EP3250928A1 - Méthodes de libération quantitative de peptides et de protéines biotinylés à partir de complexes de streptavidine - Google Patents

Méthodes de libération quantitative de peptides et de protéines biotinylés à partir de complexes de streptavidine

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Publication number
EP3250928A1
EP3250928A1 EP16701627.8A EP16701627A EP3250928A1 EP 3250928 A1 EP3250928 A1 EP 3250928A1 EP 16701627 A EP16701627 A EP 16701627A EP 3250928 A1 EP3250928 A1 EP 3250928A1
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EP
European Patent Office
Prior art keywords
streptavidin
biotinylated
biotin
protein
peptides
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.)
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Application number
EP16701627.8A
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German (de)
English (en)
Inventor
Uwe Warnken
Martina SCHNÖLZER
Eva LINDER
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Deutsches Krebsforschungszentrum DKFZ
Original Assignee
Deutsches Krebsforschungszentrum DKFZ
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Publication of EP3250928A1 publication Critical patent/EP3250928A1/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Withdrawn legal-status Critical Current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
    • G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G01N33/5735—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes co-enzymes or co-factors, e.g. NAD, ATP
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/36—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Actinomyces; from Streptomyces (G)

Definitions

  • the present invention relates to a fast, mild and quantitative method for the release of biotinylated peptides and proteins from complexes with streptavidin.
  • biotin labeling of organic and inorganic molecules Due to the extraordinary stability of the biotin- streptavidin complex, biotin labeling of organic and inorganic molecules has emerged to a widely used technique for numerous applications.
  • streptavidin or avidin
  • streptavidin-based reagents e.g. agarose beads, magnetic beads, silica pipette tips etc.
  • Chaiet and Wolf describe the crystalline protein streptavidin, isolated from fermentation filtrates of Streptomyces, as a new type of biotin-binding protein.
  • streptavidin and biotin are one of the strongest non-covalent biological bonds known today. This extraordinary stability of the biotin-streptavidin complex poses a major problem when biotinylated molecules need be recovered from such complexes.
  • the interaction between streptavidin and biotin is formed very rapidly. Once formed, the interaction is unaffected by wide extremes of pH, temperature, organic solvents and other denaturing agents. Harsh elution conditions are required to dissociate the complex using heat and chemical reagents like SDS which are not compatible with subsequent analytical techniques especially mass spectrometry.
  • Asian et al. in: Asian FM, Yu Y, Mohr SC, Cantor CR. Engineered single-chain dimeric streptavidins with an unexpected strong preference for biotin-4-fluorescein. Proc Natl Acad Sci U S A. 2005 Jun 14; 102(24):8507-12. Epub 2005 Jun 6) disclose a single-chain dimer of streptavidin (SCD) with two biotin-binding sites and random mutations introduced by error- prone PCR.
  • SCD single-chain dimer of streptavidin
  • the mutant showed a binding affinity to biotin-4-fluorescein (B4F) and K'(d) values for B4F ranged from approximately lO(-l l) to 10(-10) M, although K(d) values for biotin ranged from 10(-6) to 10(-5) M.
  • Ballikaya et al. in: Ballikaya S, Lee I, Warnken U, Schnoelzer M, Gebert J, Kopitz J. De Novo proteome analysis of genetically modified tumor cells by a metabolic labeling/azide- alkyne cycloaddition approach. Mol Cell Proteomics. 2014 Dec;13(12):3446-56. doi: 10.1074/mcp. Ml 13.036665. Epub 2014 Sep 15), amongst others, describe labeled proteins were tagged with biotin via a Click-iT chemistry approach enabling specific extraction of labeled proteins by streptavidin-coated beads.
  • the following table shows some typical elution efficiencies of the biotin- streptavidin interaction under a variety of elution conditions for commonly used magnetic beads (Dynabeads®)
  • the elution efficiencies described are for free biotin, and might differ from that of a biotinylated specific ligand.
  • Kida et al. Korean T, Sato S, Yoshida H, Teragaki A, Akashi M. 1,1, 1,3,3, 3-Hexafluoro-2- propanol (HFIP) as a novel and effective solvent to facilely prepare cyclodextrin-assembled materials.
