REVERSIBLE STREPTAVIDIN BASED ANALYTE ENRICHMENT SYSTEM FOR USE IN CROSSLINKING MASS SPECTROMETRY ANALYSIS

Abstract

It is provided a reversible streptavidin based analyte enrichment system for use in crosslinking mass spectrometry analysis, in particular for enriching at least parts of crosslinked peptides pairs in mass spectrometry analysis, and a method of enriching at least parts of crosslinked peptides pairs, in particular for use in crosslinking mass spectroscopy analysis.

Claims

1. A reversible streptavidin based analyte enrichment system for use in crosslinking mass spectrometry analysis, in particular for enriching at least parts of crosslinked peptides pairs in mass spectrometry analysis, comprising at least one crosslinking agent of the structure of general formulae (I) ##STR00026## wherein R.sup.1 is selected from a functional group comprising ester, halogen, aldehyde, isothiocyanate, isocyanate, anhydride, epoxide, acetyl, glyoxal, triazine, hydrazine, disulfide, azide, ketone, phosphine, alkene, amine, N-heterocycle, a = 0-10, preferably 1-5, more preferably 2-3; Y is selected from —O—, —S—, —S—S—, —S(═O)—, —.sup.13CH.sub.2—, —CD.sub.2—, —.sup.18O—m, in particular —O—, —S—, —S—S—; n = 0-4, preferably 0, 1 or 2, X is selected from a group comprising —N—, —C.sub.6H.sub.3(NH—), —CH(NH—)—, wherein R2 is attached to N, —CH—, —CH(CO—NH—), wherein R2 is attached to C; R.sup.2 is one of the structures of formulae (IV) comprising biotin analogues or formulae (V) comprising or desthiobiotin analogues (V), wherein biotin is excluded: ##STR00027## or ##STR00028## wherein X.sup.1, X.sup.2, X.sup.3 is selected from —NH.sub.2, —NH—, —N—Me, —N—Et, —O—., R.sup.3 is selected from the group consisting of H, optionally substituted C.sub.1-C.sub.10 alkyl, optionally substituted C.sub.3-C.sub.10 cycloalkyl, optionally substituted C.sub.2-C.sub.10 alkenyl, optionally substituted C.sub.3-C.sub.10 cycloalkenyl, optionally substituted C.sub.2-C.sub.10 alkynyl, optionally substituted C.sub.2-C.sub.10 heteroalkyl, optionally substituted C.sub.3-C.sub.10 heterocycloalkyl, optionally substituted C.sub.2-C.sub.10 heteroalkenyl, optionally substituted C.sub.3-C.sub.10 heterocycloalkenyl, optionally substituted C.sub.2-C.sub.10 heteroalkynyl, optionally substituted C.sub.6-C.sub.14 aryl, optionally substituted C.sub.5-C.sub.14 heteroaryl; Z is selected from —CO—, aryl, preferably C.sub.5-C.sub.6 such as C.sub.6-C.sub.14 aryl, C.sub.5-C.sub.14 heteroaryl, alkyl, preferably C.sub.1-C.sub.4 alkyl, triazoles, alkenes, preferably C.sub.2-C.sub.4 alkenes, alkyl tetrazines, preferably C.sub.1-C.sub.4 tetrazines, optionally substituted —CO—NH—(CH.sub.2).sub.b—, —CO—NH—(CH.sub.2).sub.b—NH—CO—, —CO—NH—(CH.sub.2).sub.b—CO—, wherein b = 1-10, preferably 2-6, optionally substituted —CO—NH—(CH.sub.2—O—).sub.c—(CH.sub.2).sub.d—CO—, CO—NH—(CH.sub.2—O—).sub.c—Triazin—(CH.sub.2).sub.d—CO—, —CO—NH—(CH.sub.2)b—(CH.sub.2—O—)c—(CH.sub.2).sub.d—NH—CO—(CH.sub.2).sub.e—CO— wherein b, c, d, e = 1-10, preferably 2-6.

