DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or
under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Acknowledgment is made of applicant’s claim for foreign priority under 35 U.S.C. 119 (a) -(d). The Certified Copy of Foreign Priority Application for CN 202211030145.7 dated 08/26/2022 has been filed on 04/08/2024. This application claims benefit of the foreign application CN 202211030145.7 filed 08/26/2022. Based on the filing receipt, the effective filing date of this application is August 26, 2022 which is the filing date of Application CN 202211030145.7 from which the benefit of priority is claimed.
Information Disclosure Statements
The information disclosure statements (IDS) filed 09/12/2024 and 04/10/2024 have been considered by the examiner.
Status of Claims
Claims 1-10 have been cancelled.
Claims 11-30 are pending and examined herein.
Claim Objections
Claims 14, 20, 22, and 28 are objected to because of the following informalities:
Claims 14, 22, and 28 all recite, “wherein the peptides suitable for the bottom-up mass
spectrometry analysis has a molecular weight less than 5 kDa”. The claims should recite, “wherein the peptides suitable for the bottom-up mass spectrometry analysis have
Claim 20 recites “a candidate target protein” in step (a), but step (e)(ii) recites “a region in the candidate protein target”. The two steps should recite the same phrase.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 14 and 20-30 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 14, 22, and 28 recite, “the peptides suitable for the bottom-up mass spectrometry analysis has [sic] a molecular weight less than 5 kDa”. It is unclear whether every peptide generated and/or isolated must be below 5 kDa, whether only the peptides that are subsequently quantified must be, or whether the limitation describes the peptide population generally (e.g., predominantly). Because proteolysis of a complex proteome necessarily yields a distribution of fragment sizes, these reading differ materially in scope. For purposes of applying prior art, the limitation is interpreted as requiring that the isolated peptides whose abundance is determined have molecular weights below 5 kDa. The specification does not ameliorate the lack of clarity. Therefore, the metes and bounds of the claim cannot be ascertained.
Claim 20 recites the limitation "contacting the protein samples" in step (b). There is insufficient antecedent basis for this limitation in the claim. Step (a) introduces “a plurality of samples”, not “protein samples”. Step (c) then recites “from the samples” and claims 24 refers back to “the plurality of samples”, further obscuring which samples are required to be contacted with the protease. The specification does not ameliorate the lack of clarity. Therefore the metes and bounds of the claim cannot be ascertained.
Claim 20 also recites “is indicative of a target protein bound by the ligand” in step (e)(i). It is unclear if the protein is referring to the protein of the preamble or the “candidate target protein” of step (a), or a different protein. The specification does not ameliorate the lack of clarity. Therefore the metes and bounds of the claim cannot be ascertained.
Claims 21-25 depend on claim 20 and are therefore also indefinite.
Claim 26 recites “the local affinity between the ligand and the protein” in step (e). There is insufficient basis for “the protein”. The claim introduces “a target protein” in the preamble and “a candidate target protein” in step (a). It is unclear which, if either, “the protein” refers, and it is also unclear how the preamble’s “target protein” relates to the “candidate target protein”. The specification does not ameliorate the lack of clarity. Therefore, the metes and bounds of the claim cannot be ascertained.
Claims 27-30 depend on claim 26 and are therefore also indefinite.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 11-30 are rejected under 35 U.S.C. 101 because the claimed inventions are directed to abstract ideas without significantly more.
Regarding independent claims 11, 20, and 26, the claims recite methods comprising “determining the abundance of the isolated peptides”, “comparing the abundance of the isolated peptides”, and “identifying a peptide from the isolated peptides”. The claims are directed at abstract ideas. Determining, comparing, and identifying are mental processes. The courts consider a mental process (thinking) that “can be performed in the human mind, or by a human using a pen and paper” to be an abstract idea. CyberSource Corp. v. Retail Decisions, Inc., 654 F.3d 1366, 1372, 99 USPQ2d 1690, 1695 (Fed. Cir. 2011). As the Federal Circuit explained, “methods which can be performed mentally, or which are the equivalent of human mental work, are unpatentable abstract ideas the ‘basic tools of scientific and technological work’ that are open to all.’” 654 F.3d at 1371, 99 USPQ2d at 1694 (citing Gottschalk v. Benson, 409 U.S. 63, 175 USPQ 673 (1972)). See also Mayo Collaborative Servs. v. Prometheus Labs. Inc., 566 U.S. 66, 71, 101 USPQ2d 1961, 1965 (2012) (“‘[M]ental processes[] and abstract intellectual concepts are not patentable, as they are the basic tools of scientific and technological work’” (quoting Benson, 409 U.S. at 67, 175 USPQ at 675)); Parker v. Flook, 437 U.S. 584, 589, 198 USPQ 193, 197 (1978) (same). See MPEP 2106.04(a)(2). These judicial exceptions are not integrated into a practical application, such as constituting an improvement in the technological field, or including steps recited in addition to the judicial exception that integrates the abstract ideas into a particular treatment/prophylaxis according to MPEP § 2106.04(d)(2). The claims do not include additional elements that are sufficient to amount to significantly more than the judicial exceptions because the additional elements fail to provide either an inventive concept or impose meaningful limits upon the method such that the invention does not preempt every observance of the abstract ideas themselves.
