WO2010109022A1 - Procede de proteomique quantitative - Google Patents

Procede de proteomique quantitative Download PDF

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WO2010109022A1
WO2010109022A1 PCT/EP2010/054109 EP2010054109W WO2010109022A1 WO 2010109022 A1 WO2010109022 A1 WO 2010109022A1 EP 2010054109 W EP2010054109 W EP 2010054109W WO 2010109022 A1 WO2010109022 A1 WO 2010109022A1
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peptides
terminal end
labelled
previous
protein
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PCT/EP2010/054109
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Bernd Thiede
Christian KÖHLER
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Universitetet I Oslo
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry

Definitions

  • the present invention relates to the field of mass-spectrometry-based quantitative proteomics and to approaches for identifying and quantifying two or more differentially labeled states using MS/MS spectra.
  • Mass spectrometry is an analytical technique for the determination of the elemental composition of a sample or molecule. Jt is also used for elucidating the chemical structures of molecules, such as peptides and other chemical compounds.
  • the MS principle consists of ionizing chemical compounds to generate charged molecules or molecule fragments and measurement of their mass-to-charge ratios.
  • Tandem mass spectrometry also known as MS/MS, involves multiple steps of mass spectrometry selection, with some form of fragmentation occurring in between the stages.
  • Proteomics is the large-scale study of proteins, particularly their structures and functions.
  • Mass spectrometry-based quantitative proteomics involves comparison of labeled peaks.
  • Many labeling techniques have been established which provide a distinct mass shift and allow quantitation by peak comparison on MS level (e.g. SILAC, ICAT).
  • Relative quantitation on MS/MS level can be performed by iTRAQ using reporter ions produced during MS/MS. Nevertheless, iTRAQ requires analysis in the low mass range which is not feasible using ion traps.
  • mass spectrometry-based quantitative proteomics of unknown proteins is usually performed with stable isotopic labeling or label-free approaches.
  • stable isotopes with defined mass differences enables the comparison of different states by relative protein quantification.
  • Stable isotope labels are most commonly introduced through metabolic and chemical approaches. Metabolic labeling techniques by e.g. stable isotope labeling by amino acids in cell culture (SILAC) requires living cells, whereas chemical labeling can be performed with any proteome.
  • SILAC stable isotope labeling by amino acids in cell culture
  • chemical labeling can be performed with any proteome.
  • chemical labeling requires quantitative derivatization preferably without any side reactions and is usually more successful with peptides than with proteins.
  • Proteomics, 2005, 5(1), pp.4-15 discloses a method based on isotope-coded protein labeling (ICPL) established in intact proteins based on isotopic labeling of all amino groups using differentially deuterated forms of N- nicotinoyloxy succinimide which enabled the early incorporation of the label. Labeling on the peptide level requires that the proteomes to be compared are purified and fractionated separately which might be an additional error source for quantification.
  • ICPL isotope-coded protein labeling
  • Isotope-coded affinity tagging was the first approach for relative proteome quantification and consisting of a cysteine-directed reactive group, a linker region including different stable isotopes, and a biotin-affinity tag for purification.
  • the ICAT- approach reduces the complexity of the sample, but is limited to cysteine-containing proteins and peptides, which is a relatively rare amino acid.
  • GIST global internal standard technology
  • Stable isotopic labeling of amines can be obtained e.g. by dimethylation or succinylation with relative quantification on the MS level.
  • isobaric tagging for relative and absolute quantification (iTRAQ) and tandem mass tagging (TMT) produce isobaric masses on MS level and specific reporter ions on MS/MS level for relative quantification, allowing the relative quantification of up to eight different samples simultaneously.
  • iTRAQ isobaric tagging for relative and absolute quantification
  • TMT tandem mass tagging
  • TMT and iTRAQ are limited to single reporter ions in the low mass range, which are difficult to detect with e.g. ion trap detectors.
  • WO 2009/141310 discloses a method for MS analysis of analytes, which may be proteins or peptides, in which the analytes are labelled with a mass label or combination of mass labels, wherein each mass label is an isobaric mass label comprising a mass spectrometrically distinct marker group. It is mentioned that TMT, iTRAQ and iPROT may be used.
