SYSTEMS AND METHODS FOR BIOMOLECULE PREPARATION
Methods for the preparation of sample polypeptide fractions are described. Sample polypeptides may be isolated from any of a variety of sources, including biological and non-biological systems. Sample polypeptides may be coupled or conjugated to other molecules to permit characterization of the sample polypeptide fractions. Sample polypeptide fractions may be prepared for analysis by a polypeptide assay.
1 . A method of forming a polypeptide array, comprising:
(a) providing a sample comprising sample polypeptides;
(b) separating the sample polypeptides from the sample in the presence of separation standard polypeptides;
(c) coupling the sample polypeptides to anchoring groups to form sample polypeptide composites, wherein the coupling occurs in the presence of coupling standard polypeptides; and
(d) attaching at least a fraction of the sample polypeptide composites, the separation standard polypeptides and the coupling standard polypeptides to a solid support, whereby each of the sample polypeptide composites, the separation standard polypeptides and the coupling standard polypeptides that is attached to the solid support comprises an address of the polypeptide array.
2 . The method of claim 1 , wherein the attaching of step (d) occurs in the presence of attachment standard polypeptides, whereby each of the attachment standards that is attached to the solid support comprises an address of the polypeptide array.
3 . The method of claim 1 , wherein the separation standard polypeptides are added to the sample, or a fraction thereof, before step (b) or before step (a).
4 . The method of claim 1 , wherein the coupling standard polypeptides are added to the sample or a fraction thereof before step (c), before step (b), or before step (a).
5 . The method of claim 1 , wherein the separation standard polypeptides and the attachment standard polypeptides are combined as an internal standard mixture before being added to the sample, or fraction thereof.
6 . The method of claim 5 , further comprising forming an internal standard mixture comprising two or more pluralities of polypeptides selected from the group consisting of a plurality of the separation standard polypeptides, a plurality of the coupling standard polypeptides, and a plurality of the attachment standard polypeptides; and adding the internal standard mixture to the sample, or fraction thereof.
7 . The method of claim 1 , wherein step (b) occurs in the presence of a first surfactant.
8 . The method of claim 7 , wherein attaching at least a fraction of the sample polypeptide composites of the plurality of sample polypeptide composites, the separation standard polypeptides, and the coupling standard polypeptides to the solid support occurs in the presence of a second surfactant.
9 . The method of claim 1 , wherein attaching at least a fraction of the sample polypeptide composites of the plurality of sample polypeptide composites, the separation standard polypeptides, and the coupling standard polypeptides to the solid support occurs in the presence of a salt.
10 . The method of claim 1 , wherein attaching at least a fraction of the sample polypeptide composites of the plurality of sample polypeptide composites, the separation standard polypeptides, and the coupling standard polypeptides to the solid support occurs due to a change in solution pH.
11 . The method of claim 1 , further comprising, after the attaching the plurality of anchoring groups to the solid support;
determining the presence or absence of an anchoring group of the plurality of anchoring groups at each address on the solid support.
12 . The method of claim 1 , further comprising:
(e) detecting the sample polypeptide composites, the separation standard polypeptides and the coupling standard polypeptides at addresses of the polypeptide array.
13 . The method of claim 12 , further comprising detecting an attachment standard polypeptide at an address of the polypeptide array.
14 . The method of claim 12 , further comprising counting the number of the addresses that comprise the sample polypeptides, separation standard polypeptides, coupling standard polypeptides of the polypeptide array.
15 . The method of claim 12 , wherein the detecting comprises acquiring a signal produced by the sample polypeptides, separation standard polypeptides, coupling standard polypeptides or attachment standard polypeptides attached to the polypeptide array.
16 . The method of claim 12 , further comprising quantifying the amount of a particular sample polypeptide species in the sample by comparing (i) a number of the addresses attached to a separation standard polypeptide with (ii) the number of the addresses attached to a sample polypeptide composite that comprise the particular sample polypeptide species.
17 . The method of claim 16 , further comprising characterizing a particular sample polypeptide species in the sample by comparing (i) a detected property of a separation standard polypeptide with (ii) a detected property of a sample polypeptide composite that comprise the particular sample polypeptide species.
