/charge ; à soumettre les ions précurseurs présentant le premier rapport masse/charge à une dissociation afin de former un ion présentant un second rapport masse/charge qui est caractéristique d'une fragmentation ayant lieu dans un site adjacent à la charge fixe ; et à détecter les ions obtenus qui présentent le second rapport masse/charge. Les ions obtenus qui présentent le second rapport masse/charge peuvent être soit des ions formés par une perte neutre de la charge fixe des ions précurseurs, soit des ions formés par une perte chargée de la charge fixe des ions précurseurs.
EP03781974A 2002-11-18 2003-11-18 Methode permettant d'analyser des acides amines, des peptides et des proteines parspectrometrie de masse de leurs derives modifies par des charges fixees Withdrawn EP1565752A2 (fr)

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SE0401293D0 (sv) * 2004-05-17 2004-05-17 Amersham Biosciences Ab Novel MS Reagents
KR100611313B1 (ko) * 2004-06-30 2006-08-10 고려대학교 산학협력단 폴리펩티드의 ｎ-말단 치환용 화합물, 이를 이용한폴리펩티드 내의 아미노산 서열분석 및 정량방법
WO2006023398A2 (fr) * 2004-08-16 2006-03-02 Ludwig Institute For Cancer Research Reactifs de spectrometrie de masse marques par des isotopes modulaires et procedes d'analyse quantitative d'acides amines, de peptides et de proteines
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US9202678B2 (en) 2008-11-14 2015-12-01 Board Of Trustees Of Michigan State University Ultrafast laser system for biological mass spectrometry
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CN110133124A (zh) * 2019-04-29 2019-08-16 天津中医药大学 疏血通注射液中18种氨基酸的含量测定方法
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CN112786105B (zh) * 2020-12-07 2024-05-07 中山大学附属第五医院 一种宏蛋白质组挖掘方法及其在获取肠道微生物蛋白水解特征中的应用

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