  • WO 2009/153577 discloses a method for MS analysis of compounds, such as peptides and proteins, which have been labelled with mass markers which may be isobaric and isotopic. The peptides are labelled in the C-terminal end or in the N-terminal end. Succinic anhydride and methoxy-4,5-dihydro-1H-imidazole may be used as mass markers.
  • EP 1 916 526 discloses a method for MS analysis of proteins which have been labelled with a combination of an isotopic and an isobaric label. During MS a low molecular fragment ion is produced.
  • WO 2003/056299 describes compounds which are useful as labels in proteomics studies. It is mentioned that methoxy-4,5-dihydro-1 H-imidazole-d4 and methoxy-4,5-dihydro-1 H- imidazole-dO may be used as labeling compounds.
  • each pair comprising a first protein sample and a second protein sample, each pair being denoted by a pair number (i),
  • step b) combining the peptides obtained in step a) from at least one of said pairs of protein samples to yield a mixture of peptides, said peptides being isobaric within each pair (i) of protein samples;
  • step b) submitting the mixture of peptides in step b) to MS/MS;
  • a x(l) is a labelling reagent A containing one stable isotope x(i) for pair (i) of protein sample
  • a y(l) is a labelling reagent A containing one stable isotope y(i) for pair (i) of protein sample
  • B x(l) is a labelling reagent B containing one stable isotope x(i) for pair (i) of protein sample
  • B y(l) is a labelling reagent B containing one stable isotope y(i) for pair (i) of protein sample
  • a x(l) , A y(l) , B x(l) and B y(0 may each be cleaved at their point of attachment to the peptide during MS/MS but are otherwise fragmentation resistant during MS/MS, for each pair (i) of protein sample x(i) is different from x(i) in any other pair (i) of protein sample, and for each pair (i) of protein sample y(i) is different from y(i) in any other pair (i) of protein sample.
  • FIG. 1 Flowchart of the IPTL method. Proteins of two different states were digested with Lys-C to produce peptides with lysine residues (K) at the C-terminus. Next, the peptides were modified at lysines with MDHI and MDHI-d4, respectively. Subsequently, isobaric peptides were generated by crosswise derivatization of the N-termini with SA-d4 and SA, respectively. After mixing of the two samples, isobaric peptide masses were detected for peptides without missed cleavage sites in MS mode. Relative quantification of the two states was achieved on MS/MS level using the fragment pairs with 4 Da differences. The most significant b- and y-ions resulted in reversed quantification values.
  • Figure 2 MALDI-MS and MS/MS spectra of a Lys-C peptide of BSA after IPTL.
  • the peptide mass fingerprint showed quantitative derivatization applying IPTL ( Figure 2, spectrum A).
  • MS/MS spectra of the isobaric peptide DLGEEHFK of BSA digested with Lys-C and subsequently crosswise labeled with MDHI-d4/SA and MDHI/SA- d4 are presented.
  • the crosswise labeled peptides were mixed together in ratios 1 :1 ( Figure 2, spectrum B), 2:1 (Figure 2, spectrum C) and 5:1 ( Figure 2, spectrum D).
  • the detected y-ion series occurred as doublets with 4 Da mass differences.
  • Figure 4 Selected MS/MS spectrum for the relative quantification of vimentin during STLC-induced apoptosis.
  • the MS/MS spectrum of the isobaric peptide NLQEAEEWYK with m/z 741.85 digested with Lys-C and subsequently crosswise labeled with MDHI- d4/SA (STLC, 24 h) and MDHI/SA-d4 (STLC, 48 h) is presented.
  • Figure 5 IsobariQ-Protein view.
  • Figure 5 shows the main window of IsobariQ where all identified proteins and their respective peptides are shown.
  • the QualPLT module (figure 6) the ratio and variability of every peptide is transfered back to this protein view where the overall protein ratio and variability is calculated.
  • This protein list can be exported to a spreadsheet application for further analysis.
  • Figure 6 IsobariQ - the QualPTL module.
