WO2016120247A1

WO2016120247A1 - Methods for a quantitative release of biotinylated peptides and proteins from streptavidin complexes

Info

Publication number: WO2016120247A1
Application number: PCT/EP2016/051525
Authority: WO
Inventors: Uwe Warnken; Martina SCHNÖLZER; Eva LINDER
Original assignee: Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
Priority date: 2015-01-26
Filing date: 2016-01-26
Publication date: 2016-08-04
Also published as: EP3250928A1

Abstract

The present invention relates to a fast, mild and quantitative method for the release of biotinylated peptides and proteins from complexes with streptavidin using a polyfluorinated alcohol.

Description

Methods for a quantitative release of biotinylated peptides and proteins from streptavi- din complexes.

The present invention relates to a fast, mild and quantitative method for the release of biotinylated peptides and proteins from complexes with streptavidin.

Background of the invention

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. Today, binding of biotin to streptavidin (or avidin) is utilized for immobilization of biotinylated molecules to a variety of streptavidin-based reagents (e.g. agarose beads, magnetic beads, silica pipette tips etc.), for tagging molecules for further functionalization, for enrichment of biotinylated molecules and for pull-down assays.

Chaiet and Wolf (in: Louis Chaiet, Frank J. Wolf The properties of streptavidin, a biotin- binding protein produced by Streptomyces Archives of Biochemistry and Biophysics, Volume 106, 1964, Pages 1-5) describe the crystalline protein streptavidin, isolated from fermentation filtrates of Streptomyces, as a new type of biotin-binding protein.

The binding between streptavidin and biotin is 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.

In addition many yet known releasing procedures are hampered by low peptide and protein recovery yields and side effects like peptide and protein destruction. To circumvent some of these restrictions in the dissociation of streptavidin-biotin complexes, cleavable linkers have been designed, which allow affinity captured proteins to be released under the linker's cleavage conditions. Currently, disulfide linkers are an essential part of many commercialized affinity assays which can be cleaved with reducing agents. However, as a consequence the experimental conditions are limited through the fact, that cleavage conditions have to be prevented before the releasing step.

Rybak et al. (in: Rybak JN, Scheurer SB, Neri D, Elia G. Purification of biotinylated proteins on streptavidin resin: a protocol for quantitative elution. Proteomics. 2004 Aug;4(8):2296-9) disclose a protocol for the quantitative elution of biotinylated proteins from streptavidin se- pharose, featuring harsh elution conditions and competition with free bio tin. The usefulness of the method was demonstrated by the quantitative recovery of biotinylated proteins from organ homogenates, obtained from mice perfused with a reactive ester derivative of biotin. Similarly, Rosli et al. (in: Christoph Rosli, Jascha-N. Rybak, Dario Neri, and Giuliano Elia; Quantitative Recovery of Biotinylated Proteins from Streptavidin-Based Affinity Chromatography Resins From: Methods in Molecular Biology, vol. 418: Avidin-Biotin Interactions, Methods and Applications Edited by: R. J. McMahon O Humana Press, Totowa, NJ) disclose that, when desired, the release of biotinylated proteins from the streptavidin-based reagents remains a major problem, due to the extraordinary stability of this complex. They present a protocol for the quantitative elution of biotinylated proteins from streptavidin sepharose, featuring harsh elution conditions and competition with free biotin.

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. 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. These results pointed to the possibility of combining an SCD streptavidin mutant with B4F derivatives to create a fluorescence-tagged affinity system.

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.

Giang et al. (Giang H. Nguyen, Jaqueline S. Milea, Anurag Rai, Cassandra L. Smith Bio- molecular Engineering Volume 22, Issue 4, October 2005, Pages 147-150) also disclose that the high affinity (k_d = ~ 10 ¹⁵ M) of streptavidin and avidin for biotin would be key to a large number of biological applications, and would be essentially irreversible unless the complex is exposed to harsh conditions (e.g. heat (100 °C for lO min)), detergents, and/or denaturants which damage macromolecules. They describe "relatively mild" conditions that release biotin and mono- and bis-biotinylated macromolecules from immobilized streptavidin on monodis- persed magnetic beads.