  • Chem Commun (Camb). 2014 Oct 21; 50(91): 14245-8) disclose that cyclodextrins (CDs) have a high solubility in l,l,l,3,3,3-hexafluoro-2-propanol (HFIP). Evaporating HFIP from CD solutions on a glass plate gave crystalline solids composed of channel-type CD assemblies, and electrospinning with an HFIP solution of CDs fabricated CD micro fibers.
  • Lu and Murphy disclose a protocol involving dissolution in mixed trifluoroacetic acid and hexafluoroisopropanol (TFA + HFIP) solvents for disaggregation of polyQ peptides. Nevertheless, in their tests, formic acid proved to be a better disaggregator and caused no oxidative damage.
  • US 6,562,952 discloses a process for forming small micron-sized (1-10 mum) protein particles is provided wherein a protein, a solvent system for the protein and an antisolvent for the protein solvent system are contacted under conditions to at least partially dissolve the protein solvent system in the antisolvent, thereby causing precipitation of the protein.
  • the solvent system is made up of at least in part of a halogenated organic alcohol, most preferably l,l,l,3,3,3-hexafluoro-2-propanol (HFIP).
  • HFIP l,l,l,3,3,3-hexafluoro-2-propanol
  • a solution of the protein in the solvent system is sprayed through a nozzle into a precipitation zone containing the antisolvent (preferably C02) under near- or supercritical conditions.
  • EP2518193 discloses methods for reversibly binding a biotin compound to a support.
  • the strong interaction between streptavidin or avidin-biotin is made much weaker by using a combination of modified streptavidin or avidin and modified biotin, like desthiobiotin or a derivative thereof, like DSB-X Biotin.
  • a protein, such as an antibody may be biotinylated with the modified biotin.
  • the proteins obtained using these methods are described to maintain their native conformation. Uses of the methods in various procedures including cell detachment procedures and techniques of detection, identification, determination, purification, separation and/or isolation of target proteins or nucleic acid molecules are also described. Overall, still a fast, simple to handle and quantitative releasing method for biotinylated proteins and peptides compatible with, for example, mass spectrometry analysis would be highly desirable, and would be a valuable tool, in particular for downstream processes.
  • This object of the present invention is solved by a fast and quantitative method for releasing a biotinylated first binding partner from a complex with streptavidin, genetically modified streptavidin, and/or avidin in a sample, comprising a step of incubating said complex with a [poly]-fluorinated alcohol under conditions that are sufficient to release said complex.
  • HFIP is used as a preferred example for a fluorinated alcohol.
  • the methods according to the invention can be performed using other fluorinated, in particular poly-fluorinated alcohols, such as, for example, lower alcohols.
  • An unexpected result of the work underlying the present invention is that neat (pure or concentrated) HFIP or concentrated solutions thereof efficiently dissociate biotin-streptavidin complexes.
  • Biotin-streptavidin complexes are completely soluble in neat HFIP or HFIP solutions spiked with low amounts of solubilizers.
  • release of biotinylated peptides and proteins from streptavidin which is immobilized on columns, beads or other surfaces can easily be performed by elution with HFIP. Release is essentially quantitative after a single elution step.
  • biotin-streptavidin complexes are dissociated by the organic solvent 1,1, 1,3,3, 3-hexafluoro-2-propanol (HFIP) or mixtures thereof with minor amounts of water and/or additional organic salts acting as protein solubilizers.
  • HFIP organic solvent 1,1, 1,3,3, 3-hexafluoro-2-propanol
  • the present inventors exemplify this approach by capture of biotinylated proteins and peptides to silica pipette tip columns with immobilized streptavidin and their release with HFIP utilizing qualitative and quantitative electrospray mass spectrometry (MS) as readout, as described herein.
  • MS electrospray mass spectrometry
  • said biotinylation of said first binding partner is selected from biotin or a chemically modified biotin such as, for example iminobiotin, or desthiobiotin, or crosslinked streptavidins.
  • a preferred method according to the invention further comprises the presence of a reducing agent, such as, for example, mercaptoethanol, dithiothreitol (DTT), or tris(2- carboxyethyl)phosphine hydrochloride (TCEP) during said releasing.
  • a reducing agent such as, for example, mercaptoethanol, dithiothreitol (DTT), or tris(2- carboxyethyl)phosphine hydrochloride (TCEP) during said releasing.
  • a more preferred method according to the invention further comprises the step of removing the solvent after releasing said complex, for example by evaporation. Due to its low boiling point of 58.2°C, HFIP can easily be removed by evaporation and hence does not interfere with downstream applications.