  • a protein has been double-clicked in the protein view (figure 5) all MS/MS spectra assigned to this protein are displayed here. All Mascot hits assigned to a given MS/MS spectrum are displayed in the top panel (A) and the annotated MS/MS spectrum is shown in the bottom panel (B).
  • all the quantification events are listed in the quantification table (C) where the user can select which ratios to include or exclude for this particular MS/MS spectrum.
  • FIG. 7A Flowchart of IPTL using succinic anhydride with different buffers for crosswise labeling.
  • An alternative to the original IPTL approach was developed using N-terminal succinylation for the first dervatisation step. Therefore, a specific buffer system for the succinylation reaction based on ammonium acetate was developed and applied. The second derivatisation was performed with another buffer system (sodium dihydrogen phosphate) to modify the C-terminal lysines.
  • Figure 7B shows N-terminal succinylation of a tryptic digest of BSA.
  • FIG 8A Flowchart of tryptic-IPTL.
  • An alternative to the original IPTL approach was developed using trypsin as enzyme (named tryptic-IPTL). Therefore, a specific buffer system for the succinylation reaction based on ammonium acetate was developed and applied.
  • the stable isotope 13 C 4 -succinic anhydride was used.
  • the second derivatisation was performed by enzymatic incorporation of two 18 O stable isotopes. 13 C and 18 O are known to have no retention time shift in contrast to deuterium.
  • FIG 8B An example for the tryptic-IPTL strategy is shown. Glyceraldehyde-3- phosphate dehydrogenase from chicken was digested with trypsin and N-terminally modified with either succinic anhydride- 13 C 4 (top) or succinic anhydride (middle). Then, the sample modified with succinic anhydride was incubated with trypsin in water-O 18 (bottom).
  • Figure 8C shows an MS/MS spectrum of a 3:1 mixture of glyceraldehyde-3-phosphate dehydrogenase from chicken.
  • Figure 8D shows a section of a MS/MS spectrum of a 3:1 mixture of glyceraldehyde-3- phosphate dehydrogenase from chicken. Mass pairs with 4 Da difference were found, which can be used for quantification.
  • the present invention provides a method for identifying and quantifying two differentially labeled states using MS/MS spectra.
  • the method which is hereinafter denominated isobaric peptide termini labeling (IPTL) is based on isobaric peptide termini labeling using crosswise dO and d4 reagents and relative quantification on the MS/MS level due to mass shifts of the fragment ions.
  • the proteins were digested with Lys-C to afford a peptide with a lysine residue
  • the produced peptides of two different states were labeled with either 2-methoxy-4,5-dihydro-1 H-imidazole (MDHI) or 2-methoxy-4,5-dihydro- 1 H-imidazole-d4 (MDHI-d4) at lysine residues following crosswise labeling with succinic anhydride-d4 (SA-d4) or SA at the N-termini.
  • digesting or “digest” as mentioned herein, refers to when the proteins or peptides are divided into two or more pieces by cleavage by an enzyme which is able to perform this digestion.
  • An endoprotease such as Lys-C is an example of an enzyme capable of digesting proteins and/or peptides. The digestion of proteins results in peptides having a specific amino acid at one end of the peptide. For example, digestion with with Lys-C affords a peptide with a lysine residue.
  • isobahc peptides refers to peptides having the same or virtually the same molecular weight.
  • isotopes of an element are atoms having nuclei with the same number of protons but different number of neutrons.
  • the present method provides more quantitation points per peptide.
  • the IPTL method was first established with standard proteins and relative quantification was achieved by the ratio of the Mascot scores obtained by searching in both directions with fixed modifications of the two different states.
  • the IPTL method allows for reducing the number of peptides and increasing the number of quantification points per peptide.
  • the novel IPTL method is strikingly different to other MS/MS-based quantification approaches such as iTRAQ and TMT, which are limited to single reporter ions in the low mass range, which are difficult to detect with e.g. ion trap detectors.
  • the IPTL approach was established by modification of Lys-C digested proteins with MDHI at C-terminal lysine residues and succinylation of the N-termini. The two required reagents yielded in quantitative modification.
