Peptide purification using mixed-phase solid phase extraction material

Abstract

The present invention relates to a method of purifying peptides and/or polypeptides, said method comprising or consisting of: (a) loading a sample comprising peptides and/or polypeptides under acidic or neutral aqueous conditions on mixed-phase solid phase extraction (SPE) material, wherein said material consists of or comprises reversed phase/ion exchange material; (b) washing said mixed-phase SPE material with (ba) an acidic or neutral composition comprising at least 50% (v/v) organic solvent; and/or (bb) an acidic or neutral aqueous solution; and (c) eluting said peptides and/or polypeptides from said mixed-phase SPE material with an alkaline composition comprising at least 50% (v/v) organic solvent.

Claims

1. A method of purifying peptides and/or polypeptides bound to mixed- phase SPE material, said method comprising: (a) loading a sample comprising peptides and/or polypeptides under acidic or neutral aqueous conditions on mixed-phase solid phase extraction (SPE) material comprising reversed phase material and/or ion exchange material; (b) washing with (ba) an acidic composition having a pH from 1 to 4 and comprising at least 50% (v/v) organic solvent; and (bb) an acidic or neutral aqueous solution; and (c) eluting the peptides and/or polypeptides from said mixed-phase SPE material with an alkaline composition comprising at least 50% (v/v) organic solvent; wherein said mixed-phase SPE material is sulfonated poly-divinyl benzene (DVB) or sulfonated poly-styrene divinyl benzene (SDB); and wherein step (a) is performed prior to step (b) which is performed prior to step (c).

2. The method of claim 1, wherein the acidic composition in step (ba) has a pH from 3 to 4.

3. The method of claim 1, wherein the acidic aqueous solution in step (bb) has a pH from 3 to 4.

4. The method of claim 1, wherein the alkaline composition in step (c) has a pH from 8 to 10.

5. The method of claim 1, wherein the organic solvent in step (ba) and the organic solvent in step (c) are independently chosen from the group consisting of: (a) alcohols; (b) alkanes; (c) halogenated hydrocarbons; (d) ethers; (e) ketones; (f) nitriles; (g) sulfoxides; and (h) esters.

6. The method of claim 1, wherein the acidic composition in step (ba) comprises at least 50% (v/v) alcohol; and/or the alkaline composition in step (c) comprises at least 50% (v/v) acetonitrile.

7. The method of claim 1, wherein said purifying comprises the reduction of the amount or the removal of one or more of the following: detergents; lipids; saccharides; polymeric or oligomeric wall material of vessels used for sample handling; and plasticizers.

8. The method of claim 1, wherein the method is performed using column chromatography.

Description

(1) The FIGURES show:

(2) FIG. 1: HeLa peptides were spiked with PEG polymers. Washing with 100% alcohol removes polymer contaminants from the sample and thereby improves sample quality and purity.

(3) The Examples illustrate the invention.

EXAMPLE 1

(4) Serum Samples

(5) Using the above disclosed method, 160 patient serum samples could be prepared and analyzed without any interruption or decrease in performance. Among the patient materials were 20 samples of hyperlipidemia, known for their very high lipid content. These samples are typically even harder to handle and cause severe interference in immune-assays.

(6) In more detail, serum samples were collected and diluted 1:10 with double-distilled water. The samples were the digested using the ‘iST’-sample preparation method (Kulak, N. A., et al., Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells, Nat Methods, 2014 March 11(3) p. 319-24). Peptides were loaded onto a sulfonated styrene-divinylbenzene matrix and washed with 100% alcohol. The matrix was then washed also with a 0.2% (v/v) TFA solution end eluted with 80% (v/v) ACN, 5% (v/v) ammonia. The peptides were measured by nanoLC-MS/MS and analyzed using the MaxQuant environment.

EXAMPLE 2

(7) Urine Samples

(8) The disclosed methods were applied to urine samples which have generally a very high content of salts and metabolites.

(9) Second-morning urine was collected from apparently healthy donors and centrifuged to remove cell debris. The supernatants were concentrated and digested using the ‘iST’-sample preparation method (Kulak et al; loc. cit.). Peptides were loaded onto a sulfonated styrene-divinylbenzene matrix and washed with 100% alcohol. The matrix was then washed also with a 0.2% (v/v) TFA solution end eluted with 80% (v/v) ACN, 5% (v/v) ammonia. The peptides were measured by nanoLC-MS/MS and analyzed using the MaxQuant environment.

(10) More than 30 samples could be prepared and analyzed without any interference and with unprecedented quality. The observations demonstrate the effective removal of undesired contaminants also from urine.

EXAMPLE 3

(11) Contaminants Introduced During Processing

(12) As noted above, enriching membrane components generates samples which are particularly challenging.

(13) Vesicles were enriched using a glucose gradient system. Each glucose fraction was then digested using the ‘iST’-sample preparation method (Kulak et al; loc. cit.). Peptides were loaded onto a sulfonated styrene-divinylbenzene matrix and washed with 100% alcohol. The matrix was then washed also with a 0.2% (v/v) TFA solution end eluted with 80% (v/v) ACN, 5% (v/v) ammonia. The peptides were measured by nanoLC-MS/MS and analyzed using the MaxQuant environment.

(14) The methods of the present invention allowed the measurement of 160 membrane enrichments on a single nano-flow column as compared to three injections which is what normally is feasible without the methods of the invention.

(15) As such, the disclosed clean-up therefore permits to significantly increase the number of injections.

EXAMPLE 4

(16) Removal of Polymers from HeLa Sample

(17) The disclosed invention relates to the removal of polymers from complex peptide mixtures. The data displayed in FIG. 1 provide evidence that washing in accordance with the invention specifically removes polymers.