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 .
Election/Restrictions
Applicant’s election of Group I, claims 1-18 in the reply filed on 02/27/2026 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Claims 19 and 20 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Claims 1-20 are pending. Claims 1-18 (claim set filed 06/01/2023) are examined on the merits herein.
Priority
This application claims benefit of provisional application 63/348,357 filed 06/02/2022. 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.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 05/09/2024 and 05/10/2024 comply with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
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 1-16 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.
Claim 15 recites: “The method of claim 19 comprising performing mass spectrometry analysis on the first set and second set of extracted biomolecules”. It appears that claim 15 has typographical error and is intended to depend on claim 1 since “the first set and second set of extracted biomolecules” does not have antecedent basis in claim 19. The scope and boundaries of claim 15 are not certain making claim 15 indefinite.
For examination purposes claim 15 is interpreted as dependent on claim 1.
Claim 1 recites: “lipids, carbohydrates, metabolites, and combinations thereof”. It is not clear if biomolecules are listed as alternatives and claim requires one of the alternatives or their combinations to be in a first portion of biomolecules or all three alternatives are required since “and” and nor “or” is present in the recitation. In the last case, if all alternatives are required, “combinations thereof” are redundant. The same issue is with the recitation: “nucleic acids, proteins, polypeptides, and combinations thereof”. It is not clear whether one of the alternatives or their combinations or all three alternatives are required. The scope and boundaries of claim 1 are not certain making claim 1 indefinite.
For examination claim 1 is interpreted as directed to one of the alternatives or their combinations.
Claims 3, 5 and 8 recite: “proteins, polypeptides, and combinations thereof” and hence have similar issue. It is not clear whether one of the alternatives or their combinations or two alternatives are required. The scope and boundaries of claims 3, 5 and 8 are not certain making claims 3, 5 and 8 indefinite.
Claims 2, 4, 6, 7 and 9-16, dependent on claims 1, 3 and 5, do not resolve the issues mentioned above and are rejected.
Claim 17 resolves the issue mentioned above for claim 1, claim 17 depends upon, by requiring lipidome and metabolome in the first portion of biomolecules and proteome in the second portion of biomolecules and therefore claim 17 is not rejected.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1, 3, 4, 9, 10, 13 and 15-17 are rejected under 35 U.S.C. 103 as being unpatentable over Coman (Coman et al. Mol. Cell. Proteomics, 2016, 15.4, 1453-1466 on record in IDS) in view of Hughes (Hughes et al. Nature Protocols, 2019, 14, 68-85 on record in IDS).
Regarding claim 1, Coman teaches simultaneous metabolite, protein and lipid extraction from one sample for quantitative investigation of lipids, metabolites and proteins. Application of this method in mass-spectrometry-based workflows allowed the simultaneous quantification of 360 lipids, 75 metabolites, and 3327 proteins from 106 cells (Abstract). Coman describes lysing cells in the presence of methanol, mixing the sample with 750 µl of cold methyl-tert-buthyl-ether (MTBE), incubating in the extraction solution for 1 hour at 4°C, inducing phase separation by adding 188 µl of water with 0.1% ammonium acetate and centrifuging the extract for 5 min. The upper phase containing lipids is collected, dried and dissolved for further analysis. Protein fraction is precipitated by adding methanol to lower phase, followed by centrifugation. The supernatant containing metabolites is dried and processed for further analysis (p. 1454, right column, last paragraph). Analysis of lipids, metabolites and proteins is performed by LC-MS/MS (p. 1455, right column, p. 1456, left column).
Coman does not teach immobilizing a second portion of biomolecules comprising nucleic acids, proteins, polypeptides and their combinations to plurality of beads.