  • Another advantage is that the deuterated forms (d4) for isobaric labeling are commercially available.
  • Trypsin is the most popular enzyme for proteomics applications because it produces peptides with positively charged termini of an optimal average size suited for detection by mass spectrometry.
  • Lys-C produces less and longer peptides than trypsin. This results in reduced number of peptides, particularly of complex samples, and thus considerably simplifies the requirements for LC separation and MS acquisition.
  • the IPTL method may also be performed using tryptic peptides.
  • Labeling of arginines may be obtained using e.g, methyfglyoxal (d ⁇ and d4, respectively), which may also be combined with N-terminal succinylation or dimethylation for crosswise isobaric labeling.
  • Mascot protein scores may be used for approximate relative quantification (Figure 3). Still, higher than 2-fold relative changes in protein amounts have been shown to be reliable to identify quantitative differences with standard proteins. Furthermore, the results obtained from complex protein samples of HeLa cells incubated with STLC for different lengths, revealed several similar regulated proteins as obtained by proteome analysis based on 2- DE and silver staining. Furthermore, many of the identified proteins have been found previously by other approaches to be linked to apoptosis, indicating the reliability of the novel IPTL approach. An approximate relative quantification can be obtained using the IPTL approach in combination with Mascot protein scoring. Moreover, the IPTL method may also be used for absolute quantification of proteins.
  • Absolute quantification of proteins has been developed using stable isotope mass-tagged synthetic peptides (e.g., 13 C and 15 N) and was named AQUA. Therefore, the synthetic reference peptides serve as internal standards and are analyzed at the same time as the corresponding natural peptide by LC-MS/MS. These peptides can be analyzed by multiple selected reaction monitoring (SRM) with highest MS sensitivity. This concept has been further developed for absolute quantification of proteomes using so-called proteotypic peptides. However, the production of the stable isotope tagged synthetic peptides is relatively expensive. Alternatively, proteotyptic peptides could be produced applying the IPTL method to synthetic peptides and absolute quantification on MS/MS level.
  • SRM selected reaction monitoring
  • the IPTL method is useful for relative quantification of proteins based on multiple fragment pairs per peptide in MS/MS mode. Quantification of isobaric peptides instead of isotopic peptides considerably reduces the number of analytes. Approximate relative quantification was achieved using Mascot protein scores.
  • a method for analysis of one or more pairs of protein samples each pair comprising a first protein sample and a second protein sample, each pair being denoted by a pair number (i)
  • step b) submitting the mixture of peptides in step b) to MS/MS;
  • a x(l) is a labelling reagent A containing one stable isotope x(i) for pair (i) of protein sample,
  • a y(l) is a labelling reagent A containing one stable isotope y(i) for pair (i) of protein sample,
  • B x(l) is a labelling reagent B containing one stable isotope x(i) for pair (i) of protein sample
  • B y(0 is a labelling reagent B containing one stable isotope y(i) for pair (i) of protein sample
  • a x(l) , A y(l) , B x(l) and B y(l) may each be cleaved at their point of attachment to the peptide during MS/MS but are otherwise fragmentation resistant during MS/MS, for each pair (i) of protein sample x(i) is different from x(i) in any other pair (i) of protein sample, and for each pair (i) of protein sample y(i) is different from y(i) in any other pair (i) of protein sample.
  • a second aspect of the invention there is provided a method according to the first aspect of the invention, wherein the total number of pairs is equal to or less than 10.000, preferably less than 1000 and more preferably less than 100.
  • a fourth aspect of the invention there is provided a method according to any previous aspect, wherein the mixture of proteins are obtained from STLC-treated HeLa cells.
  • a method according to any previous aspect wherein the enzyme is an endoprotease selected from Lys-C, trypsin, Lys-N, Asp- Is!, GIu-C and Arg-C.
  • reagent A x(l) is selected from 2-methoxy-4,5-dihydro-1 H-imidazole-d4, O- methylisourea- 13 C, 15 N 2 , succinic anhydride-d4, succinic anhydride- 13 C 4 , and water- 18 O.