  • Another aspect of the present invention relates to a method according to the present invention, further comprising at least one additional biotinylated binding partner, and/or streptavidin.
  • the present method can be simultaneously used in assays with multiple binding partners, e.g. 2, 3, 4, 5 or more biotinylated binding partners and/or different forms of streptavidin, e.g. coupled to different solid surfaces, as described herein.
  • Another aspect of the present invention then relates to a method according to the present invention, wherein said method further comprises the step of recovering said biotinylated binding partner(s).
  • Recovery involves the removal, and at least partial removal, of compounds/components other than the desired binding partner, e.g. from the elution solution, for example before a further downstream use of the binding partner.
  • the organic solvent is 1,1,1,3,3,3- hexafluoro-2-propanol (HFIP).
  • organic salts are added to the solvent as pro- tein/peptide solubilizers, such as, for example, 2% ammonium acetate.
  • said organic solvent is a mixture with small amounts of other organic solvents, such as, for example, water, such as, for example, HFIP containing 30%, preferably 10% water.
  • Neat HFIP can be used for elution as well as highly concentrated HFIP solvents containing up to 30% (v/v) water and 2% (w/v) of organic salt additives e.g. ammonium acetate, supporting solubilization of hardly soluble proteins.
  • the binding partner is selected from biological molecules that can be, directly or indirectly, be biotinylated (e.g. provided with a biotin-moiety or derivatives thereof as above being capable of binding to streptavidin or derivatives thereof), such as, for example, a protein, peptide, nucleic acid, oligosaccharide, glycoprotein, lipid, carbohydrate, hormone or toxin or derivatives thereof, antibody or a derivative thereof.
  • biotinylated e.g. provided with a biotin-moiety or derivatives thereof as above being capable of binding to streptavidin or derivatives thereof
  • a protein, peptide, nucleic acid, oligosaccharide, glycoprotein, lipid, carbohydrate, hormone or toxin or derivatives thereof, antibody or a derivative thereof such as, for example, a protein, peptide, nucleic acid, oligosaccharide, glycoprotein, lipid, carbohydrate, hormone or toxin or derivatives thereof, antibody or
  • a qualitative or quantitative assay for example utilizing qualitative and quantitative elec- trospray mass spectrometry (MS) as readout.
  • MS mass spectrometry
  • an essentially complete recovery (e.g. elution) of streptavidin-biotin bound binding partner is achieved using the method according to the present invention.
  • the person of skill will be able to determine conditions that are sufficient to release a biotinylated first binding partner from a complex with streptavidin in a sample using the fluorinated alcohol. In principle, conditions that effectively inactivate destroy and/or denaturate the binding partner will be avoided, depending from the downstream uses as intended.
  • Preferred is a method according to the present invention, wherein said conditions are selected from a temperature of between 10°C and 58.1°C, preferably between 15°C and 40°C, most preferred at ambient temperature (20°C ⁇ 5°C). Other possible temperatures are body temperature (i.e. 37°C).
  • Conditions also can include mixtures with small amounts of other organic solvents, such as, for example, water or alcohol in an amount of between 30% and 1%, preferably a maximum of 10%.
  • organic salt additives such as ammonium acetate, between 0.2 to 5, preferably a maximum of 2% (w/v) can be used.
  • the method according to the present invention wherein at least one of the binding partner or the streptavidin is coupled to a solid support, wherein said solid support is preferably selected from the group consisting of surfaces of plastic, glass, ceramics, silicone, metal, cellulose, and gels, such as beads, tubes, chips, resins, plates, wells, films, sticks or particles, more preferably wherein the solid support comprises magnetic particles.
  • the method according to the present invention can be used/performed in the context of proteomics assays.
  • these assays comprise methods like:
  • affinity tag assays for investigating protein-protein-, protein-nucleic acid-, nucleic acid-nucleic acid-crosslinked complexes
  • identifying and quantifying protein adducts with environmental toxins such as, for example, pesticides after biotin labeling and streptavidin enrichment
  • ICAT isotope coded affinity tags
  • click chemistry approaches for example for the identification of newly synthesized proteins in cells and/or organelles thereof;
  • ABSPP activity-based protein profiling
  • biotinylated peptides proteins, nucleic acids small organic molecule etc. for successive workflows utilizing the biotin moiety; protein surface structure analysis by biotin labeling of native proteins; or
  • the present releasing method of biotinylated proteins and peptides bound to streptavidin, preferably using HFIP, facilitates qualitative and quantitative analysis workflows in these state of the art proteomics assays.