2. The enrichment system according to claim 1, wherein the crosslinking agent is of an asymmetrical structure (II) ##STR00029## or a symmetrical structure (III) ##STR00030## wherein a = 1-10, preferably 2-5, more preferably 2-3. n = 1-4, preferably 1-2, more preferably 1. Y is selected from —O—, —S—, —S—S—, —S(═O)—, —.sup.13CH.sub.2—, —CD.sub.2—, —.sup.18O—.

3. The enrichment system according to claim 1, wherein R.sup.1 is selected from a group comprising N-hydroxysuccinimide esters, imidoesters, isothiocyanates, isocyanates, acyl chlorides, sulfonyl chlorides, aryl sulfonyl fluorides, acyl azides, fluorophenyl esters, anhydrides, fluorobenzene, epoxides, alpha,beta-unsaturated aldehydes, 1,3-ketoaldehydes, 1,2,3-triazines, 1,2-cyclohexanedione, 2-methoxy-3-oxindoles, phenylglyoxal, a-keto-oximes, 2-fluoro-5-nitrotropolone, O-phthalaldehyde, maleimides, halo acetyls, pyridyl disulfides, aryl azides, diazirines, benzophenones, psoralens, phosphines, alkenes, cyclooctynes, tetrazines, hydrazines, alkoxyamines.

4. The enrichment system according to claim 1, wherein R.sup.1 is a succinimide ester (NHS), phthalic-di-aldehyde, diazirine.

5. The enrichment system according to claim 1, wherein X1, X2 are in each case NH and X3 is O, NH.

6. The enrichment system according to claim 1, wherein R.sup.2 comprises a biotin derivative wherein the cyclic moiety of biotin different from that of biotin such as desthiobiotin, 2-iminobiotin, 3,4-diaminobiotin.

7. The enrichment system according to claim 1, wherein R.sup.3 is H or —CH.sub.3.

8. The enrichment system according to claim 1, wherein Z is — CO—, —CO—NH—(CH.sub.2).sub.b—, —CO—NH—(CH.sub.2).sub.b—NH—CO—, —CO—NH—(CH.sub.2).sub.b—CO—, —CO—NH—(CH.sub.2—O—).sub.c—(CH.sub.2).sub.d—CO—, —CO—NH—(CH.sub.2—O—).sub.c—Triazin—(CH.sub.2).sub.d—CO—, —CO—NH—(CH.sub.2).sub.b—(CH.sub.2—O—)c—(CH.sub.2).sub.d—NH—CO—(CH.sub.2).sub.e—CO— wherein independently from each other b, c, d, e = 1-8, preferably 2-6, more preferably 2-5.

9. The enrichment system according to claim 1, wherein the crosslinking agent is of ##STR00031## ##STR00032## ##STR00033## ##STR00034## ##STR00035## ##STR00036## ##STR00037## ##STR00038## ##STR00039## ##STR00040## .

10. (canceled)

11. A method of enriching at least parts of crosslinked peptides pairs, in particular for use in crosslinking mass spectroscopy analysis, comprising the steps of: Providing a mixture of at least one crosslinking agent as defined in one of the claims 1-9 and at least one protein to be analysed, Digesting the protein by adding at least one proteolytic enzyme to obtain a peptide mixture of crosslinked peptides and linear peptides; Applying the peptide mixture to a streptavidin support whereby the crosslinked peptides pairs will be bound to the streptavidin support and linear peptides are washed out; and Eluting the crosslinked peptides from the streptavidin support with an excess of biotin to obtain enriched crosslinked peptide pairs.

12. The method according to claim 11, wherein the crosslinked peptides are eluted from the streptavidin support by using biotin contained in an buffer system with a pH between 6 and 8, in particular at a pH of 6.5 and 7.5.

13. A kit comprising at least one crosslinking agent as defined in claim 1; Streptavidin attached to a solid support; optionally at least one proteolytic enzyme; a mobile phase / eluting agent.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0081] The solution is explained in more detail with reference to examples and figures.

[0082] FIGS. 1a-j show synthesis of different crosslinkers according to the solution.

[0083] FIG. 2 shows GST crosslinking titration using a crosslinker according to the solution.

[0084] FIGS. 3A-C show crosslinking mass spectrometry analysis of Human Serum Albumin (HSA) crosslinked wit crosslinker 3 according to the solution.