Hughes teaches solid-phase enhanced sample preparation (SP3) technology for proteomics (Abstract). Preparation includes paramagnetic bead-based approach for rapid, robust and efficient processing of protein samples for proteomic analysis (Abstract). Hughes describes that precipitated proteins bind to the surface of hydrophilic beads coated with carboxylate functional groups (p. 70, 1st paragraph). Hughes mentions that protein precipitation/aggregation can be induced by different reagents including solvents and suggests to use ethanol. The incubation time for binding is 5 minutes (p. 74, 2nd paragraph). Protein-coated beads can be separated from the solution by using magnetic rack since beads are paramagnetic (p. 78, #12). Hughes discloses that carboxylate-coated beads can capture nucleic acids and recommends to use steps designed to degrade or shear DNA and RNA such as enzymatic treatment with benzonase or sonication if bound nucleic acids interferes with protein extraction (p. 72, 1st paragraph). Hughes describes procedures for protein elution from the beads (p. 74, 4th paragraph) and digestion of protein on the beads with peptides recovery for MS analysis (p. 79). Hughes mentions that SP3 protocol is compatible with complex protein mixtures and whole cell lysates (p. 71, last paragraph). Hughes discloses that SP3 has successfully been used to facilitate examination of a broad range of sample types spanning simple and complex protein mixtures in large and very small amounts, across numerous organisms (Abstract). Hughes describes the steps and extensive considerations involved in performing SP3 for proteomics (Abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine teachings of Coman and Hughes and substitute separation of precipitated proteins in Coman teaching by immobilization of proteins on paramagnetic functionalized beads from Hughes teaching for easier proteins/polypeptides isolation in Coman method of simultaneous metabolite, protein and lipid extraction. One would have been motivated to make this combination because Hughes describes rapid, robust and efficient processing of protein samples for proteomic analysis that was successfully applied to a broad range of sample types spanning simple and complex protein mixtures in large and very small amounts, across numerous organisms and Hughes provides detailed instructions. A skilled artisan would have reasonably expected success in this combination since Coman and Hughes teach extraction of biomolecules including proteins and polypeptides from cells for mass spectrometry analysis. Thus, Coman and Hughes teachings render claim 1 obvious.
Regarding claim 2, Coman teaches extraction solution of MTBE and methanol present after cell lysis as monophasic solution since the phase separation is induced by addition of water with 0.1% ammonium acetate (p. 1454, right column, last paragraph). Thus, Coman and Hughes teachings render claim 2 obvious.
Regarding claims 3 and 17, Coman teaches extraction of lipids and metabolites (p. 1454, right column, last paragraph) that can be considered the first portion of biomolecules and Hughes teaches extraction of proteins and polypeptides by immobilization of aggregated proteins on paramagnetic beads (p. 70, 1st paragraph) and that is the second portion of biomolecules.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention that proteins bound to the beads based on Hughes teaching can be separated from the extraction solution containing lipids and metabolites as taught by Coman. One would have been motivated to expect that because Coman teaches extraction of lipids and metabolites with MTBE/methanol/water and precipitation of proteins with methanol and Hughes describes binding of aggregated proteins on paramagnetic beads that can be easily separated from the solution. Thus, Coman and Hughes teachings render claims 3 and 17 obvious.
Regarding claim 4, Hughes teaches digestion of proteins attached to the paramagnetic functionalized beads with trypsin (p. 79).
Regarding claim 13, Hughes teaches paramagnetic beads (Abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to extract proteins from a sample using paramagnetic beads and digest proteins bound to beads as taught by Hughes. One would have been motivated to do that with reasonably expected success because Hughes describes rapid, robust and efficient processing of protein samples for proteomic analysis that was successfully applied to a broad range of sample types and provides detailed instructions. Thus, Coman and Hughes teachings render claims 4 and 13 obvious.
Regarding claims 9 and 10, Coman teaches incubation of the extraction solution with the sample for 1 hour at 4°C (p. 1454, right column, last paragraph). Hughes teaches incubation of beads with the sample for protein binding for 5 min. Hughes describes that proteins can be eluted from the bead by different reagents depending on the application, however, having common conditions of treatment, i.e. 30 min at 37°C (p. 74, 4th paragraph). Therefore, the total extraction of biomolecules can be performed within 3 hours or less.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine Hughes and Coman teachings and add paramagnetic functionalized beads from Hughes teaching for protein immobilization during simultaneous extraction of lipids, metabolites and proteins taught by Coman. One would have been motivated to expect that beads can be added during extraction and therefore incubated with the extraction solution for about 1 hour and that the total extraction can be performed within 3 hours or less since Coman describes 1 hour extraction procedure and Hughes teaches 5-min binding of proteins to beads and 30-min elution of proteins from beads. Thus, Coman and Hughes teachings render claims 9 and 10 obvious.
