SEEDED PRECIPITATION OF POLYPEPTIDES

Abstract

The present invention relates to a method of processing or fractionating a sample comprising proteins, polypeptides and/or peptides, said method comprising (a) changing the physicochemical conditions of said sample; and (b) performing one or both of the following (i) and (ii): (i) adding solid particulate matter to said sample; and (ii) performing said method in a vessel with a rough surface; wherein steps (a) and (b) can be effected concomitantly or in any order; and wherein said processing or fractionating yields one or more first fractions of proteins, polypeptides and/or peptides as a precipitate on said particulate matter and/or said inhomogeneous surface, and a second fraction of proteins, polypeptides and/or peptides remaining in a supernatant.

Claims

1. A method of processing or fractionating a sample comprising proteins, polypeptides and/or peptides, said method comprising (a) changing physicochemical conditions of said sample; and (b) performing one or both of (i) and (ii): (i) adding solid particulate matter to said sample; and (ii) performing said method in a vessel with a rough surface; wherein steps (a) and (b) can be effected concomitantly or in any order; and wherein said processing or fractionating yields one or more first fractions of proteins, polypeptides and/or peptides as a precipitate on said particulate matter and/or said rough surface, and a second fraction of proteins, polypeptides and/or peptides remaining in a supernatant.

2. The method of claim 1, wherein said sample is a cell lysate or a bodily fluid, wherein optionally said cell lysate or said bodily fluid has been treated with an anticoagulant.

3. The method of claim 1, wherein step (a), step (b), or both steps (a) and (b) are performed more than once with the supernatant.

4. The method of claim 1, wherein said physicochemical conditions upon said changing: (i) do not include temperatures above 70? C., concentrations of denaturing surfactants above 0.8% (w/v), and concentrations of organic solvents above 25% (v/v); and/or (ii) are non-denaturing conditions for said proteins, polypeptides and/or peptides.

5. The method of claim 1, wherein said physicochemical conditions are one, more or all of: (i) a pH value; (ii) a temperature; (iii) a concentration of at least one organic solvent; (iv) a concentration of at least one salt; (v) a concentration of at least one surfactant; and (vi) concentration of said proteins, polypeptides and/or peptides.

6. The method of claim 5, wherein said changing of physicochemical conditions is selected from: (i) a rise in said temperature, wherein said temperature after said rise is lower than 50? C.; (ii) an increase in concentration of said at least one organic solvent, wherein said concentration of said at least one organic solvent after said increase is less than 20% (v/v); (iii) an increase in concentration of said at least one salt, wherein e at least one salt is a chaotropic salt, and wherein said concentration of said chaotropic salt after said increase is less than 0.5 M; and (iv) an increase in concentration of said at least one surfactant, where said at least one surfactant is a denaturing surfactant, and wherein said concentration of said surfactant after said increase is less than 0.5% (w/v).

7. The method of claim 1, wherein said solid particulate matter is a plurality of microparticles with a diameter between 0.4 ?m and 500 ?m.

8. The method of claim 1, wherein said rough surface is etched or porous.

9. The method of claim 1, wherein said solid particulate matter comprises one, more or all of: (i) a magnetic or magnetizable composition; (ii) floating particles; and (iii) sedimenting particles.

10. The method of claim 1, wherein said solid particulate matter or said rough surface comprises polypropylene (PP), polystyrene (PS), polystyrene divinyl benzene (PS-DVB), poly-tetrafluoro ethylene (PTFE), poly-vinyl chloride (PVC), polyoxymethylene (POM), polyethylene (PE) including high-density polyethylene (HDPE) and low-density polyethylene (LDPE), polyamide (PA), polycarbonate (PC), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), polybutylene terephthalate (PBT), acrylonitrile butadiene styrene (ABS), silica, silanol, ceramics, glass, or metal.

11. The method of claim 1, wherein said solid particulate matter carries: (i) hydrophobic C4, C8, C18 or styrene moieties; and/or (ii) hydrophilic hydroxyl, carboxyl, sulfonyl, secondary, tertiary and quaternary amine, or silanol moieties.

12. The method of claim 1, wherein said processing or fractionating is followed by one or more of: (c) separating the supernatant from the precipitate; (d) washing the precipitate, and optionally combining the washing solution with said supernatant; and (e) cleaving the proteins, polypeptides and/or peptides in the supernatant of (c) or (d) with a protease, with a chemical, or mechanically.

13. The method of claim 1, wherein said processing or fractionating is followed by one or more of: (f) separating the precipitate from the supernatant; (g) washing the precipitate; (h) solubilizing or resuspending the proteins, polypeptides and/or peptides from said precipitate; and (j) cleaving the proteins, polypeptides and/or peptides in the eluate obtained in step (h) with a protease, with a chemical, or mechanically.