[0085] FIG. 3D shows a crosslinking and enrichment procedure.

[0086] FIG. 3E shows enrichment of crosslinked peptide pairs.

[0087] FIG. 4 shows a schematic comparison of prior art approach and the approach according to the solution.

DETAILED DESCRIPTION

[0088] FIG. 1A shows the three-step synthesis and final structure of one crosslinker 4 according to the solution (called STAGEcl). The finished crosslinking reagent constitutes NHS-ester protein reactive groups (reactive predominantly with the amino groups of lysine residues and protein N-termini, but also with the hydroxyl groups of serine, threonine and tyrosine), separated by a spacer, which has a desthiobiotin moiety grafted in the middle, via a tertiary amide.

Synthesis Protocol of Crosslinker 4

General Methods

[0089] Chemicals and solvents were purchased from Fisher Scientific, Sigma-Aldrich, VWR International Ltd or TCI UK Ltd. NMR spectra were recorded at ambient temperature on a 500 MHz Bruker Avance III spectrometer. Chemical shifts are reported in parts per million (ppm) relative to the solvent peak. Rf values were determined on Merck TLC Silica gel 60 F254 plates under a 254 nm UV source. Purification was carried out by flash chromatography using commercially available Silica 60 Å, particle size 40-63 micron under positive pressure. Compounds purity was >95% pure, as measured by LC-MS using an UV-Vis 254 nm detector and Bruker ESI Micro-Tof mass spectrometer. Method was eluent A: water and trifluoroacetic acid (0.4%); eluent B: acetonitrile; A/B = 95:5 to 20:80 in 6 min, isocratic 1 min, 20:80 to 95:5 in 0.1 min, and isocratic 2 min.

Synthesis of Di-tert-butyl 3,3′-((6-(5-methyl-2-oxoimidazolidin-4-yl)hexanoyl)azanediyl) Dipropionate

##STR00023##

d-desthiobiotin (500 mg, 2.33 mmol) and 1-hydroxybenzotriazole hydrate (HOBt) (530 mg, 3.50 mmol) were dissolved in dry DCM (25 mL) under a nitrogen atmosphere. N,N-Diisopropylethylamine (DIEA) (1.1 mL, 6.99 mmol) was added dropwise and a cloudy solution was formed. Then, N,N′-diisopropylcarbodiimide (DIC) (545 L, 3.50 mmol) was added dropwise and the mixture was stirred for 10 mins. A solution of di-tert-butyl 3,3′-iminodipropionate (1, 650 L, 2.33 mmol) in dry DCM (1 mL) was then added under a nitrogen atmosphere. After the reaction was stirred overnight at room temperature, the mixture was concentrated under reduced pressure. The crude residue was dissolved in DCM and washed with water (3 × 20 mL), HCl 5% 2N (3 × 20 mL) and brine (20 mL). The organic layer was dried using anhydrous MgSO.sub.4 and the solvent was evaporated in vacuo. The resulting residue was purified by flash chromatography (2.5% MeOH in DCM) to yield 2 as a pale oil (496 mg, 46% yield). Rf = 0.44 (5% MeOH in DCM). .sup.1H NMR (500 MHz, DMSO) δ .sup.1H NMR (500 MHz, DMSO) δ 6.30 (s, 1H), 6.11 (s, 1H), 3.67 - 3.58 (m, 1H), 3.53 - 3.49 (m, 2H), 3.40 (t, J = 7.2 Hz, 2H), 2.49 (d, J = 7.1 Hz, 2H), 2.40 (t, J = 7.2 Hz, 2H), 2.29 (t, J = 7.4 Hz, 2H), 1.47 (p, J = 7.4 Hz, 2H), 1.40 (d, J = 2.9 Hz, 18H), 1.38 - 1.13 (m, 6H), 1.01 (d, J = 6.5 Hz, 2H), 0.96 (d, J = 6.4 Hz, 2H). .sup.13C NMR (126 MHz, DMSO) δ 172.40, 171.23, 170.96, 163.25, 80.70, 80.33, 55.45, 50.68, 49.07, 43.91, 41.81, 41.14, 35.13, 33.94, 32.49, 29.99, 29.21, 28.18, 28.16, 26.19, 25.17, 23.77, 15.96. HRMS (ESI) m/z [M + H].sup.+ calcd for C.sub.24H.sub.44N.sub.3O.sub.6, 470.32246; found 470.32270.