  • reagent A y(l) is selected from 2-methoxy-4,5-dihydro-1 H-imidazole-dO , O- methylisourea, succinic anhydride, and water.
  • reagent B x(l) is selected from succinic anhydride-d4 , formaldehyde-d2, propionic anhydride- 13 C 6 , and succinic anhydride.
  • reagent B y(l) is selected from succinic anhydride-d ⁇ , formaldehyde, propionic anhydride, succinic anhydride-d4, and succinic anhydride- 13 C 4 .
  • a 10 th aspect of the invention there is provided a method according to any previous aspect, wherein the enzyme is Lys-C, reagent A x(l) is 2-methoxy-4,5-dihydro-1 H-imidazole- d4, reagent B x(l) is succinic anhydride-d4, reagent A y(l) is methoxy-4,5-dihydro-1 H- imidazole-dO and reagent B y(l) is succinic anhydride -d ⁇ .
  • an 1 1 th aspect of the invention there is provided a method according to any previous aspect, wherein the method further comprises purification of the modified peptides from the first protein sample in step a) and/or purification of the modified peptides from the second protein sample in step a).
  • a 12 th aspect of the invention there is provided a method according to any previous aspect, wherein the purification is selected from liquid chromatography, isoelectric focussing and gel electrophoresis.
  • a 13 th aspect of the invention there is provided a method according to any previous aspect, wherein the ionization technique for MS/MS acquisition is MALDI-MS or ESI-MS.
  • a 14 th aspect of the invention there is provided a method according to any previous aspect, wherein the analysis comprises identification and/or relative quantification and/or absolute quantification.
  • the ratio of the isobaric peptides from any pair (i) of protein sample is determined by the intensities of the signals from the corresponding ion fragments in the MS/MS spectrum resulting from the MS/MS acquisition.
  • a 16 th aspect of the invention there is provided a method according to any previous aspect, wherein the isobaric peptides from any pair (i) of protein sample are identified using a database for protein identification.
  • an 18 th aspect of the invention there is provided use of a method according to any previous aspect for assessment of up- or downregulation of proteins.
  • a x(l) , A y(l) , B x(l) and B y(l) each have a molecular weight above 117 g/mol, 120 g/mol, 150 g/mol or 200 g/mole and may each be cleaved at their point of attachment to the peptide during MS/MS but are otherwise fragmentation resistant during MS/MS
  • a method for comparison of two or more protein samples comprising the following steps: a) separately digesting each protein sample with an enzyme to generate peptides with a specific amino acid at the N- or C-terminal end; b) modification of the peptides from the first protein sample obtained in step a) by reaction of the specific amino acid at the N- or C-terminal end of the peptides with a stable-isotope labelled reagent A denominated A x yielding peptides having a labelled N- or C-terminal end and a non-labelled N- or C-terminal end, c) modification of the peptides from the second protein sample obtained in step a) by reaction of the specific amino acid at the N- or C-terminal end of the peptides with a non-stable isotope labelled reagent A denominated A y or a stable-isotope labelled reagent A denominated A x yielding peptides having
  • peptides from the first protein sample in step b) are further modified by reaction of the specific amino acid of the non-labelled N-or C-terminal end of the peptides with a non-stable-isotope labelled reagent B denominated B y or with a stable- isotope labelled reagent denominated B x .
  • peptides from the second protein sample in step c) are further modified by reaction of the amino acid of the non-labelled N-or C-terminal end of the peptides with a stable-isotope labelled reagent B denominated B x or with a non-stable-isotope labelled reagent denominated B y .
  • peptides from the further protein sample in step d) are further modified by reaction of the amino acid of the non-labelled N-or C-terminal end of the peptides with a non-stable-isotope labelled reagent B denominated B y or by reaction of the amino acid of the non-labelled N-or C-terminal end of the peptides with a stable-isotope labelled reagent B denominated B x .
  • the comparison comprises identification and/or relative quantification and/or absolute quantification.
  • Further aspect 7 There is provided a method according to any previous further aspect wherein the enzyme is an endoprotease such as Lys-C, trypsin, Lys-N, Asp-N, GIu-C and Arg-C.