Other protocols describe the digestion of proteins directly on streptavidin surfaces. Proteolytic peptides are released but the ones that carry the biotin moiety are still attached to a remnant peptide originating from digested streptavidin. Since the masses of these adducts are unknown they escape their detection by mass spectrometry. Hence, affinity-captured proteins are identified exclusively by their non-biotinylated peptides, making it impossible to distinguish unspe- cific binders from truly biotinylated proteins.

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.

Temperature and Time Elution Solution Elution Efficiency

90°C for 10 minutes 10 mM EDTA pH 8.2 and 95% formamide 96.8%

90°C for 5 minutes 10 mM EDTA pH 8.2 and 95% formamide 96.4%

90°C for 2 minutes 10 mM EDTA pH 8.2 and 95% formamide 96.8%

65°C for 5 minutes 10 mM EDTA pH 8.2 and 95% formamide 96.4%

65°C for 2 minutes 10 mM EDTA pH 8.2 and 95% formamide 97.9%

37°C for 10 minutes 10 mM EDTA pH 8.2 and 95% formamide 41.9%

90°C for 10 minutes H₂0 7.3%

90°C for 10 minutes 10 mM EDTA pH 8.2 52.0%

90°C for 10 minutes 95% formamide 35.9%

90°C for 10 minutes 30 mM NaOAc pH 9 and 95% formamide 95.5%

90°C for 10 minutes 80 mM NaOAc pH 9 and 95%> formamide 97.3%

90°C for 10 minutes 140 mM NaOAc pH 9 and 95% formamide 95.4% This table shows that the elution efficiencies of free biotin for low temperatures (37°C), non- harsh solvent (H₂0), and lack of EDTA are very low.

Kida et al. (Kida 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 (Lu X, Murphy RM. Synthesis and disaggregation of asparagine repeat- containing peptides. J Pept Sci. 2014 Nov; 20(11):860-7) 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). Preferably, 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.

Finally, 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.

Examplary genetically modified streptavidins and avidins are described in, for example, Laitinen et al. (Laitinen OH, Hytonen VP, Nordlund HR, Kulomaa MS. Genetically engineered avidins and streptavidins. Cell Mol Life Sci. 2006 Dec;63(24):2992-3017) and the references as cited therein.

In the context of the present invention, HFIP is used as a preferred example for a fluorinated alcohol. Nevertheless, 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. Thus, 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.

In a preferred embodiment, 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. 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. In a preferred method according to the invention, 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 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. Thus, 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.

In a preferred method according to the invention, the organic solvent is 1,1,1,3,3,3- hexafluoro-2-propanol (HFIP).

In another aspect of the present invention, organic salts are added to the solvent as pro- tein/peptide solubilizers, such as, for example, 2% ammonium acetate. Thus, preferred is a method according to the present invention, wherein 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.

In a preferred method according to the present invention, 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.

Preferred is a method according to the present invention, wherein said method is performed as a qualitative or quantitative assay, for example utilizing qualitative and quantitative elec- trospray mass spectrometry (MS) as readout. In a preferred quantitative assay, an essentially complete recovery (e.g. elution) of streptavidin-biotin bound binding partner is achieved using the method according to the present invention.

In the context of the present invention, in general 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%. As organic salt additives, such as ammonium acetate, between 0.2 to 5, preferably a maximum of 2% (w/v) can be used.

Further preferred is 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. In another aspect of the present invention, the method according to the present invention can be used/performed in the context of proteomics assays. Preferably, these assays comprise methods like:

in vitro and in vivo biotinylation assays for characterization of the membrane surface proteome;

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;

drug tagging with biotin for kinetic studies;

- the use of biotinylated electrodes;

- protein/peptide labeling with isotope coded affinity tags (ICAT) for relative and absolute quantification of proteins with mass spectrometry;

click chemistry approaches, for example for the identification of newly synthesized proteins in cells and/or organelles thereof;

activity-based protein profiling (ABPP);

- multi-consecutive enrichment and release of 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

photo-induced covalent cross-linking for the analysis of bio molecular interactions.