Synthesis of 3,3′-((6-(5-methyl-2-oxoimidazolidin-4-yl)hexanoyl)azanediyl)Dipropionic Acid

##STR00024##

Compound 2 (496 mg, 1.05 mmol) was treated with a trifluoroacetic acid (TFA) solution (90% in water) for 2 h at room temperature. The mixture was then concentrated under reduced pressure. The crude was purified by SPE Cartridges Chromabond C18, 6 mL/1000mg (30% MeOH in H.sub.2O) to yield 3 as a pale oil (292 mg, 78% yield)..sup.1H NMR (500 MHz, DMSO) δ 6.29 (s, 1H), 6.10 (s, 1H), 3.60 (ddt, J = 9.6, 6.3, 3.5 Hz, 1H), 3.52 (t, J = 7.4 Hz, 2H), 3.40 (t, J = 7.4 Hz, 2H), 2.53 - 2.51 (m, 1H), 2.47 (s, 1H), 2.40 (t, J = 7.3 Hz, 2H), 2.29 (t, J = 7.4 Hz, 2H), 1.47 (p, J = 7.4 Hz, 2H), 1.37 - 1.16 (m, 6H), 1.00 (d, J = 6.5 Hz, 1H), 0.96 (d, J = 6.4 Hz, 3H). .sup.13C NMR (126 MHz, DMSO) δ 182.56, 182.22, 181.44, 172.32, 64.48, 59.71, 53.11, 51.00, 43.21, 41.97, 41.41, 39.00, 38.22, 35.19, 34.18, 32.78, 24.98. HRMS (ESI) m/z [M + H].sup.+ calcd for C.sub.16H.sub.28N.sub.3O.sub.6, 358.19726; found 358.19680.

NHS Activation of 3,3′-((6-(5-methyl-2-oxoimidazolidin-4-yl)hexanoyl)azanediyl) Dipropionic Acid

[0090] ##STR00025##

Compound 3 (292 mg, 0.82 mmol) and N-hydroxysuccinimide (190 mg, 1.64 mmol) were dissolved in dry DMF (5 mL) under a nitrogen atmosphere, followed by addition of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.Math.HCl) (289 mg, 1.64 mmol) dissolved in dry DMF (1 mL). The mixture was stirred for 24 h at room temperature. The solvent was evaporated in vacuo and the solid was dissolved in EtOAc (10 mL), which was then washed with sat. NaHCO.sub.3 (2 × 10 mL), 10% citric acid (2 × 10 mL) and brine (10 mL). The organic layer was dried using anhydrous MgSO.sub.4 and the solvent was evaporated in vacuo to yield 4 as a colourless oil (303 mg, 68%). Rf = 0.50 (10% MeOH in DCM). .sup.1H NMR (500 MHz, DMSO) δ 6.28 (s, 1H), 6.10 (s, 1H), 3.70 (t, J = 6.9 Hz, 2H), 3.60 - 3.56 (m, 2H), 3.51 - 3.47 (m, 1H), 3.07 (t, J = 6.9 Hz, 2H), 2.93 (t, J = 7.2 Hz, 2H), 2.82 (s, 8H), 2.35 (t, J = 7.5 Hz, 2H), 1.49 (p, J = 7.5 Hz, 2H), 1.41 - 1.14 (m, 6H), 1.01 (d, J = 6.5 Hz, 1H), 0.96 (d, J = 6.4 Hz, 3H). .sup.13C NMR (126 MHz, DMSO) δ 172.94, 170.61, 170.57, 168.01, 167.88, 163.26, 162.77, 55.46, 50.69, 43.56, 41.54, 41.14, 36.25, 32.47, 31.25, 30.54, 29.99, 29.39, 29.17, 26.22, 25.92, 25.01, 23.77, 15.97. HRMS (ESI) m/z [M + H].sup.+ calcd for C.sub.24H.sub.34N.sub.5O.sub.10, 552.23002; found 552.22970.