Regarding claim 15, Coman teaches mass spectrometry analysis of all extracted biomolecules, i.e. lipids, metabolites and proteins (p. 1455, right column, p. 1456, left column). Thus, Coman and Hughes teachings render claim 15 obvious.
Regarding claim 16, Coman teaches lysing cells, i.e. OP9 mesenchymal cell line, and extracting biomolecules from the whole lysate (p. 1454, right column, 2nd and last paragraph). Thus, Coman and Hughes teachings render claim 16 obvious.
Claims 5-8 are rejected under 35 U.S.C. 103 as being unpatentable over Coman (Coman et al. Mol. Cell. Proteomics, 2016, 15.4, 1453-1466 on record in IDS) in view of Hughes (Hughes et al. Nature Protocols, 2019, 14, 68-85 on record in IDS) as applied to claim 1 above, and further in view of Switzar (Switzar et al., J. Proteome Res., 2013, 12, 1067-1077) as evidenced by Havlis (Havlis et al. Anal. Chem., 2003, 75, 12300-1306).
The teachings of Coman and Hughes have been set forth above.
Coman and Hughes do not teach digestion of attached to immobilizing beads proteins and polypeptides for 30-60 or 35-45 min at temperature of 40-80°C or 55-65°C.
Regarding claims 5-8, Switzar teaches digestion of proteins into peptides for LC-MS or MALDI-TOF-MS analysis and strategies for acceleration of the digestion process (Abstract). Switzar describes that “reductive methylation of trypsin enhances the rigidity of its secondary structure and thereby increases its thermostability. This modification shifts the temperature for optimal catalytic activity to 50−60 °C, thereby allowing for faster digestion at elevated temperature” (p. 1070, right column, 3rd paragraph). The temperature of 50−60 °C reads on limitations in claims 5, 7 and 8. Switzar refers to prior art of Halvis that teaches digest with modified trypsin at 58°C for 30 min (p. 1302, left column, 1st paragraph). 30 min reads of time limitation in claim 5 and is very close to lower limit of time limitation in claims 6 and 8, i.e. 35 min. It is noted that where the claimed ranges "overlap or lie inside the ranges disclosed by the prior art" and even when the claimed ranges and prior art ranges do not overlap but are close enough that one skilled in the art would have expected them to have similar properties, a prima facie case of obviousness exists (See MPEP 2144.05 I).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the trypsin, used by Hughes for on-bead digestion of proteins and requiring 18 h incubation, with modified trypsin and perform digest at 58-60 °C for 30 min as taught by Switzar and as evidenced by Halvis. One would have been motivated to do that because Switzar describes that modified trypsin has optimal activity at 50−60 °C allowing faster digestion at elevated temperature and refers to Halvis using 30-min digest at 58°C. A skilled artisan would have reasonably expected success in this combination since Coman provides method of simultaneous extraction of lipids, proteins and metabolites, Hughes teaches easier protein separation by immobilization of paramagnetic beads and Switzar and Halvis provide modified trypsin for accelerated digestion of proteins and polypeptides for MS analysis.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention that digestion with modified trypsin can be performed for at least 30 min or close to 30 min, e.g. 35 min. One would have been motivated to do that to achieve complete digest. A skilled artisan would have reasonably expected success in that because optimization of the reaction time is within skills of the artisan in the field. Thus, Coman, Hughes and Switzar teachings as evidenced by Halvis render claims 5-8 obvious.
Claim 11 is rejected under 35 U.S.C. 103 as being unpatentable over Coman (Coman et al. Mol. Cell. Proteomics, 2016, 15.4, 1453-1466 on record in IDS) in view of Hughes (Hughes et al. Nature Protocols, 2019, 14, 68-85 on record in IDS) as applied to claim 1 above, and further in view of Alshehry (Alshehry et al. Metabolites, 2015, 5, 389-403) and Konig (Konig et al. J. Phys. Chem., 2018, 122, 6975-6988 on record in IDS).
The teachings of Coman and Hughes have been set forth above.
Coman and Hughes do not teach extraction solvent to comprise 20-80% of n-butanol.