14. A method of modulating or enhancing precipitation of protein(s), polypeptide(s) and/or peptide(s), comprising processing or fractionating a sample comprising said protein(s), polypeptides and/or peptide(s) in a vessel with a rough surface and/or with solid particular matter.

15. Solid particulate matter with at least one agent adhered thereto, wherein said at least one agent is a trigger of precipitation of protein(s), polypeptide(s) and/or peptide(s).

16. The solid particulate matter of claim 15, wherein (i) said at least one agent is the one agent; (ii) said at least one agent is an acid or base; (iii) said at least one agent is a non-denaturing surfactant; and/or (iv) said at least one agent is a kosmotropic.

17. A prefilled reaction vessel, said vessel comprising solid particulate matter of claim 15.

18. A prefilled reaction vessel, said vessel comprising solid particulate matter of claim 16.

19. The method of claim 7, wherein said plurality of microparticles is identical in composition.

Description

[0120] The Figures show:

[0121] FIG. 1: Device used to roughen the inner surface of LoBind Eppendorf tubes.

[0122] FIG. 2: LoBind eppendorf tube with the roughened surface (left) and regular tube (right).

[0123] The Example illustrates the invention.

EXAMPLE 1: SEEDED PRECIPITATION USING PARTICLES

Materials and Methods

[0124] The precipitation on seeds method in accordance with the invention may be used in conjunction with the iST procedure (Kulak et. al. 2014) utilizing the iST buffers and iST-cartridge from the iST-kit which can be bought from PreOmics GmbH (Planegg, Germany). It adds n additional step prior to the iST process as described below.

[0125] Step 1 (additional step): In general, as a first step the precipitation seedtypically magnetic beads with silanol surface chemistry, diameter 3 ?m, if not mentioned otherwisewas washed three times with 100 ?l ddH.sub.2O ending with the dry seeds. Then, 80 ?l of an incubation buffer was added followed by adding 20 ?l human blood plasma which corresponds to roughly 1000 ?g of protein content. All components were mixed thoroughly using a vortexer, followed by incubation at room temperature while mixing at 1.000 rpm for 30 minutes. After incubation, the fluid was separated from the seeds using a suitable separation technique, followed by washing the seeds three times with the incubation buffer as above, resulting in dry seeds with precipitated protein on them.

[0126] Step 2: Then 50 ?l of LYSE-BCT buffer was added followed by heating and shaking for 10 minutes at 95? C. and 1.000 rpm. After cooling to room temperature, 50 ?l of resuspended DIGEST was added and incubated at 37? C. for 60 minutes while shaking at 500 rpm.

[0127] The digest reaction was quenched adding 100 ?l of STOP buffer. The resulting suspension was thoroughly mixed and transferred on an iST-cartridge and then centrifuged for one minute at 3.800 rcf. 200 ?l of WASH1 was added followed by centrifugation as just described. This was repeated as before using WASH2. The flow-through was discarded and the cartridge was transferred to a fresh collection tube.

[0128] 100 ?l of ELUTE buffer was added to the cartridge, followed by centrifugation at 3.800 rcf for one minute. The step was repeated once for a total of two elutions gathered in one collection tube.

[0129] The eluate was transferred into in an Eppendorf Concentrator under vacuum at 45? C. until completely dry. Typically, 10 ?l of the LC-LOAD buffer of the iST-kit were added for resuspending the dried peptides and resuspending properly by shaking and/or vortexing. The concentration of peptides was measured with a NanoDrop instrument.

[0130] For analysis of protein and peptide identifications, the solution was injected into a LTQ Orbitrap XL mass spectrometer using a Thermo easy nLC 1200 liquid chromatography system. For the MS measurement, a 45 minute gradient from 5% buffer A (0.1% formic acid in water) to 95% buffer B (0.1% formic acid in 80% acetonitrile) was applied for all experiments.

