US20020146743A1 - Stable isotope, site-specific mass tagging for protein identification - Google Patents

Stable isotope, site-specific mass tagging for protein identification Download PDF

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US20020146743A1
US20020146743A1 US10/043,965 US4396502A US2002146743A1 US 20020146743 A1 US20020146743 A1 US 20020146743A1 US 4396502 A US4396502 A US 4396502A US 2002146743 A1 US2002146743 A1 US 2002146743A1
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Xian Chen
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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
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/15—Non-radioactive isotope labels, e.g. for detection by mass spectrometry

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  • the present invention relates generally to protein identification using mass spectrometry and, more specifically, to the stable isotope mass tagging of selected amino acids which are incorporated into proteins in a sequence-specific manner during cell culturing to enable protein identification from the characteristic patterns in the mass spectra of proteolytic peptides.
  • proteomics is a newly emerging field in the post-genomics era 1 .
  • a major activity of proteomics is the identification of unique proteins in cellular complexes in a high throughput mode 2 .
  • Peptide mass mapping followed by database searching is a major approach towards the identification of a protein using mass spectrometry (MS).
  • MS mass spectrometry
  • the most commonly used method is an in-gel digestion of the protein spots separated by two dimensional polyacrylamide gel electrophoresis (2D PAGE) for analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS 5, 6 .
  • Mass accuracy and precision are of prime importance to ensure specificity of the search for a target protein in database searches.
  • Stable isotope 13 C/ 15 N-labeled nucleotides have successfully been incorporated as internal markers to determine the nucleotide composition of PCR products 23 .
  • the method for identifying a protein hereof includes the steps of: separating the protein from other proteins; digesting the protein, thereby forming first proteolytic peptides; acquiring the monoisotopic mass distribution spectrum of the first proteolic peptides and acquiring the m/z values therefor; incorporating an amino acid 100% labeled with a stable isotope into the protein in a sequence-specific manner; separating the protein bearing the labeled amino acid from other proteins; digesting the protein bearing the labeled amino acid, thereby forming second proteolytic peptides; acquiring the monoisotopic mass distribution spectrum of the second proteolytic peptides and acquiring the m/z values therefor; comparing the monoisotopic mass distribution spectrum of the second proteolytic peptides with the monoisotopic mass distribution spectrum of the first proteolytic peptides to determine the amino acid composition of
  • the step of incorporating the 100% labeled amino acid into the protein in a sequence-specific manner further includes the steps of: introducing the 100% labeled amino acid into a cell capable of expressing the protein; and inducing the cell to express the protein.
  • the method for identifying a protein hereof includes the steps of: incorporating an amino acid 100% labeled with a stable isotope into the protein in a sequence-specific manner at a variable number of the sites for that amino acid in the protein, forming thereby a mixture of partially labeled proteins; separating the mixture of partially labeled proteins from other proteins; digesting the mixture of partially labeled proteins, thereby forming proteolytic peptides; and acquiring the monoisotopic mass distribution spectrum of the proteolytic peptides and acquiring the m/z values therefor, whereby the protein is identified from the m/z values of the proteolytic peptides and the amino acid composition of the proteolytic peptides.
  • the step of incorporating the 100% labeled amino acid into the protein in a sequence-specific manner at a variable number of sites for that one amino acid in the protein further includes the steps of: introducing the 100% labeled amino acid and a chosen amount of an unlabeled same amino acid into a cell capable of expressing the protein; and inducing the cell to express the protein.
  • FIG. 1 shows delayed-extraction MALDI mass spectra of tryptic digests of the unlabeled UBL1.
  • FIG. 2 a shows monoisotopic patterns of peptides at m/z of 896.67 Da (M + ) and 1001.75 Da (M + ) from tryptic digestion of (A) unlabeled UBL1; (B) Met-d 3 labeled UBL1; and (C) a mixture of the Met-d 3 labeled and unlabeled UBL1,
  • FIG. 2 b shows monoisotopic patterns of peptides at m/z of 896.67 Da (M + ) and 1001.75 Da (M + ) from tryptic digestion of: (A) unlabeled UBL1; and (B) a mixture of Gly-d 2 labeled and unlabeled UBL1, and FIG.