  • an endoprotease such as Lys-C, trypsin, Lys-N, Asp-N, GIu-C and Arg-C.
  • reagent B is selected from SA or formaldehyde.
  • HeLa cells were grown as a monolayer in RPMI supplemented with 10% foetal bovine serum and maintained in a humid incubator at 37°C in a 5% CO 2 environment.
  • Cells were treated with 5 ⁇ M S-trityl-L-cysteine (STLC) from a 5 mg/mL stock in DMSO.
  • STLC S-trityl-L-cysteine
  • Cells were trypsinized after 24 h, and 48 h, harvested, resuspended in 1 ml PBS and centrifuged again at 10.000 rpm. Cell pellets were frozen in liquid nitrogen and stored at -20 0 C.
  • Fetuin (bovine), lactoglobulin (bovine), transferrin (human), and serum albumin (bovine) were purchased form Sigma-Aldrich (Oslo, Norway).
  • the proteins were dissolved in Lys-C buffer (25 mM Tris pH 8.5, and 1 mM EDTA) and digested with Lys-C (enzyme to protein ratio 1 :50 ) for 16 h at 37°C. The digestion was stopped by adding formic acid to a final concentration of 0.8%.
  • MALDI-TOF/TOF-MS An Ultraflex Il (Bruker Daltonics, Bremen, Germany) MALDI-TOF/TOF mass spectrometer was used with a mass accuracy of 50 ppm after external calibration with kemptide, bradykinin, substance P, glu-fibrinopeptide B, and dynorphin A 2-17 (Sigma-AIdrich, Oslo, Norway or Bachem, Basel, Switzerland). The samples were analyzed in the MS mode for the generation of peptide mass fingerprints as well as in the TOF/TOF mode for fragmentation analysis of chosen peaks.
  • Nano-LC-LTQ Orbitrap mass spectrometry The dried peptides were dissolved in 10 ⁇ l 1% formic acid in water and 3 ⁇ l were injected onto an LC/MS system consisting of an Ultimate 3000 nano-LC system (Dionex, Sunnyvale CA, USA) connected to a linear quadrupole ion trap-orbitrap (LTQ Orbitrap XL) mass spectrometer (ThermoScientific, Bremen, Germany) equipped with a nanoelectrospray ion source.
  • An Acclaim PepMap 100 column (C18, 3 ⁇ m, 100 A) (Dionex, Sunnyvale CA, USA) with a capillary of 12 cm bed length was used for separation by liquid chromatography.
  • the flow rate used was 300 nL/min for the nano column, and the solvent gradient used was 7% B to 50% B in 45 minutes.
  • Solvent A was 0.1% formic acid, whereas aqueous 90% acetonitrile in 0.1 % formic acid was used as solvent B.
  • the method used allowed sequential isolation of the most intense ions, up to six, depending on signal intensity, for fragmentation on the linear ion trap using collisionally induced dissociation at a target value of 100,000 charges.
  • the lock mass option was enabled in MS mode and the polydimethyicyclosiloxane (PCM) ions generated in the electrospray process from ambient air were used for internal recalibration during the analysis.
  • Target ions already selected for MS/MS were dynamically excluded for 60 seconds.
  • General mass spectrometry conditions were: electrospray voltage, 1.5 kV; no sheath and auxiliary gas flow.
  • Ion selection threshold was 500 counts for MS/MS, and an activation Q-value of 0.25 and activation time of 30 ms were also applied for MS/MS.
  • Raw LTQ Orbitrap XL data were processed using DTA supercharge software to generate mgf files. Then, a database search was performed by tandem mass spectrometry ion search algorithms from the Mascot in-house version 2.2.1 by database comparisons with mammalian (63892 sequences) or human entries (20411 sequences) from Swiss-Prot (20081212). Lys-C was selected as enzyme without any missed cleavage sites and tolerance of 10 ppm for the precursor ion and 0.6 Da for the MS/MS fragments was applied. Moreover, methionine oxidation was allowed as variable modification. Fixed modifications were set to the two corresponding modifications SA/MDHI-d4 or SA- d4/MDHI, respectively.