[0091] FIGS. 1b-j show the synthesis and final structure of further crosslinkers 5-13 according to the solution.

[0092] FIG. 2 shows the SDS-PAGE resulting from crosslinking of Glutathione S-Transferase (GST) dimer with increasing amounts of STAGEcl (crosslinker 4). GST runs on SDS-PAGE as a 25 kDa monomer under denaturing conditions. Crosslinked dimer can be observed as a result of crosslinking and increasing the ratio crosslinker:protein results in increased formation of crosslinked GST dimer.

[0093] GST in 2 .Math.g aliquots was crosslinked (0.33 .Math.g/.Math.L) in crosslinking buffer (20 mM HEPES, 20 mM NaCl, 5 mM MgCl.sub.2, pH 7.8) using different crosslinker:protein ratios (w/w): 0.15:1, 0.44:1, 1.3:1 and 4:1. Crosslinking was carried out for 1 h at room temperature, after which the reaction was quenched with 50 mM ABC, with incubation for 20 mins at room temperature. Crosslinked protein samples were separated by SDS-PAGE on a 1 mm thick NuPAGE 4-12% Bis-Tris SDS-PAGE gel, using MES running buffer and Coomassie blue stain.

[0094] FIG. 3A shows a crosslinked network resulting from STAGEcl crosslinking mass spectrometry analysis of human serum albumin (HSA) with crosslinker 4. The outer circular line represents the protein sequence. Links shown are at a 5% FDR level.

[0095] HSA (200 .Math.g, 0.5 .Math.g/.Math.L) in crosslinking buffer (20 mM HEPES, 20 mM NaCl, 5 mM MgCl.sub.2, pH 7.8) was crosslinked using STAGEcl (crosslinker 4) in a crosslinker:protein (w/w) ratio of 1.64:1, for 1 h at room temperature. The reaction was then quenched with 50 mM ABC. Crosslinked protein was separated by SDS-PAGE on a 1.5 mm thick NuPAGE 4-12% Bis-Tris SDS-PAGE gel using MES running buffer and Coomassie blue stain.

[0096] FIG. 3B shows a high-resolution fragmentation spectrum of a matched STAGEcl (crosslinker 4) crosslinked peptide pair. The sequences of the matched crosslinked peptides, Peptide 1 (red, upper line) and Peptide 2 (black, lower line), are given by the single letter amino acid code. The crosslinking site (K-K) is represented by the solid black line between residues, connecting both peptide sequences. Crosslinked peptide pairs are matched by database search according to precursor m/z (obtained by an MS1 (survey) scan), and the combination of m/z fragments identified in the mass spectrometer following MS2 fragmentation. The fragment ions detected for each peptide are indicated by vertical lines between amino acid residues. Fragment ions belong to either a y-series (indicated by a top tick on the vertical line) or b-series (indicated by a bottom tick on the vertical line). The presented fragmentation spectrum shows the m/z of b- and y-ions identified according to the matched peptide sequence.

[0097] FIG. 3C shows links (indicated by red lines) between residue pairs (5% FDR) fitted to the solved x-ray crystal structure (PDB 1A06, cartoon representation in grey). The histogram shows the observed Ca-Ca distance distribution (in angstroms) for observed linked residue pairs (red), against the random distance distribution (grey) where all crosslinked distances are considered. STAGEcl has an estimated upper distance constraint for crosslinked K-K residues of 27 Å. The majority of crosslinked residues have Ca-Ca distance <20 Å, falling well within the expected distance distribution. The crosslinked residue pairs that exceed this estimated distance fall within the range of expected false positive matches according to the FDR estimation.