Eligibility Step 1:
Claims 11-30 are directed to methods comprising determining the abundance of isolated peptides, comparing the abundance of isolated peptides, and identifying a peptide from the isolated peptides. Methods are one of the eligible statutory categories for invention (STEP 1: YES). However, eligibility of the claims is not self-evident, and therefore analysis must proceed to Step 2.
Eligibility Step 2A, Prong One:
The abstract ideas to which the claims are directed are mental processes. In addition, claim 30 recites, “calculating the local affinity”. Calculating is an abstract idea, specifically, a mathematical concept. Regarding the mathematical concepts, “the discovery of [a mathematical formula] cannot support a patent unless there is some other inventive concept in its application.”’ Flook, 437 U.S. at 594, 198 USPQ at 199. More recent opinions of the Supreme Court, however, have affirmatively characterized mathematical relationships and formulas as abstract ideas. See, e.g., Alice Corp. Pty. Ltd. v. CLS Bank Int’l, 573 U.S. 208, 218, 110 USPQ2d 1976, 1981 (2014) (describing Flook as holding “that a mathematical formula for computing ‘alarm limits’ in a catalytic conversion process was also a patent-ineligible abstract idea.”); Bilski v. Kappos, 561 U.S. 593, 611-12, 95 USPQ2d 1001, 1010 (2010)”).
Regarding the mental processes, the courts do not distinguish between mental processes that are performed entirely in the human mind and mental processes that require a human to use a physical aid (e.g., pen and paper or a slide rule) to perform the claim limitation. See, e.g., Benson, 409 U.S. at 67, 65, 175 USPQ at 674-75, 674 (noting that the claimed “conversion of [binary-coded decimal] numerals to pure binary numerals can be done mentally,” i.e., “as a person would do it by head and hand.”); Synopsys, Inc. v. Mentor Graphics Corp., 839 F.3d 1138, 1139, 120 USPQ2d 1473, 1474 (Fed. Cir. 2016) (holding that claims to a mental process of “translating a functional description of a logic circuit into a hardware component description of the logic circuit” are directed to an abstract idea, because the claims “read on an individual performing the claimed steps mentally or with pencil and paper”).
Eligibility Step 2A, Prong Two:
According to Step 2A, Prong Two, set forth in MPEP 2106.04 II A (2), the claims are next evaluated with respect to whether the judicial exception is integrated into a practical application. This analysis turns to the additional steps/elements recited within the claims. In independent claims 11, 20, and 26, the additional steps are “providing a plurality of samples” (claims 20 and 26), contacting samples with a protease (claims 11, 20, and 26), and “isolating the peptides” (claims 11, 20, and 26). These claim limitations are insufficient to integrate the judicial exception into a practical application because they do not go beyond insignificant presolution activity, i.e., a mere data gathering step necessary to use the abstract ideas.
Regarding the additional steps cited in claims 11, 20, and 26, the “determining the abundance of the isolated peptides” is recited at a high level of generality and is not tied, for example to any particular machine or apparatus.
There are no additional elements that reflect an actual improvement within the technical field. For example, there are no additional elements that apply the abstract idea judicial exceptions to a particular treatment or which utilize a particular machine; there are no additional elements that effect a transformation; and, there are no additional elements that apply the judicial exception in some other meaningful way beyond generally linking it to a field, namely, proteomics. In this way the claims, as drafted, do not integrate the judicial exception into a practical application that would overcome monopolizing the exceptions. (STEP 2A, Prong Two: NO).
Eligibility Step 2B:
Lastly, the additional elements, individually and as an ordered combination, do not amount to significantly more than the exceptions, because they are well-understood, routine, and conventional in the filed before the effective filing date. See MPEP 2106.05(d).