Regarding claim 11, Alshehry teaches that 1-butanol/methanol 1:1 mixture extracts all the major lipid classes (Abstract). Alshehry discloses that 1-butanol/methanol (1:1) lipid extraction method shows high correlation with established chloroform/methanol extraction methods (p. 393, 2nd paragraph) and provides good reproducibility (p. 394, 1st paragraph).
Konig teaches that 1-butanol is efficient for extractions of polar compounds from aqueous medium. Konig discloses that the high affinity of polar molecules to the wet 1-butanol phase is associated with its nanostructure, i.e. because the small inverse micelles of water are able to accommodate polar solutes and locally mimic an aqueous environment (Abstract). Konig describes extraction of cyclohexane-1,2-diol with 1:1 mixture of 1-butanol/water (p. 6979, left column, 4th paragraph) with higher concentration of cyclohexane-1,2-diol found in 1-butanol phase (p. 6979, right column, 1st paragraph). Konig mentions that amphiphilic molecules are more soluble in wet 1-butanol since they have capacity to interact both with water, the hydroxyl groups of 1-butanol and with the aliphatic part of 1-butanol (p. 6981, left column, 1st paragraph). Konig shows that hydrophobic molecules have preference for the aliphatic part of 1-butanol (p. 6981, right column, 2nd paragraph). Konig tested extraction of cyclohexane-1,2-diol from cell lysate and whole cells and determined the isolated yields of 98% and 94% respectively (p. 6981, right column, last paragraph). Thus, Konig teaching shows that 1-butanol/water mixture can extract polar and non-polar compounds.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow Alshehry teaching and expect that n-butanol would extract various lipids. One would have been motivated to do that with reasonably expected success because Alshehry teaches that 1-butanol/methanol 1:1 mixture extracts all the major lipid classes.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow Konig teaching that 50%/50% n-butanol/water mixture would extract not only lipids but also polar molecules (metabolites) and use that mixture for extraction of lipids and metabolites in the method of biomolecules extraction based on Coman and Hughes teachings. One would have been motivated to do that because Konig shows that 50%/50% n-butanol/water mixture extracts polar molecules due to wet n-butanol nanostructure and non-polar molecules interacting with aliphatic part of 1-butanol. A skilled artisan would have reasonably expected success in that since Coman, Hughes, Alshehry and Konig teach extraction of biomolecules from the sample. Thus, Coman, Hughes, Alshehry and Konig teachings render claim 11 obvious.
Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over Coman (Coman et al. Mol. Cell. Proteomics, 2016, 15.4, 1453-1466 on record in IDS) in view of Hughes (Hughes et al. Nature Protocols, 2019, 14, 68-85 on record in IDS) and further in view of Alshehry (Alshehry et al. Metabolites, 2015, 5, 389-403) and Konig (Konig et al. J. Phys. Chem., 2018, 122, 6975-6988) as applied to claims 1and 11 above, and further in view of Blum (Blum et al. Mol. Omics, 2018, 14, 307-319) and Johnson (Johnson et al. bioRxiv, 2021, 1-23).
The teachings of Coman, Hughes, Alshehry and Konig have been set forth above.
Coman, Hughes, Alshehry and Konig do not teach extraction solvent to comprise 55-65% of n-butanol, 15-25% acetonitrile and 15-25% of water.
Regarding claim 12, Blum teaches multi-omics profiling approach for obtaining different molecules measurement from a single biological sample (Abstract). Blum discloses that hydrophilic metabolites are extracted with isopropanol, ethanol, methanol, acetonitrile, water or their mixtures (p. 309, right column, 1st paragraph).