[0098] FIG. 3D shows a scheme of the protein crosslinking, digestion and enrichment process. Following the crosslinking reaction, the crosslinked proteins can be proteolytically digested, the resulting peptide mixture applied to solid-supported streptavidin in a PBS buffer, and crosslinked peptides bound to the solid-support with high specificity and efficiency. Linear modified peptides are then washed from the solid-support, and competitive elution with an excess of biotin is achieved, also in PBS buffer, pH 7.4. The resulting enriched crosslinked peptide pairs can then be either analysed directly by mass spectrometry, or subjected to further chromatographic fractionation, including (but not limited to) SCX, SEC and hSAX.

[0099] FIG. 3E shows the level of enrichment following the enrichment procedure on a STAGEcl (crosslinker 4) crosslinked E.coli lysate digest. The three columns show the relative intensity of matched MS1 precursors for non-crosslinker and crosslinker modified peptides (comprised of mono-linked peptides and crosslinked peptide pairs) at different stages of enrichment: (1) input to beads (without enrichment), (FT) the flow-through from beads during enrichment and (E) eluate from beads after enrichment. The bars representing the input (1) and flow-through (FT) were the result of the respective samples following an additional SEC fractionation. The bar representing the eluate (E) was the result of enrichment (affinity enrichment-SCX-SEC).

Cell Production

[0100] A single clone of E.coli K12 strain (BW25113, DSMZ, Germany) grown on agar plates was selected for inoculation of lysogeny broth (LB)-media. Fermentation was initiated in a Biostat A Plus Bioreactor (Sartorius, Göttingen, Germany) using a preculture aliquot in LB medium, with 0.5% (w/v) glucose, at 37° C. Growth was monitored frequently by taking optical density measurements at 600 nm. Fermentation was stopped at an optical density of 10 by rapidly cooling the culture in stirred ice water followed by biomass harvesting by centrifugation at 5000 g, 4° C. for 15 mins. Cell pellets were snap-frozen in liquid nitrogen and stored at -80° C.

Cell Lysis

[0101] Cell pellets were resuspended in ice-cold lysis buffer (50 mM HEPES, pH 7.4 at RT, 50 mM KCl, 50 mM NaCl, 1.5 mM MgCl.sub.2, 5% (v/v) glycerol, 1 mM dithiothreitol (DTT), a spatula tip of chicken egg white lysozyme (Sigma-Aldrich, St. Louis, MO, USA) and complete EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland)). Cells were lysed by sonication on ice at 30% amplitude, 30 s on/off for 10 cycles (total time 5 mins), using a Branson Digital Sonifier. After sonication, 125 units of Benzonase (Merck, Darmstadt, Germany) were added. Liquid was collected in a centrifuge tube (upper foaming with DNA proteins was discarded) and sample was clarified by centrifugation at 15,500 rpm for 30 mins at 4° C. DTT was added to 2 mM. The cleared lysate was then subjected to ultracentrifugation using a 70 Ti fixed-angle rotor for 1h at 106,000 g at 4° C., after which the supernatant was concentrated 10x using ultrafiltration with Amicon spin filters (15 kDa molecular weight cut-off) to achieve a total protein concentration of 10 mg/mL, determined by microBCA protein assay (Thermo Fisher Scientific, Waltham, MA, USA).

E.Coli Lysate Crosslinking

[0102] Cell lysate was diluted to 1 mg/mL protein concentration with crosslinking buffer (50 mM HEPES, 50 mM NaCl, 50 mM KCl, 1.5 mM MgCl.sub.2, 5% glycerol, pH 7.4). Cell lysate was then crosslinked with 1 mM STAGEcl (crosslinker 4) for 1 h at room temperature. After this time the crosslinking reaction was quenched with 50 mM ABC, with incubation for 30 mins on ice. Crosslinked proteins were acetone-precipitated overnight at -20° C. Protein was solubilised in 6 M urea, 2 M thiourea, 100 mM ABC. Protein sample was then subjected to proteolysis with LysC added at a 1:100 (m/m) ratio followed by incubation for 4 h at 37° C. After 1:5 dilution with 100 mM ABC, trypsin was added at a ratio of 1:25 (m/m) and the digestion allowed to continue for 16 h at 37° C., after which digestion was stopped with the addition of TFA to 1% (v/v). Digests were desalted using SPE cartridges according to the manufacturer’s instruction and eluates dried, aliquoted and stored at -20° C. until further use.