The additional elements include: wherein the change in the energy state is indicative of an interaction with a ligand (claim 12), isolating the peptides based on a difference in hydrophobicity (claims 13), wherein the peptides suitable for bottom-up mass spectrometry have a molecular weight less than 5 kDa (claims 14, 22, and 28), wherein the samples comprise a purified protein, or a protein mixture originated from a cell or tissue extract from a human (claim 15), wherein the protease comprises trypsin, proteinase K, or thermolysin (claims 16, 23, and 29), wherein the protease weight ratio to total protein ranges from 1/1 to 1/50 (claims 17, 24, and 30), wherein the sample is contacted with the protease for 0.5 to 60 minutes (claim 18), wherein determining the abundance of the isolated peptides by a quantitative mass spectrometry-based assay (claims 19 and 25), and wherein the ligand is a drug (claims 21 and 27).
The additional elements are shown to be well-understood, routine, and conventional in the field by Piazza (cited below) and Ma (cited below), demonstrated below.
Therefore, the steps/elements recited in addition to the judicial exception do not add significantly more (STEP 2B: NO).
The claimed steps/elements recited in addition to the judicial exception, alone or in combination, do not make an inventive contribution over the methods that were known in the art prior to filing because the judicial exceptions themselves cannot form the basis of eligibility.
For all of these reasons, the claimed subject matter is ineligible under 35 U.S.C. 101 because the claims are directed to abstract idea judicial exceptions without significantly more.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 11-13, 15-16, 18-21, 23, 25-27, and 29 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Piazza (“A machine learning-based chemoproteomic approach to identify drug targets and binding sites in complex proteomes”, published 2020-08-21, cited in IDS filed 04/10/2024).
With respect to claim 11, Piazza teaches a method for detecting a change in the energy state of a protein, comprising: (a) contacting a plurality of samples, each comprising the protein, with a protease in an amount sufficient to generate peptides suitable for a bottom-up mass spectrometry analysis; (b) isolating the peptides suitable for the bottom-up mass spectrometry analysis from the plurality of samples; (c) determining the abundance of the isolated peptides; and (d) performing step (i) or (ii): (i) comparing the abundance of the isolated peptides between the plurality of samples, wherein a difference in the abundance of one or more of the isolated peptides between the plurality of samples is indicative of a change in the energy state of the protein; or (ii) identifying a peptide from the isolated peptides that has a different abundance between the plurality of samples; and determining the location of the identified peptide in the protein, wherein the location is indicative of a region that has a change in the energy state of the protein (see, e.g., step (a) – p. 9, col. 2, under “Lysate and cell treatment for LiP-Quant in native conditions”: “Cell lysates from at least three independent biological replicates were aliquoted in equivalent volumes containing 100 µg of proteome sample and incubated for 10 min at 25 °C with the drug of interest. Proteinase K from Tritirachium album (Sigma Aldrich) was added simultaneously to all the proteome metabolite samples with the aid of a multichannel pipette, at a proteinase K: substrate mass ratio of 1:100, and incubated at 25 °C for 4 min”, and p. 9, col. 2, under “Mass spectrometric acquisition”; step (b) – p. 9, col. 2, under “Proteome preparation in denaturing conditions”: “Samples were further digested for 16 h at 37°C with trypsin (1:100 enzyme: substrate ratio). Deoxycholate was precipitated by addition of formic acid to a final concentration of 1.5% and centrifuged at 16,000g for 10 min. After transferring the supernatant to a new Eppendorf tube an equal volume of formic acid was added again and the centrifugation repeated”; step (c) – p. 9, col. 2, under “Mass spectrometric acquisition”, and p. 10, col. 1, under “Mass spectrometric data analysis”; step (d)(i) – p. 3, under “Fig. 1”, under panel “a”, and p. 10, col. 1, under “Machine learning-based training of the LiP-Quant classifier”, and p. 10, col. 2, under “Automated peptide/protein ranking of LiP-Quant experiments”; step (d)(ii) – p. 11, col. 1, under “Approximating drug binding site from LiP-Quant data”, and p. 8, under “Fig. 4”, under panel “e”).
With respect to claim 12, Piazza teaches wherein the change in the energy state is indicative of an interaction with a ligand (see, e.g., p. 1, under abstract, and p. 3, under “Fig. 1”).
With respect to claim 13, Piazza teaches wherein step (b) comprises isolating the peptides suitable for the bottom-up mass spectrometry analysis based on a difference in hydrophobicity (see, e.g., p. 9, col. 2, under “Proteome preparation in denaturing conditions”: “an equal volume of formic acid was added again and the centrifugation repeated. Digests were desalted using C18 MacroSpin columns (The Nest Group), or Sep-Pak C18 cartridges or into 96-well elution plates (Waters)”).