  • 2 c shows the characteristic isotopic patterns of the large tryptic digest at m/z of 3644.88 (M + ) for: (A) unlabeled UBL1; (B) Met-d 3 labeled UBL1; and (C) a mixture of the Met-d 3 labeled and unlabeled UBL1.
  • FIG. 3 a shows the PSD fragment ion mass spectra of the fragment of 64 FLFEGQ 70 R containing unlabeled glycine residue
  • FIG. 3 b shows postsource decay fragment ion mass spectra of the fragment of 64 FLFEGQ 70 R containing the labeled glycine residue, Gly-d 2 .
  • FIG. 4 a shows delayed-extraction MALDI mass spectra of tryptic digests of 50% Gly-d 2 labeled E. coli cell lysate
  • FIG. 4 b shows delayed-extraction MALDI mass spectra of tryptic digests of 50% Met-d 3 labeled E. coli cell lysate.
  • FIG. 5 shows the delayed-extraction MALDI-TOF spectrum of the tryptic digests of the complex of interacting proteins of UBL1 and UBC9.
  • the present invention includes the incorporation of stable isotope-labeled amino acid residue(s) in proteins to “mass-tag” some proteolytic peptides according to their content of these labeled residue(s).
  • Stable isotope labeling of proteins are specific for particular amino acid residues 24-26 .
  • Particular labeled amino acid are incorporated into proteins during cell growth or in an in vitro transcription/translation system 26 in a manner that provides residue-specific mass-labeled proteins without scrambling of the label to other types of residues 24 .
  • a comparison of the masses of the peptides generated from proteolytic digestion of the residue-specific labeled protein with those of an unlabeled control assists in identifying the mass-tagged peptides, because modern mass spectrometry, including MALDI-TOF MS, permits the accurate determination of these changes with monoisotopic resolution 27,28 .
  • This provides an additional constraint of the amino acid identity of mass tagged peptides to enable accurate peptide identification.
  • the magnitude of the mass shifts for peptides reflect the content of particular amino acid residue(s). A smaller number of identified mass-tagged peptides is then used for more effective protein identification. It should be mentioned that other mass spectrometers, such as electrospray mass spectrometers, can effectively be employed in accordance with the teachings of the present invention.
  • partial amino acid sequences of selected peptides can be obtained by postsource decay (PSD) experiments 29,30 , many precursor ions obtained by delayed-extraction (DE) MALDI do not produce sufficient PSD fragmentation to allow the identification of even short sequence tags 30 .
  • the characteristic monoisotopic distribution pattern(s) of labeled amino acid residues provide internal marker(s) for the assignment of PSD derived peptides.
  • the incorporation of mass labels into specific proteolytic fragments significantly increase datasearch specificity, efficiency and accuracy for peptide sequencing and protein identification.
  • B. E. coli strains for residue-specific labeling of proteins 21 strains of bacteria, each containing a different genetic defect closely linked to a selectable transposon marker were used to construct strains of E. coli with effective genotypes for residue-specific, selective labeling of proteins with almost any stable isotope-labeled amino acid.
  • strains which have been modified to contain the appropriate genetic lesions to control amino acid biosynthesis dilution of the isotope label by endogenous amino acid biosynthesis and scrambling of the label to other types of residues was avoided.
  • Clearly other cell lines can be generated to perform the same task.
  • E. coli strain CT2 was constructed by transduction of the BL21(DE3) strain to tetR with a P1 lysate from MF14, and then screening for the gly-phenotype 26 .
  • This derivative of BL21(DE3) was used for the selective labeling of proteins with the stable isotope-labeled glycine.
  • CT13 was constructed by transducing BL21(DE3) to tetR with a P1 lysate from MF 21, and then screening for the met- phenotype (metA-).
  • metA- met- phenotype
  • C Residue-specific labeling of proteins and purification.
  • the expression plasmid of UBL1 was transformed into both CT2 BL21(DE3) and CT13 BL21(DE3).