  • the method according to the present invention can be used/performed in the context of a technique of detecting, identifying, determining, separating and/or isolating or purifying of cells from a mixture of components, in particular in the analysis or diagnosis of a sample.
  • kits comprising (i) streptavidin, optionally attached to a solid support; (ii) a reagent for biotinylation of a binding partner; (iii) at least one fluorinated organic solvent; and (iv) optionally, free biotin or derivatives or fragments thereof for performing a method according to the present invention.
  • instructions for use can be included in the form of a manual as well.
  • kits according to the present invention wherein said solid support comprises magnetic particles.
  • Figure 1 shows a protein view of biotinylated and non-biotinylated peptides matching BSA identified by Orbitrap mass spectrometry analysis.
  • Protein database search was performed with the MS/MS data of the tryptic digest after elution of in vitro biotinylated BSA mixed with a 100-fold excess of native ovalbumin. The mixture was applied to a streptavidin coated D.A.R.T.'S silica column (Thermo-Scientific), streptavidin bound proteins were released with HFIP. Matched peptides are indicated in bold gray. Sequence coverage is 87%. (SEQ ID No. 52)
  • Figure 2 shows the mass spectrometry analysis of in vitro biotinylated BSA digested with trypsin. Number of detected biotinylated and non-biotinylated peptides before (A) and after (6) capture and release with HFIP from streptavidin silica gel D.A.R.T.'S columns (Thermo- Fisher). Singly and doubly biotinylated peptides are highly enriched after streptavidin D.A.R.T.'S capture and HFIP release.
  • Figure 3 shows that a mixture of both biotinylated (3 A) and non-biotinylated synthetic peptides (3B) (about 200 fmol each) was analyzed by mass spectrometry without D.A.R.T.'S extraction. Shown are the extracted ion chromatograms of all eight peptides.
  • FIG. 4 shows that a mixture of both biotinylated and non-biotinylated synthetic peptides (about 200 fmol each) was analyzed by mass spectrometry after D.A.R.T.'S extraction (experiment 1). Shown are the extracted ion chromatograms of all eight peptides. Biotinylated peptides were recovered in high amounts (B) whereas non-biotinylated peptides were not detectable or were identified at the limit of detection (A).
  • Figure 5 shows that D.A.R.T.'S columns were saturated with free biotin prior to the addition of the mixture of both biotinylated and non-biotinylated synthetic peptides as described above (experiment 2). Shown are the extracted ion chromatograms of all eight peptides. Neither biotinylated (B) nor non-biotinylated (A) peptides could be detected.
  • Figure 6 shows measurements of peptide recovery.
  • Four synthetic biotinylated light BSA peptides were captured on streptavidin D.A.R.T.'S, and subsequently eluted with HFIP. Mass spectrometric quantification of each peptide was performed by adding equal amounts of their corresponding [ 13 C, 15 N] leucine labeled heavy peptide prior to mass spectrometry analysis.
  • Peptide recoveries were in the range of 85-96 %.
  • Figure 7 shows the amounts of non-absorbed biotinylated peptides in the binding buffer after streptavidin D.A.R.T.'S affinity binding of four synthetic biotinylated BSA peptides.
  • Mass spectrometry quantification of each peptide was performed by adding equal amounts of their corresponding [ 13 C, 15 N] leucine labeled heavy peptide prior to mass spectrometry analysis. Amounts of residual peptides were ranging from 2-26 %, corresponding to the deficits as shown in Figure 6.
  • Figure 8 shows the ID-PAGE analysis of streptavidin D.A.R.T.'S HFIP eluates from example 6.
  • Lane 1 shows the molecular weight marker set and lane 2 shows a distinct spot of streptavidin at 14 kDa eluted with HFIP from streptavidin D.A.R.T.'S. Streptavidin was removed almost completely from the HFIP eluates after C18 ZipTip purification, as demonstrated in lanes 3 and 4.
  • Figure 9 shows the MALDI-ToF mass spectrometry analysis of four biotinylated BSA peptides, eluted with HFIP from a streptavidin D.A.R.T.'S column followed by C18 ZipTip purification as described in example 6 (B). No differences in the mass spectra could be observed if compared with the unpurified control sample (A).