With respect to claim 15, Piazza teaches wherein the plurality of samples comprise a purified protein, or a protein mixture originated from a cell from a human (see, e.g., p. 9, col. 1, under “Whole-proteome preparation for MS analysis”, and p. 9, col. 2, under “Lysate and cell treatment for LiP-Quant in native conditions”).
With respect to claim 16, Piazza teaches wherein the protease comprises trypsin and proteinase K (see, e.g., p. 9, col. 2, under “Lysate and cell treatment for LiP-Quant in native conditions”: “Proteinase K from Tritirachium album (Sigma Aldrich) was added simultaneously to all the proteome metabolite samples with the aid of a multichannel pipette”, and p. 9, col. 2, under “Proteome preparation in denaturing condition”: “Samples were further digested for 16 h at 37°C with trypsin”).
With respect to claim 18, Piazza teaches wherein step (a) comprises contacting the plurality of samples with the protease for 0.5 to 60 minutes (see, e.g., p. 9, col. 2, under “Lysate and cell treatment for LiP-Quant in native conditions”: “Proteinase K from Tritirachium album (Sigma Aldrich) was added simultaneously to all the proteome metabolite samples with the aid of a multichannel pipette, at a proteinase K: substrate mass ratio of 1:100, and incubated at 25 °C for 4 min”).
With respect to claim 19, Piazza teaches wherein step (c) comprises determining the abundance of the isolated peptides by a quantitative mass spectrometry-based assay (see, e.g., p. 9, col. 2, under “Mass spectrometric acquisition”, and p. 10, col. 1, under “Mass spectrometric data analysis”).
With respect to claim 20, Piazza teaches a method of identifying a target protein that is bound by a ligand, comprising: (a) providing a plurality of samples, each comprising a candidate target protein, wherein (1) two or more of the plurality of samples further comprise the ligand at different concentrations, (2) at least one of the plurality of samples further comprises the ligand and at least one of the plurality of samples does not comprise the ligand, or (3) both (1) and (2); (b) contacting the protein samples with a protease in an amount sufficient to generate peptides suitable for a bottom-up mass spectrometry analysis; (c) isolating the peptides suitable for the bottom-up mass spectrometry analysis from the samples; (d) determining the abundance of the isolated peptides; and (e) performing step (i) or (ii):(i) comparing the abundance of the isolated peptides between the plurality of samples, wherein a difference in the abundance of one or more of the isolated peptides between the plurality of samples is indicative of a target protein bound by the ligand; or (ii) identifying a peptide from the isolated peptides that has a different abundance between the plurality of samples; and determining the location of the identified peptide in the candidate target protein, wherein the location is indicative of a region in the candidate protein target bound by the ligand (see, e.g., step (a) – p. 9, col. 2, under “Lysate and cell treatment for LiP-Quant in native conditions”: “Cell lysates from at least three independent biological replicates were aliquoted in equivalent volumes containing 100 µg of proteome sample and incubated for 10 min at 25 °C with the drug of interest”, and p. 9, col. 2, under “Mass spectrometric acquisition”; step (b) – p. 9, col. 2, under “Proteome preparation in denaturing conditions”: “Samples were further digested for 16 h at 37°C with trypsin (1:100 enzyme: substrate ratio)”; step (c) – p. 9, col. 2, under “Proteome preparation in denaturing conditions”: “Deoxycholate was precipitated by addition of formic acid to a final concentration of 1.5% and centrifuged at 16,000g for 10 min. After transferring the supernatant to a new Eppendorf tube an equal volume of formic acid was added again and the centrifugation repeated”; step (d) – p. 9, col. 2, under “Mass spectrometric acquisition”, and p. 10, col. 1, under “Mass spectrometric data analysis”; step (e)(i) – p. 3, under “Fig. 1”, under panel “a”, and p. 10, col. 1, under “Machine learning-based training of the LiP-Quant classifier”, and p. 10, col. 2, under “Automated peptide/protein ranking of LiP-Quant experiments”; step (e)(ii) – p. 11, col. 1, under “Approximating drug binding site from LiP-Quant data”, and p. 8, under “Fig. 4”, under panel “e”).
With respect to claim 21, Piazza teaches wherein the ligand is a drug (see, e.g., p. 7, under “Fig. 3”).