  • the CT2 BL21(DE3) cells were grown in M9 minimum media supplemented with 0.2 g per liter of the L-Methionine-99.9%-d 3 , 0.02 g per liter of unlabeled cysteine, and 0.2 g per liter of each of other unlabeled amino acids.
  • the CT13 BL21(DE3) cells were fed with a similar mixture that contained the labeled precursor, 0.2 g of Glycine-99.9%-2,2-d 2 . These cells were induced with 1 mM isopropylthiogalactoside (IPTG) for protein expression. It is clear that other amino acids than Methionine and Glycine can be labeled and used in accordance with the teachings of the present invention. Moreover, other inducing agents than IPTG can be employed. The corresponding unlabeled protein was expressed in regular LB media. The His-tagged proteins were purified in a buffer of 150 mM ammonium acetate (NH 4 OAC), pH 7.0 with a gradient of 0-150 mM imidazole.
  • IPTG isopropylthiogalactoside
  • Mass spectrometry experiments were carried out on a PE Voyager DE-STR Biospectrometry workstation equipped with a N 2 laser (337 nm, 3-ns pulse width, and 20-Hz repetition rate) in both linear and reflectron mode (PE Biosystems, Framingham, Mass.).
  • the mass spectra of the tryptic digests were acquired in the reflectron mode with delayed extraction (DE).
  • the m/z values of proteolytic peptides were calibrated with Calimix 2 including Angiotensin I at 1297.51 Da (M + ) and Insulin at 5734.59 Da (M + ).
  • E. Mass tagging in an E. coli strain and the target protein identification The E. coli BL21(DE3) cell strain containing the UBL1 expression vector was cultured in M9 media supplemented with a mixture of amino acids including 50% labeled amino acid precursors (Gly-d 2 or Met-d 3 ) respectively. The cells were then induced with 1 mM IPTG. An aliquot of the cell culture was collected 30 min. after the IPTG induction when the target protein did not overwhelm the proteins in the total cell extract. After centrifugation of the cell aliquot, the resulting pellet was resuspended and sonicated in a buffer of 1 mM DTT and 20 mM NH 4 HCO 3 at pH 8.0.
  • the supernatant of the cell extract was treated with trypsin (10 ⁇ g/ml) overnight without purification.
  • the cell extract containing the tryptic digests was then desalted by C18 ZipTip (Millipore) and analyzed using MALDI-TOF MS.
  • E. coli BL21(DE3) cell strains containing the UBL1 and UBC9 expression vectors were mixed in the same copy numbers and grown in M9 media supplemented with a mixture of amino acids that included 50% deuterium-labeled glycine (Gly-d 2 ). Both UBC9 and UBL1 were readily expressed and labeled with Gly-d 2 at all glycine residues in the E. coli strains upon IPTG induction.
  • the Pharmacia Biotech FPLC with a gel filtration mini-column (Superdex 75, 1.0 cm ⁇ 10 cm, Pharmacia Biotech) was used to isolate the complex of UBL1 and UBC9 from the cell lysate.
  • the same buffer of 1 mM DTT and 20 mM NH 4 HCO 3 at pH 8.0 was used for the protein elution.
  • the fraction containing the complex was lyophilized and then treated with trypsin (10 ⁇ g/ml in 10 mM NH 4 HCO 3 , pH 8) overnight.
  • PSD Post-source decay
  • the protein, UBL1-Met-d 3 was extracted from E.coli strain BL21(DE3) CT13 cells transformed with the UBL expression vector and had the 2 H-labeled precursor, methionine-99.9%-S-methyl-d 3 (Met-d 3 ), incorporated at all of the methionine sites of the protein.
  • the glycine-specific labeled protein, UBL1-Gly-d 2 extracted from E.coli BL21(DE3) CT2 cells, had the 2 H-labeled precursor, glycine-99.9%-2,2-methene-d 2 (Gly-d 2 ), incorporated at all glycine sites.
  • Gly-d 2 2-methene-d 2
  • FIG. 1 shows the mass spectrum obtained from a tryptic digest of the unlabeled UBL1.