  • the peptides SLGKbioVGTR (SEQ ID NO. 63), LSQKbioFPK (SEQ ID NO. 64), ALKbioAWSVAR (SEQ ID NO. 65), and FKbio- DLGEEHFK (SEQ ID NO. 66) were detected based on their monoisotopic masses of 1042.5625 Da, 1072.5775 Da, 1226.6645 Da and 1474.6957 Da, respectively.
  • Figure 10 shows the ID-PAGE analysis of streptavidin D.A.R.T.'S HFIP eluates from example 7.
  • Lane 1 shows the molecular weight marker set and lane 2 shows a distinct spot of strep- tavidin at 14 kDa eluted with HFIP from streptavidin D.A.R.T.'S. Streptavidin was removed almost completely from the HFIP eluates after MICROCON YM3 3 kDa molecular sieve filtration, as demonstrated in lanes 3 and 4.
  • Figure 11 shows the MALDI-ToF mass spectrometry analysis of four biotinylated BSA peptides, eluted with HFIP from a streptavidin D.A.R.T.'S column followed by MICROCON YM3 3 kDa molecular sieve filtration as described in example 7 (B).
  • SEQ ID NO. 1 to 51 show the peptide sequences as depicted in table 1.
  • SEQ ID NO. 52 shows the peptide sequence as depicted in Figure 1.
  • SEQ ID NO. 53 to 62 show the peptide sequences as depicted in table 2.
  • SEQ ID NO. 63 to 66 show peptides according to example 6 and 7.
  • Example 1 Capture and release of biotinylated bovine serum albumin from streptavidin coated silica columns
  • Bovine serum albumin was biotinylated with sulfo-NHS-biotin and, after removal of excess sulfo-NHS-biotin, mixed with a 100-fold excess of ovalbumin. The mixture was applied to a streptavidin coated D.A.R.T.'S silica column (Thermo -Scientific).
  • the column was washed four times ((i) 2 M ammonium acetate in 10 % acetonitrile, (ii) 10 % acetonitrile, (iii) 2 % SDS, and (iv) 10 % acetonitrile) for removal of unspecific binders and eluted two times with HFIP containing 10% water and 2% ammonium acetate. After evaporation of HFIP, proteins were digested with trypsin and applied to mass spectrometry analysis.
  • Bovine serum albumin was identified as the major component with minor amounts of ovalbumin. As shown in figure 1, the sequence coverage of serum albumin is more than 80% with high amounts of singly and doubly biotinylated BSA peptides (Table 1). It is an unexpected finding, that highly biotinylated BSA linked to steptavidin could be released simply by elution with concentrated HFIP solution.
  • Mr(calc) calculated monoisotopic peptide mass
  • ppm peptide mass accuracy in parts per million calculated from the difference between Mr(e and Mr(calc).
  • Score Mascot peptide score, scores >30, significant (>99%) probability of peptide identification.
  • Peptide identified peptide a acid sequence and assigned post translational modification derived from the mass spectrum. Remarkable is the high amount of BSA recovered f the streptavidin column, despite the high degree of biotinylation and thus, strong binding to streptavidin.
  • Example 2 Tryptic digest of biotinylated BSA, streptavidin D.A.R.T.'S enrichment and HF1P elution, followed by MS identification.
  • Bovine serum albumin was biotinylated with sulfo-NHS-biotin and subsequently digested with trypsin. 3.3 ⁇ g of the tryptic digest of biotinylated BSA was mixed with 2.5 ⁇ g trypsin- digested ovalbumin. Biotinylated peptides were affinity captured using the streptavidin D.A.R.T.'S . Binding was performed for 20 minutes, followed by washing steps with 10% acetonitrile for the removal of non-biotinylated peptides. The streptavidin column was eluted two times with 100 ⁇ HFIP. After evaporation, one half of the eluate was applied to mass spectrometry analysis.
  • Example 3 Streptavidin capture and release experiments with biotinylated and non- biotinylated synthetic peptides.
  • experiment 1 and 2 indicate that biotinylated peptides are bound specifically to streptavidin immobilized on the D.A.R.T.'S and, moreover, that the biotinylated peptides can be eluted in a very simple and efficient way with HFIP.
  • Example 4 Enrichment and quantitative elution of biotinylated peptides from D.A.R.T.'S streptavidin coated silica columns.