With respect to claim 23, Piazza teaches wherein the protease comprises trypsin and proteinase K (see, e.g., p. 9, col. 2, under “Lysate and cell treatment for LiP-Quant in native conditions”: “Proteinase K from Tritirachium album (Sigma Aldrich) was added simultaneously to all the proteome metabolite samples with the aid of a multichannel pipette”, and p. 9, col. 2, under “Proteome preparation in denaturing condition”: “Samples were further digested for 16 h at 37°C with trypsin”).
With respect to claim 25, Piazza teaches wherein step (d) comprises determining the abundance of the isolated peptides by a quantitative mass spectrometry-based assay (see, e.g., p. 9, col. 2, under “Mass spectrometric acquisition”, and p. 10, col. 1, under “Mass spectrometric data analysis”).
With respect to claim 26, Piazza teaches a method for determining the local affinity between a target protein and a ligand, comprising:(a) providing a plurality of samples, each comprising a candidate target protein, wherein (1) two or more of the plurality of samples further comprise the ligand at different concentrations, (2) at least one of the plurality of samples further comprises the ligand and at least one of the plurality of samples does not comprise the ligand, or (3) both (1) and (2); (b) contacting the plurality of samples with a protease in an amount sufficient to generate peptides suitable for a bottom-up mass spectrometry analysis; (c) isolating the peptides suitable for bottom-up mass spectrometry analysis from the plurality of samples; (d) determining the abundance of the isolated peptides; and (e) calculating the local affinity between the ligand and the protein based on the difference in the abundance of the isolated peptides between the plurality of samples (see, e.g., step (a) – p. 9, col. 2, under “Lysate and cell treatment for LiP-Quant in native conditions”: “Cell lysates from at least three independent biological replicates were aliquoted in equivalent volumes containing 100 µg of proteome sample and incubated for 10 min at 25 °C with the drug of interest”, and p. 9, col. 2, under “Mass spectrometric acquisition”; step (b) – p. 9, col. 2, under “Proteome preparation in denaturing conditions”: “Samples were further digested for 16 h at 37°C with trypsin (1:100 enzyme: substrate ratio)”; step (c) – p. 9, col. 2, under “Proteome preparation in denaturing conditions”: “Deoxycholate was precipitated by addition of formic acid to a final concentration of 1.5% and centrifuged at 16,000g for 10 min. After transferring the supernatant to a new Eppendorf tube an equal volume of formic acid was added again and the centrifugation repeated”; step (d) – p. 9, col. 2, under “Mass spectrometric acquisition”, and p. 10, col. 1, under “Mass spectrometric data analysis”; step (e) – p. 11, col. 1, para. 1: “Every LiP-Quant peptide has an EC50 value assigned, which corresponds to the inferred dose of drug necessary to observe half-maximum of the relative peptide intensity variation between drug and vehicle”).
With respect to claim 27, Piazza teaches wherein the ligand is a drug (see, e.g., p. 7, under “Fig. 3”).
With respect to claim 29, Piazza teaches wherein the protease comprises trypsin and proteinase K (see, e.g., p. 9, col. 2, under “Lysate and cell treatment for LiP-Quant in native conditions”: “Proteinase K from Tritirachium album (Sigma Aldrich) was added simultaneously to all the proteome metabolite samples with the aid of a multichannel pipette”, and p. 9, col. 2, under “Proteome preparation in denaturing condition”: “Samples were further digested for 16 h at 37°C with trypsin”).
Claims 14, 22, and 28 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Piazza (cited above) as evidenced by Tsiatsiani (“Proteomics beyond trypsin”, published 2015-03-30).
With respect to claims 14, 22, and 28, Piazza teaches the peptides suitable for the bottom-up mass spectrometry analysis has a molecular weight less than 5 kDa (see, e.g., p. 9, col. 2, under “Proteome preparation in denaturing conditions”: “Samples were further digested for 16 h at 37°C with trypsin (1:100 enzyme: substrate ratio)”). Tsiatsiani teaches that tryptic peptides are bottom-up analysis and are less than 5 kDa (see, e.g., Tsiatsiani, col. 2, para. 3: “This bottom-up or shotgun analysis of short tryptic peptides (typically 0.5–3 kDa) has made a tremendous contribution to the study of proteomes”).
Claims 11-13, 15-21, 23-27, and 29-30 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Ma (“Chemo-Selection Strategy for Limited Proteolysis Experiments on the Proteomic Scale”, published 2018-12-04, cited in IDS filed 04/10/2024).