  • the PE Voyager-DE STR MALDI-TOF MS has a mass resolution, M/ ⁇ M, of 5000 which is sufficient to resolve monoisotopic peaks of all the tryptic peptides of masses up to 5000 daltons (Da).
  • Inset A shows an expanded view of the monoisotopic distribution pattern corresponding to the relative abundance of isotopes, M + :(M+1) + :(M+2) + . . .
  • M refers to the mass corresponding to the most abundant isotope
  • M + ion the less abundant isotopes such as 13 C, 15 N or 2 H also increase, so that at a higher m/z the isotopic pattern is more pronounced as shown in inset B (the m/z of M + ion is at 3644.91 Da).
  • Ion fragments having m/z values of 1895.39, 2198.66, 2275.92, 2614.04 and 3155.54 probably derive from incomplete digestion and impurities were not assigned to the protein.
  • FIG. 2 a shows the MALDI-TOF mass spectra of two proteolytic peptides from: (A) unlabeled UBL1; (B) UBL1-Met-d 3 ; and (C) a mixture of (A) and (B) in a 1:2 ratio. It was observed that the monoisotopic M + ion at 1004.85 Da from UBL1-Met-d 3 (B) was 3 Da heavier than that of the unlabeled UBL1 (1001.75 Da) (A) because of the presence of the labeled methionine (FIG.
  • the mass tag of a labeled methionine residue is 3 Da
  • M + ion the mass tag of a labeled methionine residue
  • M + ion the mass tag of a labeled methionine residue
  • the monoisotopic distribution patterns of these labeled peptides are essentially unchanged when compared to the unlabeled peptides. This is because only a few protons are replaced by deuterium in the labeled precursors.
  • FIG. 2 b shows monoisotopic patterns of peptides at m/z of 896.67 Da (M + ) and 1001.75 Da (M + ) from tryptic digestion of: (A) unlabeled UBL1; and (B) a mixture of the Gly-d 2 labeled and unlabeled UBL1 (2:1 molar ratio).
  • the incorporation of a Gly-d 2 label can be recognized by the 2-Da split between the monoisotopic peaks of the unlabeled and labeled peptides.
  • FIG. 2 c shows the characteristic isotopic patterns of the large tryptic digest at m/z of 3644.88 (M + ) for: (A) unlabeled UBL1; (B) Met-d 3 labeled UBL1; and (C) a mixture of the Met-d 3 labeled and unlabeled UBL1 (2:1 molar ratio).
  • the incorporation of a Met-d 3 label can be recognized by the 3-Da split between the monoisotopic peaks of the unlabeled and labeled peptides. Changes in isotopic distribution patterns (FIG.
  • FIG. 2 c (C) shows the mass spectrum of a mixture of the unlabeled and Met-d 3 labeled peptide of 3644.88 Da.
  • the fragment of 4521.99 Da is from the incomplete digestion of the last two fragments at the C-terminal of the protein; that is, 71 IADNHTP 78 K and 79 ELG M EEEDVIEVYQEQTGGHSTVLEHHHHH 107 H (bold type indicates the labeled Gly, while the labeled Met is underlined).
  • the hydrolysis of the fragment of 71-107 results from the addition of a water molecule at C-terminal of the fragment of 71-78 to form the fragments 71-78 (the M + ion at 895.46 Da) and 79-107 (the M + ion at 3645.10 Da).
  • FIG. 3 a shows the PSD fragment ion mass spectrum of the fragment of 64 FLFEGQ 70 R containing unlabeled glycine residue.
  • FIG. 3 b shows the PSD fragment ion mass spectra of the fragment of 64 FLFEGQ 70 R containing 50% labeled glycine residue, Gly-d 2 .
  • the M + ion of 50% Gly-d 2 at 896.67 Da (FIG. 3 b, inset B) was selected as a PSD precursor because the characteristic mass-split pattern indicates the location of the labeled glycine residue in the progressively produced fragment ions through PSD.
  • the gate width was adjusted for the full isotopic distribution pattern of the PSD fragments.