Johnson teaches inert glass beads as the means of protein aggregation capture and as alternative to SP3 magnetic beads (Abstract). Johnson describes that organic solvents induce denaturation and strong non-covalent interactions between proteins causing precipitation through aggregation (p. 1, right column, 3rd paragraph). Johnson discloses that acetonitrile causes protein precipitation through aggregation. Johnson mentions that acetonitrile is also highly compatible with protein preparation, digestion and LC-MS (p. 1, right column, 3rd paragraph).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow Blum and Johnson teachings and try to add acetonitrile to the extraction solution of 50%/50% n-butanol/water during isolation of biomolecules based on Coman, Hughes, Alshehry and Konig teachings by replacing part of water, for instance 25%, and apply that extraction solution for extraction of lipids and metabolites. One would have been motivated to do that because is shown to extract lipids and polar molecules as discussed above, acetonitrile is used for extraction of metabolites as taught by Blum and causes protein precipitation as described by Johnson and additionally acetonitrile is highly compatible with protein preparation, digestion and LC-MS as taught by Johnson. Therefore, acetonitrile can facilitate both metabolites extraction and immobilization of precipitated proteins on beads. A skilled artisan would have reasonably expected success in that since Coman provides method of simultaneous extraction of biomolecules, Hughes and Johnson describe protein extraction by immobilization of aggregated proteins on beads and Alshehry, Konig and Blum describe reagents for extraction of lipids and metabolites.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to optimize the ratio of n-butanol/acetonitrile/water in extraction solution. One would have been motivated to do that to achieve high level of extraction of lipids and metabolites. A skilled artisan would have reasonably expected success in that because optimization of the concentration of the reagents is within skills of the artisan in the field. Thus, Coman, Hughes, Alshehry, Konig, Blum and Johnson teachings render claim 12 obvious.
Claim 14 is rejected under 35 U.S.C. 103 as being unpatentable over Coman (Coman et al. Mol. Cell. Proteomics, 2016, 15.4, 1453-1466 on record in IDS) in view of Hughes (Hughes et al. Nature Protocols, 2019, 14, 68-85 on record in IDS) as applied to claim 1 above, and further in view of Johnson (Johnson et al. bioRxiv, 2021, 1-23).
The teachings of Coman and Hughes have been set forth above.
Coman and Hughes do not teach immobilizing beads to be unmodified silica beads.
Regarding claim 14, Johnson teaches inert glass beads (silica beads) as the means of protein aggregation capture and as alternative to SP3 magnetic beads (Abstract, 9. 3, left column, 3rd paragraph). Johnson describes that organic solvents induce denaturation and strong non-covalent interactions between proteins causing precipitation through aggregation (p. 1, right column, 3rd paragraph). Johnson discloses that aggregation interactions observed between functionalized carboxylate beads and proteins result in highly similar proteomes to the interactions during protein-protein aggregation and precipitation alone and inert surfaces are also capable of promoting protein aggregation capture (p. 11, left column, 2nd paragraph). Johnson mentions that functionalized carboxylate beads of SP3 can reduce protein yields by binding protease and phosphatase inhibitors and inert beads have advantage of preventing unwanted interactions with certain chemical additives (p. 2, 2nd paragraph).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the carboxylate functionalized paramagnetic beads of Hughes teaching used for extraction of proteins and polypeptides based on combine Coman and Hughes teachings with inert silica beads as taught by Johnson. One would have been motivated to do that because Johnson describes that inert beads have similar effectively in capturing aggregated proteins as functionalized beads, provide similar proteomes and prevent unwanted binding on the bead surface. A skilled artisan would have reasonably expected success in this combination since Johnson and Hughes teach capturing of aggregated proteins on beads for mass spectrometry. Thus, Coman, Hughes and Johnson teachings render claim 14 obvious.
Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over Coman (Coman et al. Mol. Cell. Proteomics, 2016, 15.4, 1453-1466 on record in IDS) in view of Hughes (Hughes et al. Nature Protocols, 2019, 14, 68-85 on record in IDS), Alshehry (Alshehry et al. Metabolites, 2015, 5, 389-403), Konig (Konig et al. J. Phys. Chem., 2018, 122, 6975-6988) and Switzar (Switzar et al., J. Proteome Res., 2013, 12, 1067-1077) as evidenced by Havlis (Havlis et al. Anal. Chem., 2003, 75, 12300-1306).