18 . The method of claim 12 , further comprising quantifying the amount of a particular sample polypeptide species in the sample by comparing (i) a number of addresses attached to a coupling standard polypeptide with (ii) a number of addresses attached to a sample polypeptide composite that comprise the particular sample polypeptide species.
19 . The method of claim 18 , further comprising characterizing a particular sample polypeptide species in the sample by comparing (i) a detected property of a coupling standard polypeptide with (ii) a detected property of a sample polypeptide composite that comprise the particular sample polypeptide species.
20 . The method of claim 12 , further comprising quantifying the amount of a particular sample polypeptide species in the sample by comparing (i) a number of addresses attached to an attachment standard polypeptide with (ii) a number of addresses attached to a sample polypeptide composite that comprise the particular sample polypeptide species.
21 . The method of claim 20 , further comprising characterizing a particular sample polypeptide species in the sample by comparing (i) a detected property of an attachment standard polypeptide with (ii) a detected property of a sample polypeptide composite that comprise the particular sample polypeptide species.
22 . The method of claim 12 , wherein step (e) comprises:
(i) binding a first set of binding reagents to at least one of the sample polypeptide composites, at least one of the separation standard polypeptides, at least one of the coupling standard polypeptides and at least one of the attachment standard polypeptides in the array,
(ii) detecting the first set of binding reagents in the array,
thereby detecting the sample polypeptide composites, the separation standard polypeptides and the coupling standard polypeptides at addresses of the polypeptide array.
23 . The method of claim 12 , wherein step (e) further comprises
(iii) removing binding reagents of the first set from the array,
(iv) binding a second set of binding reagents to at least one of the sample polypeptide composites, at least one of the separation standard polypeptides, at least one of the coupling standard polypeptides and at least one of the attachment standard polypeptides in the array, wherein binding reagents in the second set are different from binding reagents in the first set, and
(v) detecting the second set of binding reagents in the array,
thereby detecting the sample polypeptide composites, the separation standard polypeptides and the coupling standard polypeptides at addresses of the polypeptide array.
24 . A composition, comprising:
a. a solid support comprising a plurality of addresses, wherein each address of the plurality of addresses is spatially resolvable from each other address of the plurality of addresses, and wherein each address of the plurality of addresses is configured to couple a polypeptide; and
b. a sample polypeptide mixture, wherein the sample polypeptide mixture comprises a plurality of sample polypeptides, a plurality of separation standard polypeptides, and a plurality of coupling standard polypeptides, and wherein each polypeptide of the sample polypeptide mixture is coupled to an anchoring group, wherein the anchoring group is configured to couple a polypeptide to an address of the plurality of addresses.
25 . The composition of claim 24 , wherein the sample polypeptide mixture further comprises a plurality of fiducial elements.
26 . The composition of claim 25 , wherein each address of the plurality of addresses comprises one and only one polypeptide of the sample polypeptide mixture, wherein the plurality of addresses comprises a first subset of addresses, a second subset of addresses, a third subset of addresses, and a fourth subset of addresses, wherein each address of the first subset of addresses is coupled to a sample polypeptide, wherein each address of the second subset of addresses is coupled to a coupling standard polypeptide, wherein each address of the third subset of addresses is coupled to a separation standard polypeptide, and wherein each address of the fourth subset of addresses is coupled to a fiducial element.
27 . The composition of claim 26 , wherein one or more of the first subset of addresses, the second subset of addresses, the third subset of addresses, and the fourth subset of addresses comprises a random spatial distribution.
28 . The composition of claim 24 , wherein the sample polypeptide mixture further comprises a ladder of standard polypeptides, wherein the ladder of standard polypeptides comprises a plurality of polypeptides comprising a range of a polypeptide property.
29 . The composition of claim 24 , wherein the sample polypeptide mixture further comprises a fragment library, where in the fragment library comprises a plurality of unique fragments of a sample polypeptide of the plurality of sample polypeptides.
30 . The composition of claim 24 , wherein the sample polypeptide mixture further comprises a proteoform library, where in the proteoform library comprises a plurality of unique proteoforms of a sample polypeptide of the plurality of sample polypeptides.