With respect to claim 11, Ma teaches a method for detecting a change in the energy state of a protein, comprising: (a) contacting a plurality of samples, each comprising the protein, with a protease in an amount sufficient to generate peptides suitable for a bottom-up mass spectrometry analysis; (b) isolating the peptides suitable for the bottom-up mass spectrometry analysis from the plurality of samples; (c) determining the abundance of the isolated peptides; and (d) performing step (i) or (ii): (i) comparing the abundance of the isolated peptides between the plurality of samples, wherein a difference in the abundance of one or more of the isolated peptides between the plurality of samples is indicative of a change in the energy state of the protein; or (ii) identifying a peptide from the isolated peptides that has a different abundance between the plurality of samples; and determining the location of the identified peptide in the protein, wherein the location is indicative of a region that has a change in the energy state of the protein (see, e.g., step (a) – p. 4, under “Cell Culture and Lysis”: “For ligand-binding experiments using PP, the yeast lysate was divided into two equal aliquots. One aliquot was spiked with a solution of the test ligand (CsA or geldanamycin) prepared in DMSO and the other one was spiked with DMSO as vehicle. The concentration of drug in each cell lysate aliquot was 500 µM and 50 µM in the CsA and geldanamycin binding experiments, respectively. The final concentration of DMSO in each cell lysate aliquot was 5% v/v in all experiments”, and p. 4, under “STEPP-PP Analysis”: “The proteolysis reaction was initiated by adding thermolysin to the protein and protein–ligand samples in each denaturant-containing buffer. The thermolysin to total protein ratio was approximately 1:10 (w/w)”; step (b) – p. 5, under “STEPP-PP Analysis”: “The resulting samples were dried, re-dissolved in 2% v/v TFA solution, and desalted using C18 columns according to the manufacturer’s protocol. The desalted samples were dried, re-dissolved in 100 µL of 0.1 M TEAB solution (pH 8.5), and digested with trypsin overnight at 37 °C. NHS-activated agarose resin and 50 µL of 0.5M NaCl were added to the trypsin-digested samples such that the NHS-activated agarose resin to total peptide ratio was approximately 150:1 (w/w). The tryptic peptides mixture was allowed to react with the NHS-activated agarose resin for 1.5 hours at room temperature before the samples were acidified with 2% v/v TFA and the soluble peptides desalted using C18 columns”; step (c) – p. 5, under “Quantitative LC-MS/MS Analyses”; step (d)(i) - p. 5, under “Quantitative LC-MS/MS Analyses”).
With respect to claim 12, Ma teaches wherein the change in the energy state is indicative of an interaction with a ligand (see, e.g., p. 8, under “STEPP-PP Analysis of CsA and Geldanamycin Binding to Proteins in a Yeast Cell Lysate”, para. 2-3).
With respect to claim 13, Ma teaches wherein step (b) comprises isolating the peptides suitable for the bottom-up mass spectrometry analysis based on a difference in hydrophobicity (see, e.g., p. 5, para. 1: “The tryptic peptides mixture was allowed to react with the NHS-activated agarose resin for 1.5 hours at room temperature before the samples were acidified with 2% v/v TFA and the soluble peptides desalted using C18 columns. The desalted samples were subject to the LC MS/MS analysis described below”).
With respect to claim 15, Ma teaches wherein the plurality of samples comprise a purified protein, or a protein mixture originated from a cell from a human (see, e.g., p. 4, under “Cell Culture and Lysis”: “The MCF-7 and MCF-10A cell lines were purchased from American Type Culture Collection (ATCC) (Manassas, VA) and cultured according to ATCC guidelines”).
With respect to claim 16, Ma teaches wherein the protease comprises thermolysin (see, e.g., p. 4, under “STEPP-PP Analysis”: “The proteolysis reaction was initiated by adding thermolysin to the protein and protein–ligand samples in each denaturant-containing buffer. The thermolysin to total protein ratio was approximately 1:10 (w/w). The thermolysin proteolysis reactions were allowed to proceed for 1 min at room temperature before each reaction was quenched”).
With respect to claim 17, Ma teaches wherein step (a) comprises contacting the plurality of samples with the protease at a weight ratio of protease to total protein ranging from 1/1 to 1/50 (see, e.g., p. 4, under “STEPP-PP Analysis”: “The proteolysis reaction was initiated by adding thermolysin to the protein and protein–ligand samples in each denaturant-containing buffer. The thermolysin to total protein ratio was approximately 1:10 (w/w)”).