[0131] This generic procedure has been implemented in a number of different manners as indicated in the following: [0132] Experiment 1 (SJ150): The incubation buffer was modified. Sample 1+2: water, sample 3+4: 0.2?PBS solution, sample 5+6: 0.4?PBS solution, sample 7+8: 0.6?PBS solution, sample 9+10: 0.8?PBS solution, sample 11+12: 1?PBS solution [0133] Experiment 2 (SJ154): The incubation buffer was modified. Sample 1+2: water, sample 3+4: 2% ethanol, sample 5+6: 4% ethanol, sample 7+8: 6% ethanol, sample 9+10: 8% ethanol, sample 11+12: 10% ethanol [0134] Experiment 3 (SJ155): The incubation buffer was modified. Sample 1+2: water, sample 3+4: 2% acetonitrile, sample 5+6: 4% acetonitrile, sample 7+8: 6% acetonitrile, sample 9+10: 8% acetonitrile, sample 11+12: 10% acetonitrile [0135] Experiment 4 (SJ156): The incubation buffer was modified. Sample 1+2: pH=2.4, sample 3+4: pH=4.2, sample 5+6: pH=6.0, sample 7+8: pH=6.92, sample 9+10: pH=9.0, sample 11+12: pH=10.94, [0136] Experiment 5 (SJ164): Incubation buffer at pH 10.94 (as Experiment 4, sample 11+12). Here different seeds have been used. Sample 1+2: C18 magnetic beads, sample 3+4: COOH magnetic beads, sample 5+6: tosyl magnetic beads, Sample 1+2: NH2 magnetic beads, sample 9+10: protein G magnetic beads, sample 11+12 epoxy magnetic beads, sample 13-15: internal control (repeat Experiment 4, sample 11+12) [0137] Experiment 6 (SJ165): Incubation buffer at pH 10.94 (as Experiment 4, sample 11+12). Here, the effect of transferring the seeds to new flasks after the precipitation step was tested. Sample 1-3: as Experiment 4, sample 11+12 (internal control), sample 4-6: transfer seeds to new flask after precipitation [0138] Experiment 6 (SJ169): Incubation buffer at pH 10.94 (as Experiment 4, sample 11+12). Here the influence of porosity of seeds was tested. Sample 1-3: 3 zeolith beads were used as seeds, porous material. Sample 4-6: non-porous ceramic beads as seeds [0139] Experiment 7 (SJ174): Incubation buffer at pH 10.94 (as Experiment 4, sample 11+12). Here the influence of the seed surface was tested. Sample 1-3: glass beads were used as seeds, diameter 100-200 ?m. Sample 4-6: korund F120 was used as seeds, diameter 90-125 ?m. Sample 7-9: silicium F120 was used as seeds, diameter 90-125 ?m. These materials are typical blasting abrasives. [0140] Control 1 (SJ150, 13-15): iST-BCT procedure as described above but without any precipitation (step 1). 2 ?l human plasma was used as educt. [0141] Control 2 (SJ130, 1+2; SJ119, 3+4): Step 1 was modified as follows: for sample 1 and 2, 800 ?l Blue Sepharose 6 Fast Flow (Merck, GE17-0948-01) was added to a column containing a polyethylene frit. The liquid from the blue Sepharose slurry was pressed through using a syringe. Then, 0.8 ml 0.2?PBS solution was added and pressed through again. This was repeated twice for a total of three times. 64 ?l human plasma was filled up to 100 ?l using 0.2?PBS and added to the washed blue Sepharose beads and incubated for 1 h with 360? rotation. Then 400 ?l 0.2?PBS was added and pressed through with syringe. The flow-through was collected and dried in an Eppendorf Concentrator. Half of the dried pellet was then processed as described in Step 2. For sample 3 and 4, 50 ?l blue Sepharose beads were washed three times with 100 ?l ddH2O, equilibrated 3? with 100 ?l equilibration buffer (0.05 m KH.sub.2PO.sub.4, as described by the manufacturer) using quick vortexing (15 sec) and centrifugation (5 sec) and taking off the supernatant. Then 50 ?l binding buffer (0.05 m KH.sub.2PO.sub.4, as described by the manufacturer) was added to the relatively dry beads and carefully transferred onto 10 ?l human plasma. Incubation was time 10 minutes while shaking at 1.000 rpm for efficient binding. Sample was spun down and the supernatant was transferred to a new reaction flask. 25 ?l 2?LYSE-BCT (PreOmics GmbH) was added and Step 2 was performed. [0142] Control 3 (SJ134): Sample 1 and 2: 100 ?l Protein-A magnetic beads (ReSyn Biosciences (Pty) Ltd, South Africa) were washed three times with 300 ?l PBS, using magnetic separation to discard the fluid. Then 90 ?l binding buffer (PBS, as described by manufacturer) was added to 10 ?l human plasma, mixed and added to the washed, dried beads from above. The suspension was incubated for 30 minutes while shaking at 1.000 rpm. After incubation, the supernatant was taken off using magnetic separation and added to 100 ?l Protein G beads (ReSyn Biosciences (Pty) Ltd, South Africa), which were washed and dried above. Incubation was performed as above, followed by separation and another round of incubation with Protein G beads. Finally the supernatant was dried in an Eppendorf Concentrator. When dry, Step 2 was performed. Samples 3+ and were treated as above but using Protein A beads (Magtivio) only once.