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.

In yet another aspect of the present invention, 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.

Another aspect of the present invention then relates to the use of a kit 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. Optionally, instructions for use can be included in the form of a manual as well.

Preferred is the use of the kit according to the present invention, wherein said solid support comprises magnetic particles.

The invention will now be described further in the following examples with reference to the accompanying figures, nevertheless, without being limited to these examples. For the purposes of the present invention, all references as cited herein are incorporated in their entireties.

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.

Figure 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 [¹³C, ¹⁵N] 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 [¹³C, ¹⁵N] 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). The peptides SLGK- bioVGTR (SEQ ID NO. 63), LSQKbioFPK (SEQ ID NO. 64), ALKbioAWSVAR (SEQ ID NO. 65), and FKbioDLGEEHFK (SEQ ID NO. 66) were detected based on their monoisotop- ic masses of 1042.5625 Da, 1072.5775 Da, 1226.6645 Da and 1474.6957 Da. All biotinylated peptides were identified in high amounts in the molecular sieve filtrate as demonstrated with the mass spectrum of the unpurified control sample (A).

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.

EXAMPLES

The following examples have been produced using HFIP as an example for a fluorinated alcohol. Nevertheless, the methods can be performed using other fluorinated, in particular poly- fluorinated, as alcohols, for example, lower alcohols.

Example 1: Capture and release of biotinylated bovine serum albumin from streptavidin coated silica columns

Bovine serum albumin (BSA) 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.

Table 1 (see below): Intact biotinylated BSA was captured and released from streptavidin D.A.R.T.'S column and tryptically digested. BSA tides were identified by Orbitrap mass spectrometry. Shown are the Mascot protein database search results, Start-End: start and end point o peptide sequence in the BSA protein, Mz(Observed): detected ion mass to charge ratio, Mr(expt); experimental monoisotopic peptide

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.

19 549-557 507.8109 1013.6072 1013.6121 -4.80 44 K. QTAL VELLK.H

20 89-100 710.3466 1418.6787 1418.6864 -5.44 51 K. SLHTLFGDELCK. V

21 76-100 1030.7797 3089.3171 3089.3351 -5.82 63 K.TCVADESHAGCEKSLHTLFGDELCK.V + Biotin (K)

22 490-498 607.8239 1213.6333 1213.6376 -3.61 59 K.TPVSEKVTK.C + Biotin (K)

23 569-580 700.3466 1398.6786 1398.6853 -4.86 66 K.T VMENF VAFVDK. C

24 264-280 780.6553 2338.9440 2338.9551 -4.77 54 K.VHKECCHGDLLECADDR.A + Biotin (K)

25 438-451 756.4208 1510.8270 1510.8355 -5.65 44 K. VPQ VSTPTLVE VSR. S

26 496-507 846.8920 1691.7695 1691.7793 -5.81 54 K.VTKCCTESLVNR.R + Biotin (K)

27 286-297 722.3209 1442.6272 1442.6347 -5.26 66 K.YICDNQDTISSK.L

28 286-299 955.9480 1909.8814 1909.8914 -5.19 86 K.YICDNQDTISSKLK.E + Biotin (K)

29 161-167 464.2484 926.4823 926.4861 -4.17 47 K.YLYEIAR.R

30 184-197 874.8444 1747.6743 1747.6818 -4.29 59 K.YNGVFQECCQAEDK.G + Deamidated (NQ)

31 281-297 1084.4980 2166.9815 2166.9925 -5.07 88 R.ADLAKYICDNQDTISSK.L + Biotin (K)

32 233-241 614.3341 1226.6536 1226.6594 -4.66 44 R.ALKAWSVAR.L + Biotin (K)

33 223-232 711.3332 1420.6518 1420.6591 -5.14 53 R.CASIQKFGER.A + Biotin (K)

34 460-468 696.7858 1391.5570 1391.5632 -4.46 43 R.CCTKPESER.M + Biotin (K)

35 25-34 473.8962 1418.6668 1418.6725 -4.00 41 R.DTHKSEIAHR.F + Biotin (K)