  • biotinylated BSA peptides biotinylated unlabeled peptides
  • 13 C and 15 N labeled counterparts biotinylated heavy labeled peptides
  • Biotinylated unlabeled peptides (20 pmol each) was bound to the streptavidin column in the presence of an excess of non-biotinylated peptides originating from 100 ⁇ g of trypsin-digested HCT-116 protein lysate. Biotinylated peptides were captured using the streptavidin D.A.R.T.'S columns. Binding was performed for 1 h, followed by four washing steps with (i) to remove non-specifically bound peptides.
  • the AUCs of the extracted ion chromatograms were used for quantification of the peptides. As shown in Figure 6 84-96 %of the different biotinylated light peptides were recovered in the eluate. Only a weak background of non-biotinylated peptides was observed. In order to determine the amount of biotinylated peptides which have escaped binding to streptavidin D.A.R.T.'S the binding solution after extraction of the biotinylated peptides was quantified in the same manner as the eluates. Only for one sequence the amount of unbound biotinylated peptide was remarkably high (25 %). In the other cases less than 5 % remained unbound.
  • Example 5 Affinity separation and release of biotinylated peptides from a click chemistry approach for the detection of newly synthesized proteins in cell culture.
  • HCT-116-cells were cultured in suitable growth medium, containing azidohomoalanine (AHA) for four hours in the absence of methionine in order to label newly synthesized proteins.
  • Cells were lysed with RIP A buffer.
  • Newly synthesized AHA containing proteins were labelled with biotin-alkyne applying the Click-iTTM Protein Reaction Buffer Kit (Life Technologies) according to the manufacturer's instructions.
  • proteins were digested with trypsin. In total, 100 ⁇ g peptides (biotinylated and non-biotinylated) were enriched using the streptavidin D.A.R.T.'S columns. Binding was performed for 2 h followed by four washing steps for removal of non- specifically bound peptides. Elution was performed in two steps with HFIP. Eluates were combined, evaporated and entirely used for mass spectrometry analysis on an LTQ-Orbitrap.
  • Streptavidin is a homotetramer protein with a molecular mass of around 60 kDa made of four streptavidin subunits with each 16.8 kDa.
  • the homotetramer complex of streptavidin is resolved partially or completely likewise, except the streptavidin monomer is bound covalently to a stationary phase, for instance silica, agarose etc.
  • a set of biotinylated BSA peptides was prepared; affinity captured and released with HFIP from streptavidin D.A.R.T.'S columns in the same way as described in example 6.
  • the evaporated HFIP eluate was resolved in water added with 0.1% trifluoroacetic acid.
  • the samples were filtered through MICROCON YM3 (Millipore) 3 kDa molecular sieve centrifuge filter devices as described according to the manufacturers protocol and washed 2 times with 0.1 % trifluoroacetic acid.
  • experiment 6 and 7 indicate that streptavidin elution is accompanied with the HFIP release of biotinylated peptides immobilized on streptavidin. Moreover, the resolved streptavidin can be removed in very simple and efficient way with CI 8 reversed phase chromatography or 3 kDa molecular sieve filtration.
  • Table 2 List of newly synthesized proteins in cell culture identified by a click-chemistry approach and mass spectrometry analysis after streptavi din-biotin affinity capture and release of biotinylated peptides with HFIP. Shown are the Mascot protein database search results, Protein acc: Uni prot accession number, Protein name: name of the identified protein, Mass: molar mass of the identified protein, Mz(obs): mass spectrometricall detected ion mass to charge ratio, Mr(expt); experimental monoisotopic peptide mass, Mr(calc): calculated monoisotopic peptide mass. Score: Mas cot peptide score, scores >30 indicate significant (>99%) probability of peptide identification. Peptide: identified peptide amino acid sequence as signed azidohomoalanine biotin alkyne modification of methionine. In contrast to previous approaches, proteins were identified directly by thei biotinylated peptides.

Abstract

La présente invention concerne un procédé quantitatif, rapide et doux pour la libération de peptides et de protéines biotinylés à partir de complexes avec de la streptavidine à l'aide d'un alcool polyfluoré.
EP16701627.8A 2015-01-26 2016-01-26 Méthodes de libération quantitative de peptides et de protéines biotinylés à partir de complexes de streptavidine Withdrawn EP3250928A1 (fr)

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