  • 67 Glu, and 69 Gln have been identified as the closest amino acids to the Gly-d 2 , and the peak of 343.27 Da was assigned to the fragment ion of 67 EGQ. From this core residue of 68 Gly-d 2 , the sequence of the M + fragment of 896.67 Da has been determined.
  • FIG. 4 a shows the delayed-extraction MALDI mass spectra of tryptic digests of the cell lysates for the 50% Gly-d 2 labeled E. coli cell lysate, while FIG. 4 b shows that for the 50% Met-d 3 labeled E. coli cell lysate.
  • FIG. 5 shows the MALDI-TOF spectrum of the tryptic digest of the complex which shows the peak pairs with 2 ⁇ n Da mass-split (“n” represents the number of glycine residues) with about a 1:1 intensity ratio resulting from specific-labeled glycine-containing peptides.
  • n represents the number of glycine residues
  • Three such characteristic peak pairs have been observed in the mass spectrum from the pool of tryptic digests. They are the peak pairs at 896.67 Da (M + ion) and 1001.75 Da (M + ion) each with the characteristic 2 Da mass-split, and a pair of M + ions at 1092.25 Da and 1098.31 Da with a 6 Da in spacing.
  • the former two peak sets are mass-tagged peptides of UBL1 protein.
  • the latter pair indicates that the fragment ion contains three glycines.
  • the matched peptide is the GTPWEGGLFK (the theoretical m/z value of the M + ion is 1091.55 Da) of UBC9 protein.
  • the ratio of unlabeled to labeled amino acid precursors was varied.
  • the change of the relative intensity of 1092.25 Da (M + ion) to 1098.31 Da (M + ion) was in agreement with this assignment. Therefore, not only from their matched m/z values, but also from their amino acid compositions, the above assigned peptides provide “fingerprints” for the identifications of UBL1 and UBC9.
  • Mass calibration was performed externally using the calibration standard, Calmix 2 (PE Biosystem). Typical observed mass errors were ⁇ 0.2 to ⁇ 0.4 Da compared to the theoretically calculated masses for most peptides, which is expected for routine MALDI-TOF measurements.
  • the use of absolute m/z values of measured peptides with such large errors (about 250 ppm) in database searching can result in the identification of a number of proteins other than the target protein.
  • An advantage of the mass-tagging method of the present invention is that the mass of the tags requires only relative measurements; that is, the mass difference between the labeled and unlabeled peptides. For example, whereas the absolute m/z value of an ion peak is in error by 0.4 Da in the spectrum of FIG.
  • Mass tagging provides another parameter for unique protein identification.
  • the present method is also generally applicable for the identification of unique proteins in a complex. Residue-specific labeling in E. coli -expressed proteins using genetically engineered E. coli cell strains has been demonstrated. We have also examined isotopic scrambling of the residue-specific labeling of the protein, UBL1, with proteins of the E. coli BL21(DE3) cell host. In the M9 media enriched with the 20 amino acids, the stable isotope enriched amino acids, L-Methionine-99.9%-d 3 (Met-d 3 ) and Glycine-99.9%-2,2-d 2 (Gly-d 2 ), were used as the mass-tagging precursors for the methionine and glycine sites respectively.
  • Residue-specific mass tagging is particularly useful for the direct analysis of large protein complexes, when a denatured and reduced protein complex is first digested to peptide fragments in a sequence-specific manner, followed by liquid chromatography separation and MS analysis 4 .
  • the experimentally measured m/z values of mass-tagged peptides can be compared with the calculated m/z values of a proteolytic peptide library derived from the predicted digestion of proteins translated from the genomic sequence databases.
  • the mass-tagged peptides identified from the matches will be selected for the search and identification of unique proteins present in the translated genomic databases.
  • both the m/z values of peptides and the mass tags of certain peptides can be utilized in selective database searches for the unique identification of different proteins in complex mixtures. The specificity and accuracy of protein identification will be significantly increased by this analytical methodology of residue-specific mass tagging.

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WO2014197754A1 (fr) * 2013-06-07 2014-12-11 Pierce Biotechnology, Inc. Quantification absolue de protéines et de modifications de protéines par spectrométrie de masse à étalons internes multiplexés
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