36 106-117 739.7616 1477.5086 1477.5160 -4.96 48 R.ETYGDMADCCEK.Q

37 106-122 1172.4462 2342.8778 2342.8912 -5.72 68 R.ETYGDMADCCEKQEPER.N + Biotin (K)

38 35-44 417.2105 1248.6097 1248.6139 -3.36 44 R.FKDLGEEHFK.G

39 35-44 492.5683 1474.6831 1474.6915 -5.65 53 R.FKDLGEEHFK.G + Biotin (K)

40 361-371 642.3563 1282.6980 1282.7034 -4.20 58 R.HPEYAVSVLLR.L

41 169-183 630.3105 1887.9098 1887.9195 -5.16 52 R.HPYFYAPELLYYANK.Y

42 437-451 547.3145 1638.9217 1638.9305 -5.36 48 R.KVPQVSTPTLVEVSR.S

43 437-451 933.5063 1864.9981 1865.0081 -5.33 47 R.KVPQVSTPTLVEVSR.S + Biotin (K)

44 372-386 1020.9520 2039.8894 2039.9002 -5.30 55 R. L AKEYE ATLEECC AK. D + Biotin (K)

45 483-495 883.4479 1764.8812 1764.8902 -5.13 58 R.LCVLHEKTPVSEK.V + Biotin (K)

46 242-256 1102.0624 2202.1102 2202.1217 -5.22 51 R.LSQKFPKAEFVEVTK.L + 2 Biotin (K)

47 469-482 862.9166 1723.8187 1723.8273 -5.00 111 R.MPCTEDYLSLILNR.L

48 360-371 480.6060 1438.7960 1438.8045 -5.86 65 R.RHPEYAVSVLLR.L

49 168-183 682.3440 2044.0101 2044.0206 -5.14 49 R.RHPYFYAPELLYYANK.Y

50 508-528 899.7570 2696.2490 2696.2614 -4.60 46 R.RPCFSALTPDETYVPKAFDEK.L + Biotin (K)

51 452-459 522.2842 1042.5538 1042.5593 -5.32 43 R.SLGKVGTR.C + Biotin (K)

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 μg of the tryptic digest of biotinylated BSA was mixed with 2.5 μg 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 μΐ HFIP. After evaporation, one half of the eluate was applied to mass spectrometry analysis. The results of the Mascot (Matrix Science) protein mass spectrometry database search for BSA before and after extraction are shown in figure 2. The proportion of biotinylated peptides is substantially higher after the D.A.R.T.'S extraction compared to untreated biotinylated BSA. Additionally, in the HFIP eluates only very small amounts of ovalbumin were found.

Example 3: Streptavidin capture and release experiments with biotinylated and non- biotinylated synthetic peptides.

A set of synthetic biotinylated BSA peptides was applied to different streptavidin D.A.R.T.'S affinity capture and release experiments. The goal of these experiments was to demonstrate whether or not binding of biotinylated peptides to D.A.R.T.'S columns is specific.

To this end, a mixture of 20 pmol each of four biotinylated and four non-biotinylated synthetic peptides was applied to D.A.R.T.'S column which were subsequently washed four times ((i) 2 M ammonium acetate in 10% acetonitrile, (ii) 10 % acetonitrile, (iii) 2 % SDS, and (iv) 10 % acetonitrile) to remove non-biotinylated peptides. Finally biotinylated peptides were released by elution with HFIP and the eluate was analyzed by mass spectrometry. In comparison, the untreated mixture without extraction on D.A.R.T.'S columns was also applied to mass spectrometry. The extracted ion chromatograms of the untreated mixture are shown in figure 3.

In experiment 1, untreated streptavidin D.A.R.T.'S were used for extraction. Washing steps were performed as described above. As shown in figure 4, biotinylated peptides were present in high amounts in the eluate, whereas non-biotinylated peptides were either not detectable or identified at the limit of detection. In experiment 2, streptavidin D.A.R.T.'S were blocked with a 300-fold excess of free biotin prior to the peptide capture step. Blockage with biotin should prevent subsequent binding of biotinylated peptides in case the binding is biotin specific. As expected, only negligible amounts of biotinylated peptides were found in the eluate (see figure 5). Neither biotinylated nor non-biotinylated peptides were detected after washing with 2% SDS elution with HFIP and mass spectrometry analysis. Thus, it could be demonstrated, that binding of biotinylated peptides to D.A.R.T.'S columns is not an unspecific event due to hydrophobic effects.