Regarding claim 18, Coman teaches simultaneous metabolite, protein and lipid extraction from one sample for quantitative investigation of lipids, metabolites and proteins. Application of this method in mass-spectrometry-based workflows allowed the simultaneous quantification of 360 lipids, 75 metabolites, and 3327 proteins from 106 cells (Abstract). Coman describes lysing cells in the presence of methanol, mixing the sample with 750 µl of cold methyl-tert-buthyl-ether (MTBE), incubating in the extraction solution for 1 hour at 4°C, inducing phase separation by adding 188 µl of water with 0.1% ammonium acetate and centrifuging the extract for 5 min. The upper phase containing lipids is collected, dried and dissolved for further analysis. Protein fraction is precipitated by adding methanol to lower phase, followed by centrifugation. The supernatant containing metabolites is dried and processed for further analysis (p. 1454, right column, last paragraph). Analysis of lipids, metabolites and proteins is performed by LC-MS/MS (p. 1455, right column, p. 1456, left column).
Coman does not teach immobilizing a second portion of biomolecules comprising nucleic acids, proteins, polypeptides and their combinations to plurality of beads, extraction solvent to comprise 20-80% of n-butanol and digestion of attached to immobilizing beads proteins and polypeptides for 35-45 min at temperature of 55-65°C.
Hughes teaches solid-phase enhanced sample preparation (SP3) technology for proteomics (Abstract). Preparation includes paramagnetic bead-based approach for rapid, robust and efficient processing of protein samples for proteomic analysis (Abstract). Hughes describes that precipitated proteins bind to the surface of hydrophilic beads coated with carboxylate functional groups (p. 70, 1st paragraph). Hughes mentions that protein precipitation/aggregation can be induced by different reagents including solvents and suggests to use ethanol. The incubation time for binding is 5 minutes (p. 74, 2nd paragraph). Protein-coated beads can be separated from the solution by using magnetic rack since beads are paramagnetic (p. 78, #12). Hughes describes procedures for protein elution from the beads (p. 74, 4th paragraph) and digestion of protein on the beads with peptides recovery for MS analysis (p. 79). Hughes mentions that SP3 protocol is compatible with complex protein mixtures and whole cell lysates (p. 71, last paragraph). Hughes discloses that SP3 has successfully been used to facilitate examination of a broad range of sample types spanning simple and complex protein mixtures in large and very small amounts, across numerous organisms (Abstract). Hughes describes the steps and extensive considerations involved in performing SP3 for proteomics (Abstract).
Alshehry teaches that 1-butanol/methanol 1:1 mixture extracts all the major lipid classes (Abstract). Alshehry discloses that 1-butanol/methanol (1:1) lipid extraction method shows high correlation with established chloroform/methanol extraction methods (p. 393, 2nd paragraph) and provides good reproducibility (p. 394, 1st paragraph).
Konig teaches that 1-butanol is efficient for extractions of polar compounds from aqueous medium. Konig discloses that the high affinity of polar molecules to the wet 1-butanol phase is associated with its nanostructure, i.e. because the small inverse micelles of water are able to accommodate polar solutes and locally mimic an aqueous environment (Abstract). Konig describes extraction of cyclohexane-1,2-diol with 1:1 mixture of 1-butanol/water (p. 6979, left column, 4th paragraph) with higher concentration of cyclohexane-1,2-diol found in 1-butanol phase (p. 6979, right column, 1st paragraph). Konig mentions that amphiphilic molecules are more soluble in wet 1-butanol since they have capacity to interact both with water, the hydroxyl groups of 1-butanol and with the aliphatic part of 1-butanol (p. 6981, left column, 1st paragraph). Konig shows that hydrophobic molecules have preference for the aliphatic part of 1-butanol (p. 6981, right column, 2nd paragraph). Konig tested extraction of cyclohexane-1,2-diol from cell lysate and whole cells and determined the isolated yields of 98% and 94% respectively (p. 6981, right column, last paragraph). Thus, Konig teaching shows that 1-butanol/water mixture can extract polar and non-polar compounds.
Switzar teaches digestion of proteins into peptides for LC-MS or MALDI-TOF-MS analysis and strategies for acceleration of the digestion process (Abstract). Switzar describes that “reductive methylation of trypsin enhances the rigidity of its secondary structure and thereby increases its thermostability. This modification shifts the temperature for optimal catalytic activity to 50−60 °C, thereby allowing for faster digestion at elevated temperature” (p. 1070, right column, 3rd paragraph). The temperature of 50−60 °C reads on limitation in claim 18. Switzar refers to prior art of Halvis that teaches digest with modified trypsin at 58°C for 30 min (p. 1302, left column, 1st paragraph). 30 min is very close to lower limit of time limitations in claim 18, i.e. 35 min. It is noted that where the claimed ranges "overlap or lie inside the ranges disclosed by the prior art" and even when the claimed ranges and prior art ranges do not overlap but are close enough that one skilled in the art would have expected them to have similar properties, a prima facie case of obviousness exists (See MPEP 2144.05 I).
First, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine teachings of Coman and Hughes and substitute separation of precipitated proteins in Coman teaching by immobilization of proteins on paramagnetic functionalized beads from Hughes teaching for easier proteins/polypeptides isolation in Coman method of simultaneous metabolite, protein and lipid extraction. One would have been motivated to make this combination because Hughes describes rapid, robust and efficient processing of protein samples for proteomic analysis that was successfully applied to a broad range of sample types spanning simple and complex protein mixtures in large and very small amounts, across numerous organisms and Hughes provides detailed instructions. A skilled artisan would have reasonably expected success in this combination since Coman and Hughes teach extraction of biomolecules including proteins and polypeptides from cells for mass spectrometry analysis.
Second, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow Alshehry teaching and expect that n-butanol would extract various lipids. One would have been motivated to do that with reasonably expected success because Alshehry teaches that 1-butanol/methanol 1:1 mixture extracts all the major lipid classes.
Third, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow Konig teaching that 50%/50% n-butanol/water mixture would extract not only lipids but also polar molecules (metabolites) and use that mixture for extraction of lipids and metabolites in the method of biomolecules extraction based on Coman and Hughes teachings. One would have been motivated to do that because Konig shows that 50%/50% n-butanol/water mixture extracts polar molecules due to wet n-butanol nanostructure and non-polar molecules interacting with aliphatic part of 1-butanol. A skilled artisan would have reasonably expected success in that since Coman, Hughes, Alshehry and Konig teach extraction of biomolecules from the sample.
Forth, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the trypsin, used by Hughes for on-bead digestion of proteins and requiring 18 h incubation, with modified trypsin and perform digest at 58-60 °C for 30 min as taught by Switzar and evidenced by Halvis. One would have been motivated to do that because Switzar describes that modified trypsin has optimal activity at 50−60 °C allowing faster digestion at elevated temperature and refers to Halvis using 30-min digest at 58°C. A skilled artisan would have reasonably expected success in this combination since Coman provides method of simultaneous extraction of lipids, proteins and metabolites, Hughes teaches easier protein separation by immobilization of paramagnetic beads and Switzar and Halvis provide modified trypsin for accelerated digestion of proteins and polypeptides for MS analysis.
Fifth, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention that digestion with modified trypsin can be performed for at least 30 min or close to 30 min, e.g. 35 min. One would have been motivated to do that to achieve complete digest. A skilled artisan would have reasonably expected success in that because optimization of the reaction time is within skills of the artisan in the field.
Sixth, regarding the first and the second set of extracted biomolecules, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention that extracted lipids and metabolites can be considered the first set of biomolecules and extracted and digested proteins and polypeptides can be considered the second set of biomolecules. One would have been motivated to expect that because Hughes describes binding of aggregated proteins on paramagnetic beads that can be easily separated from the solution and Switzar and Halvis provide accelerated digestion method for proteins. The remaining solution will contain lipids and metabolites extracted based on Coman, Alshehry and Konig teachings.
Last, regarding performing steps a) through d) within three hours or less, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention that the total extraction of biomolecules can be performed within 3 hours or less based on combination of Coman, Hughes, Alshehry, Konig, Switzar and Halvis teachings. One would have been motivated to expect that because: Coman teaches incubation of the extraction solution with the sample for 1 hour at 4°C (p. 1454, right column, last paragraph); Hughes teaches incubation of beads with the sample for protein binding for 5 min (p. 74, 2nd paragraph) and Switzar and Halvis describes digestion of proteins with modified trypsin for 30-min (p. 1302, left column, 1st paragraph). Therefore, the total extraction of biomolecules can be performed within 3 hours or less.
Thus, Coman, Hughes, Alshehry, Konig and Switzar teachings as evidenced by Halvis render claim 18 obvious.
Conclusion
No claims are allowed.
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/L.G.K./Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653
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