  • STLC is a reversible inhibitor of kinesin Eg5 and inhibits tumor growth. STLC blocks cells in the M phase of the cell cycle and subsequently leads to apoptosis.
  • Peptides from corresponding slices were pooled and analysed by LCMS. The resulting MS data were searched against the SwissProt database using Mascot setting either MDHI-d4/SA or MDHI/SA-d4 as constant modifications.
  • the protein names, Swiss Prot accession numbers, the Mascot protein score (Score) and the number of matched queries (QM) searching the respective set of fixed modifications are displayed Incubation of HeIa cells with STLC for 24 h to induce mitotic arrest corresponded to MDHI-d4/SA and for 48 h to trigger apoptosis to MDHI/SA-d4
  • the relative protein score (ReI score) was calculated if the protein was identified with both sets of fixed modifications (MDHI-d4/SA)/(MDHI/SA-d4) Most of the proteins have previously been identified to be linked to apoptosis
  • the ratio and variability of the peptide is transferred back to the protein table where also the overall protein ratio and variability is calculated.
  • the protein list and all its quantification information can be exported to a spreadsheet application via a tab-separated values (tsv) file. From the file menu choose "Save As..” and give the file a unique name ending with .tsv.
  • the protein list can be opened and processed for post-quantification analysis like ratio normalization and significance determination.
  • ICAT isotope-coded affinity tagging
  • ICPL isotope-coded protein labeling
  • IPTL isobaric peptide termini labeling
  • iTRAQ isobaric tagging for relative and absolute quantification
  • MDHI methoxy-4,5-dihydro-1 H-irnidazole
  • SA succinic anhydride
  • SILAC stable isotope labeling by amino acids in cell culture
  • STLC S-trityl-L-cysteine
  • TMT Tandem mass tagging.
  • EDTA ethylenediaminetetraacetic acid
  • LTQ linear trap quadrupole MALDI/TOF MS : matrix assisted laser desorption ionization / time of flight mass spectroscopy
  • PCM polydimethylcyclosiloxane

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Abstract

L'invention concerne un procédé de quantification relative ou absolue d'analyse d'au moins deux échantillons protéiques. Les échantillons protéiques sont modifiés de façon à produire des peptides isobares qui sont par la suite soumis à la spectrométrie de masse en tandem. Le procédé implique la digestion des échantillons protéiques et l'étiquetage transversal d'extrémités N et C terminales des peptides ainsi obtenus.
PCT/EP2010/054109 2009-03-27 2010-03-29 Procede de proteomique quantitative WO2010109022A1 (fr)

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CN102707001A (zh) * 2012-05-21 2012-10-03 天津大学 一种微藻蛋白组的分析方法
WO2014066284A1 (fr) * 2012-10-22 2014-05-01 President And Fellows Of Harvard College Protéomique quantitative multiplexe précise et sans interférence faisant appel à la spectrométrie de masse
US10145818B2 (en) 2012-10-22 2018-12-04 President And Fellows Of Harvard College Accurate and interference-free multiplexed quantitative proteomics using mass spectrometry
CN103884806A (zh) * 2012-12-21 2014-06-25 中国科学院大连化学物理研究所 结合二级质谱和机器学习算法的蛋白质组无标记定量方法
CN103884806B (zh) * 2012-12-21 2016-01-27 中国科学院大连化学物理研究所 结合二级质谱和机器学习算法的蛋白质组无标记定量方法
US11085927B2 (en) 2016-06-03 2021-08-10 President And Fellows Of Harvard College Techniques for high throughput targeted proteomic analysis and related systems and methods
GB2607197A (en) * 2018-06-06 2022-11-30 Bruker Daltonics Gmbh & Co Kg Targeted protein characterization by mass spectrometry
GB2607197B (en) * 2018-06-06 2023-04-26 Bruker Daltonics Gmbh & Co Kg Targeted protein characterization by mass spectrometry
CN109142561A (zh) * 2018-07-17 2019-01-04 上海师范大学 同时定量蛋白质丰度与半胱氨酸氧化水平的方法及其应用

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