Taken together, 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.

A set of synthetic biotinylated BSA peptides (biotinylated unlabeled peptides) and their ¹³C and ¹⁵N labeled counterparts (biotinylated heavy labeled peptides) were used in the quantification experiment aiming to estimate the recovery rate of the streptavidin binding and HFIP elution workflow.

A mixture of four 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 μg 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.

Elution was performed in two steps with HFIP. The eluates were combined and biotinylated heavy labeled peptides (20 pmol each) were added as internal standards, thus allowing quantification of their unlabeled counterparts. The combined eluates were evaporated and 1/50 was applied to Orbitrap-M5 analysis.

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-iT™ Protein Reaction Buffer Kit (Life Technologies) according to the manufacturer's instructions.

After removal of excess biotin-alkyne reagent by precipitation, proteins were digested with trypsin. In total, 100 μg 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.

Example 6: Removal of streptavidin contamination originated from elution with HFIP

Streptavidin is a homotetramer protein with a molecular mass of around 60 kDa made of four streptavidin subunits with each 16.8 kDa. As a consequence of the elution of for example bio- tin, biotinylated peptides or proteins etc. from streptavidin applying HFIP, 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. Simple methods for the removal of streptavidin from biotinylated peptides are introduced here, which could be used combination with the release of biotinylated peptides or other small biotinylated molecules from complexes with streptavidin applying HFIP.

A set of synthetic biotinylated BSA peptides was applied to streptavidin D.A.R.T.'S affinity capture and releasing experiments using HFIP. Biotinylated peptides as well as streptavidin were released from the D.A.R.T.'S columns. The eluates were supplied to different experi- ments, the first was a CI 8 reversed phase separation and the second a molecular sieve filtration cleanup method. The goal of these experiments was to demonstrate, whether or not resolved streptavidin could be separated from mixtures with biotinylated peptides following affinity capture and release experiments using HFIP.

A mixture of four synthetic biotinylated peptides SLGKbioVGTR, LSQKbioFPK (2), ALK- bioAWSVAR, and FKbioDLGEEHF, (0.5 μg each) was bound using streptavidin D.A.R.T.'S columns. Binding was performed for 1 h, followed by four washing steps with ((i) 2 M ammonium acetate in 10% acetonitrile, (ii) 10 % acetonitrile, (iii) 2 % SDS, and (iv) 10 % ace- tonitrile) to remove non-specifically bound contaminations. Elution was performed in two steps with HFIP. The eluates were combined, evaporated and resolved in water added with 0.1% trifluoroacetic acid. The sample was bound to a reversed phase CI 8 Zip Tip (Millipore) pipette chromatography column and eluted according to the manufacturer's protocol. 1/10 was applied to MALDI-ToF mass spectrometry for the analysis of biotinylated peptides and 9/10 were applied to one dimensional polyacrylamide gel electrophoresis analysis (ID- PAGE) and coomassie staining for streptavidin analysis. The ID-PAGE image is shown in figure 8 and the MALDI-ToF mass fingerprint spectra of recovered biotinylated peptides are shown in figure 9.

Example 7:

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. The combined filtrates were evaporated and after solvation, 1/10 was applied to MALDI-ToF mass spectrometry for the analysis of biotinylated peptides and 9/10 were applied to one dimensional polyacrylamide gel electrophoresis analysis (ID-PAGE) and coomassie staining for streptavidin analysis. The ID-PAGE image is shown in figure 10 and the MALDI-ToF mass spectrum of the recovered biotinylated peptides are shown in figure 11.

Taken together, 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.

Protein Protein description/SEQ ID NO. Mz(obs) Mr(expt) Mr(calc) Score Peptide

acc.