Results

Number of Identified Proteins

[0143]

TABLE-US-00001 Protein Sam- Sam- Sam- Sam- Sam- Sam- Sam- Sam- Sam- Sam- Sam- Sam- Sam- Sam- Sam- identifications ple 1 ple 2 ple 3 ple 4 ple 5 ple 6 ple 7 ple 8 ple 9 ple 10 ple 11 ple 12 ple 13 ple 14 ple 15 Experiment 1 196 208 202 205 220 214 227 216 222 218 223 213 N/A N/A N/A Experiment 2 186 204 200 197 193 191 198 194 200 199 195 191 N/A N/A N/A Experiment 3 200 194 196 201 188 195 197 199 198 197 190 192 N/A N/A N/A Experiment 4 175 185 176 183 157 166 183 182 226 231 267 279 N/A N/A N/A Experiment 5 255 251 226 203 267 261 178 231 164 161 276 284 273 284 288 Experiment 6 261 232 251 260 250 254 N/A N/A N/A N/A N/A N/A N/A N/A N/A Experiment 7 228 226 241 168 216 213 307 314 300 182 163 177 N/A N/A N/A Protein identifications Sample 1 Sample 2 Sample 3 Sample 4 Control 1 145 148 137 Control 2 128 124 137 132 Control 3 106 101 127 128

Discussion

[0144] For all experiments shown a significant increase in protein identifications compared to all controls can be observed. This demonstrates that the precipitation on the seeds in accordance with the invention has the advantages disclosed herein above.

EXAMPLE 2: PRECIPITATION USING ROUGH SURFACES

Materials and Methods

[0145] In this example, no particles have been used. Instead, a vessel with a rough surface has been used, thereby illustrating an alternative implementation of the present invention. Otherwise, materials and methods are those of Example 1 if not specified otherwise. [0146] Experiment 8 (SJ221): The wall of LoBind Eppendorf tubes were roughend using a commercially available Dremel instrument (see FIGS. 1 and 2) and compared to the bead based workflow (as in Example 1, Experiment 4, sample 11+12) and the iST workflow. The last two triplicates serve as control within this experiment. [0147] Experiment 9 (SJ216): The plasma itself was harvested using different tubes (Sarstedt Monovette EDTA KE 9 ml, Sarstedt Monovette Na Citrate 2.9 ml, Sarstedt Monovette Li Heparin 7.5 ml, Sarstedt Monovette No anticoagulant, BD P100 K2EDTA Proprietary Protein Stabilizers 2 mL), centrifuged at 150 g for 10 minutes at room temperature. After centrifugation the supernatant was taken off for further processing. The plasma from different donors of the same anticoagulant was pooled. The samples were processed using the bead based workflow (as in Example 1, Experiment 4, sample 11+12) but measured on a Bruker TimsTOF Pro with a Thermo easy nLC 1200 liquid chromatography attached with the same gradient setting as described.

Results

[0148] Results are given in the following tables. The term bead based workflow refers to Example 1.

Experiment 8:

[0149]

TABLE-US-00002 Experiment Proteins identified Rough Surface 166 Rough Surface 139 Rough Surface 163 Bead based workflow 215 Bead based workflow 156 Bead based workflow 194

Experiment 9:

[0150]

TABLE-US-00003 Proteins Tube used for harvesting identified Sarstedt Monovette EDTA KE 9 mL 2390 Sarstedt Monovette EDTA KE 9 mL 2420 Sarstedt Monovette EDTA KE 9 mL 2267 Sarstedt Monovette Na Citrate 2.9 mL 1564 Sarstedt Monovette Na Citrate 2.9 mL 1688 Sarstedt Monovette Na Citrate 2.9 mL 1720 Sarstedt Monovette Li Heparin 7.5 mL 1429 Sarstedt Monovette Li Heparin 7.5 mL 1327 Sarstedt Monovette Li Heparin 7.5 mL 1431 Sarstedt Monovette No anticoagulant (Serum) 533 Sarstedt Monovette No anticoagulant (Serum) 600 BD P100 K2EDTA Proprietary Protein Stabilizers 2 mL 1937 BD P100 K2EDTA Proprietary Protein Stabilizers 2 mL 2078 BD P100 K2EDTA Proprietary Protein Stabilizers 2 mL 2017

Discussion

[0151] Experiment 8 indicates that the rough surface increases the number of proteins identified (protein IDs) as compared to the regular iST workflow.

[0152] Experiment 9 shows the stabilizing effect of the use of an anticoagulant for harvesting plasma.