With respect to claim 18, Ma teaches wherein step (a) comprises contacting the plurality of samples with the protease for 0.5 to 60 minutes (see, e.g., p. 4, under “STEPP-PP Analysis”: “The thermolysin proteolysis reactions were allowed to proceed for 1 min at room temperature before each reaction was quenched”).
With respect to claim 19, Ma teaches wherein step (c) comprises determining the abundance of the isolated peptides by a quantitative mass spectrometry-based assay (see, e.g., p. 5, under “Quantitative LC-MS/MS Analyses”).
With respect to claim 20, Ma teaches a method of identifying a target protein that is bound by a ligand, comprising: (a) providing a plurality of samples, each comprising a candidate target protein, wherein (1) two or more of the plurality of samples further comprise the ligand at different concentrations, (2) at least one of the plurality of samples further comprises the ligand and at least one of the plurality of samples does not comprise the ligand, or (3) both (1) and (2); (b) contacting the protein samples with a protease in an amount sufficient to generate peptides suitable for a bottom-up mass spectrometry analysis; (c) isolating the peptides suitable for the bottom-up mass spectrometry analysis from the samples; (d) determining the abundance of the isolated peptides; and (e) performing step (i) or (ii):(i) comparing the abundance of the isolated peptides between the plurality of samples, wherein a difference in the abundance of one or more of the isolated peptides between the plurality of samples is indicative of a target protein bound by the ligand; or (ii) identifying a peptide from the isolated peptides that has a different abundance between the plurality of samples; and determining the location of the identified peptide in the candidate target protein, wherein the location is indicative of a region in the candidate protein target bound by the ligand (see, e.g., step (a) – p. 4, under “Cell Culture and Lysis”: “For ligand-binding experiments using PP, the yeast lysate was divided into two equal aliquots. One aliquot was spiked with a solution of the test ligand (CsA or geldanamycin) prepared in DMSO and the other one was spiked with DMSO as vehicle. The concentration of drug in each cell lysate aliquot was 500 µM and 50 µM in the CsA and geldanamycin binding experiments, respectively. The final concentration of DMSO in each cell lysate aliquot was 5% v/v in all experiments”, and p. 4, under “STEPP-PP Analysis”: “The proteolysis reaction was initiated by adding thermolysin to the protein and protein–ligand samples in each denaturant-containing buffer. The thermolysin to total protein ratio was approximately 1:10 (w/w)”; step (b) – p. 5, under “STEPP-PP Analysis”: “The proteolysis reaction was initiated by adding thermolysin to the protein and protein–ligand samples in each denaturant-containing buffer. The thermolysin to total protein ratio was approximately 1:10 (w/w)”; step (c) – p. 5, under “STEPP-PP Analysis”: “The resulting samples were dried, re-dissolved in 2% v/v TFA solution, and desalted using C18 columns according to the manufacturer’s protocol. The desalted samples were dried, re-dissolved in 100 µL of 0.1 M TEAB solution (pH 8.5), and digested with trypsin overnight at 37 °C. NHS-activated agarose resin and 50 µL of 0.5M NaCl were added to the trypsin-digested samples such that the NHS-activated agarose resin to total peptide ratio was approximately 150:1 (w/w). The tryptic peptides mixture was allowed to react with the NHS-activated agarose resin for 1.5 hours at room temperature before the samples were acidified with 2% v/v TFA and the soluble peptides desalted using C18 columns”; step (d) - p. 5, under “Quantitative LC-MS/MS Analyses”; step (e)(i) - p. 5, under “Quantitative LC-MS/MS Analyses”).
With respect to claim 21, Ma teaches wherein the ligand is a drug (see, e.g., p. 8, under “STEPP-PP Analysis of CsA and Geldanamycin Binding to Proteins in a Yeast Cell Lysate”, para. 2-3).
With respect to claim 23, Ma teaches wherein the protease comprises thermolysin (see, e.g., p. 4, under “STEPP-PP Analysis”: “The proteolysis reaction was initiated by adding thermolysin to the protein and protein–ligand samples in each denaturant-containing buffer. The thermolysin to total protein ratio was approximately 1:10 (w/w). The thermolysin proteolysis reactions were allowed to proceed for 1 min at room temperature before each reaction was quenched”).
With respect to claim 24, Ma teaches wherein step (b) comprises contacting the plurality of samples with the protease at a weight ratio of protease to total protein ranging from 1/1 to 1/50 (see, e.g., p. 4, under “STEPP-PP Analysis”: “The proteolysis reaction was initiated by adding thermolysin to the protein and protein–ligand samples in each denaturant-containing buffer. The thermolysin to total protein ratio was approximately 1:10 (w/w)”).