P62736 Actin, aortic smooth muscle/53 842.9522 1683.8899 1683.8865 40 EITALAPSTMK AHA BioAlky (M)

P62736 Actin, aortic smooth muscle/54 847.9280 1693.8414 1693.8392 31 HQGVMVGMGQK AHA BioAlky (M)

P14866 Heterogeneous nuclear ribonucleoprotein L/55 721.3531 1440.6917 1440.6892 42 MAAAGGGGGGGR AHA BioAlky (M)

P62937 Peptidyl-prolyl cis-trans isomerase A/56 1061.5132 2121.0118 2121.0136 40 IIPGFMCQGGDFTR AHA BioAlky (M)

P14174 Macrophage migration inhibitory factor/57 905.9856 1809.9567 1809.9560 37 PMFIVNTNVPR AHA BioAlky (M)

Heterogeneous nuclear ribonucleoprotein

Q32P51 A1L2/58 871.4626 1740.9107 1740.9080 35 IEVIEIMTDR AHA BioAlky (M)

P62258 14-3-3 protein epsilon/59 985.9980 1969.9815 1969.9779 34 VAGMDVELTVEER AHA BioAlky (M)

P62805 Histone H4 OS/60 917.4945 1832.9744 1832.9706 32 TVTAMDVVYALK AHA BioAlky (M)

P13639 Elongation factor 2/61 873.4541 1744.8936 1744.8931 32 MVNFTVDQIR AHA BioAlky (M)

P11021 78 kDa glucose-regulated protein/62 870.9576 1739.9006 1739.8988 31 DAGTIAGLNVMR AHA BioAlky (M)

Claims

1. A method for releasing a biotinylated first binding partner from a complex with strep- tavidin, 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.

2. The method according to claim 1, wherein said biotinylation of said first binding partner is selected from biotin, chemically modified biotin, such as, for example iminobiotin or desthiobiotin.

3. The method according to claim 1 or 2, further comprising the presence of a reducing agent, such as, for example, mercaptoethanol, dithiothreitol (DTT), or tris(2-carboxyethyl)phosphine hydrochloride (TCEP) during said releasing.

4. The method according to any of claims 1 to 3, wherein said method further comprises the step of removing the solvent after releasing said complex, for example by evaporation.

5. The method according to any of claims 1 to 4, further comprising at least one additional biotinylated binding partner, and/or streptavidin.

6. The method according to any of claims 1 to 5, wherein said method further comprises the step of recovering said biotinylated binding partner(s).

7. The method according to any of claims 1 to 6, wherein said organic solvent is 1,1,1,3,3,3- hexafluoro-2-propanol (HFIP).

8. The method according to any of claims 1 to 7, wherein organic salts are added as pro- tein/peptide solubilizers, such as, for example, 2% ammonium acetate.

9. The method according to any of claims 1 to 8, wherein 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.

10. The method according to any of claims 1 to 9, wherein said binding partner is selected from biological molecules, such as, for example, a protein, peptide, nucleic acid, oligosaccharide, glycoprotein, lipid, carbohydrate, hormone or toxin or derivatives thereof, antibody or a derivative thereof.

11. The method according to any of claims 1 to 10, wherein said method is performed as a qualitative or quantitative assay, for example utilizing qualitative and quantitative elec- trospray mass spectrometry (MS) as readout.

12. The method according to any of claims 1 to 11, 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)

13. The method according to any of claims 1 to 12, 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.

14. The method according to any of claims 1 to 13, wherein said method is performed in the context of a proteomics assay comprising in vitro and in vivo biotinylation assays for characterization of the membrane surface proteome; 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; drug tagging with biotin for kinetic studies; biotinylated electrodes; protein labeling with isotope coded affinity tags (ICAT) for relative and absolute quantification of proteins with mass spectrometry; or click chemistry approaches, for example for the identification of newly synthesized cellular proteins.

15. The method according to any of claims 1 to 14, wherein said method is 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.

16. Use of a kit 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 any of claims 1 to 15.

17. Use according to claim 16, wherein said solid support comprises magnetic particles.