With respect to claim 25, Ma teaches wherein step (d) comprises determining the abundance of the isolated peptides by a quantitative mass spectrometry-based assay (see, e.g., p. 5, under “Quantitative LC-MS/MS Analyses”).
With respect to claim 26, Ma teaches a method for determining the local affinity between a target protein and a ligand, comprising:(a) providing a plurality of samples, each comprising a candidate target protein, wherein (1) two or more of the plurality of samples further comprise the ligand at different concentrations, (2) at least one of the plurality of samples further comprises the ligand and at least one of the plurality of samples does not comprise the ligand, or (3) both (1) and (2); (b) contacting the plurality of samples with a protease in an amount sufficient to generate peptides suitable for a bottom-up mass spectrometry analysis; (c) isolating the peptides suitable for bottom-up mass spectrometry analysis from the plurality of samples; (d) determining the abundance of the isolated peptides; and (e) calculating the local affinity between the ligand and the protein based on the difference in the abundance of the isolated peptides between the plurality of samples (see, e.g., step (a) – p. 4, under “Cell Culture and Lysis”: “For ligand-binding experiments using PP, the yeast lysate was divided into two equal aliquots. One aliquot was spiked with a solution of the test ligand (CsA or geldanamycin) prepared in DMSO and the other one was spiked with DMSO as vehicle. The concentration of drug in each cell lysate aliquot was 500 µM and 50 µM in the CsA and geldanamycin binding experiments, respectively. The final concentration of DMSO in each cell lysate aliquot was 5% v/v in all experiments”, and p. 4, under “STEPP-PP Analysis”: “The proteolysis reaction was initiated by adding thermolysin to the protein and protein–ligand samples in each denaturant-containing buffer. The thermolysin to total protein ratio was approximately 1:10 (w/w)”; step (b) – p. 5, under “STEPP-PP Analysis”: “The proteolysis reaction was initiated by adding thermolysin to the protein and protein–ligand samples in each denaturant-containing buffer. The thermolysin to total protein ratio was approximately 1:10 (w/w)”; step (c) – p. 5, under “STEPP-PP Analysis”: “The resulting samples were dried, re-dissolved in 2% v/v TFA solution, and desalted using C18 columns according to the manufacturer’s protocol. The desalted samples were dried, re-dissolved in 100 µL of 0.1 M TEAB solution (pH 8.5), and digested with trypsin overnight at 37 °C. NHS-activated agarose resin and 50 µL of 0.5M NaCl were added to the trypsin-digested samples such that the NHS-activated agarose resin to total peptide ratio was approximately 150:1 (w/w). The tryptic peptides mixture was allowed to react with the NHS-activated agarose resin for 1.5 hours at room temperature before the samples were acidified with 2% v/v TFA and the soluble peptides desalted using C18 columns”; step (d) - p. 5, under “Quantitative LC-MS/MS Analyses”; step (e)(i) - p. 10, under “Dissociation Constant Determination in STEPP-PP”: “An inherent advantage of PP over other energetics-based approaches such as the thermal proteome profiling (TPP) approach,24 is that it can be used to evaluate the dissociation constants (Kd) of protein-ligand complexes”).
With respect to claim 27, Ma teaches wherein the ligand is a drug (see, e.g., p. 8, under “STEPP-PP Analysis of CsA and Geldanamycin Binding to Proteins in a Yeast Cell Lysate”, para. 2-3).
With respect to claim 29, Ma teaches wherein the protease comprises thermolysin (see, e.g., p. 4, under “STEPP-PP Analysis”: “The proteolysis reaction was initiated by adding thermolysin to the protein and protein–ligand samples in each denaturant-containing buffer. The thermolysin to total protein ratio was approximately 1:10 (w/w). The thermolysin proteolysis reactions were allowed to proceed for 1 min at room temperature before each reaction was quenched”).
With respect to claim 30, Ma teaches wherein step (b) comprises contacting the plurality of samples with the protease at a weight ratio of protease to total protein ranging from 1/1 to 1/50. (see, e.g., p. 4, under “STEPP-PP Analysis”: “The proteolysis reaction was initiated by adding thermolysin to the protein and protein–ligand samples in each denaturant-containing buffer. The thermolysin to total protein ratio was approximately 1:10 (w/w)”).
Conclusion
No claims are allowed.
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/MICHAEL CAMERON SVEIVEN/ Examiner, Art Unit 1678
/GREGORY S EMCH/ Supervisory Patent Examiner, Art Unit 1678
