The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
This office action is in response to Applicants’ amendments/remarks received January 28, 2026.
Rejections and/or objections not reiterated from previous office actions are hereby withdrawn.
Claims 1-35 are canceled. Claims 36-44 are under consideration.
Priority: This application claims benefit of provisional application 62/901022, filed September 16, 2019.
Objections and Rejections
Claims 36, 38, 43 are objected to because of the following informalities: in claim 36, line 13, the plural “booster wells” should be amended to the singular “booster well”; in claim 38, the term “pre-fractionate” should be amended to “pre-fractionating”; in claim 43, the term “LC” should be spelled out in full the first time that it is recited in the claim. Appropriate correction is required.
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 36-44 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 36, line 6 recites hold liquid volume > “uL”. There is no number recited with the “uL” unit. Therefore, it is not clear what the volume is. Further clarification and/or correction is requested.
Claim 42 recites trademark/trade names, including Triton X-100, Tween-20, Tween-80. In the present case, the trademark/trade name has not been properly presented or recited in proper form. Further correction is requested. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982).
Claims 37-41, 43-44 are included in this rejection because they are dependent on the above claim(s) and fail to cure its defects.
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 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 36, 38, 43-44 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Dou et al. (2019 Anal Chem 91: 13119-13127, published September 11, 2019; previously cited). Dou et al. teach a method of performing proteomic analysis comprising combining the nanoPOTS (nanodroplet processing in one-pot for trace samples) approach with TMT (tandem mass tag) isobaric labeling (p. 13119-13120). Dou et al. teach the nanoPOTS approach comprises single cell proteomic sample processing and analysis using ultrasensitive nanoLC-MS/MS (p. 13119-13120). Dou et al. teach preparing proteomic samples in nanodroplets comprising placing a cell sample into a nanowell disposed within a chip; adding or loading a peptide mixture into a boosting channel or well; performing cell lysis, protein extraction, disulfide reduction, including digesting the protein in the nanowell; labeling the peptides in the nanowell and the peptide mixture in the boosting well using TMT labels; combining the TMT labeled peptides from the nanowells with the labeled boosting peptides in the booster well; separating the mixed sample; and acquiring data from the separated sample by nanoflow LC/MS-MS analysis for single cell samples (at least p. 13120-13121, also Fig. 1). Dou et al. teach the nanowells have diameters of 1.2 mm and the booster wells have diameters of 1.8 mm (at least p. 13122, also Fig. 1). Therefore, Dou et al. can be deemed to anticipate instant claim 36.
Regarding instant claim 38, Dou et al. teach fractionating the mixed sample by nanoflow LC.
Regarding instant claim 43, Dou et al. teach performing one round of nanowell wash to increase recovery and the wash solution was combined into the booster well prior to LC separation (at least p. 13120).
Regarding instant claim 44, Dou et al. teach alkylation of sulfhydryl groups during protein extraction (at least p. 13120).
Reply: Applicants’ remarks have been considered but they are not persuasive.
Applicants filed a request to correct inventorship by adding inventor Maowei Dou on January 12, 2026. The request to correct inventorship was accepted January 15, 2026.
However, the Dou et al. reference still names additional authors in addition to the instant inventors and it is not readily apparent from the publication that it is an inventor-originated disclosure. The instant application claims benefit of provisional application 62/901022, filed September 16, 2019. The Dou et al. reference has a publication date of September 11, 2019. Therefore, the Dou et al. reference qualifies as prior art under 35 U.S.C. 102(a)(1) since it is a printed publication available to the public before the effective filing date of the claimed invention.
MPEP 2153.01(a) indicates that if the application names fewer joint inventors than a publication (e.g., the application names as joint inventors A and B, and the publication names as authors A, B and C), it would not be readily apparent from the publication that it is an inventor-originated disclosure and the publication would be treated as prior art under AIA 35 U.S.C. 102(a)(1) unless there is evidence of record that an exception under AIA 35 U.S.C. 102(b)(1) applies.
The Office has provided a mechanism for filing an affidavit or declaration (under 37 CFR 1.130) to establish that a disclosure is not prior art under AIA 35 U.S.C. 102(a) due to an exception in AIA 35 U.S.C. 102(b). See MPEP § 717. In the situations in which it is not apparent from the grace period disclosure itself or the patent application specification that the disclosure is an inventor-originated disclosure, the applicant may establish that the AIA 35 U.S.C. 102(b)(1)(A) exception applies by way of an affidavit or declaration under 37 CFR 1.130(a). MPEP § 2155.01 discusses the use of affidavits or declarations to show that a disclosure was an inventor-originated disclosure made during the grace period.
In this instance, Applicants still need to provide an affidavit or declaration under 37 C.F.R. 1.130 that is sufficient to overcome the Dou et al. reference.
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.
Claims 36-44 are rejected under 35 U.S.C. 103 as being unpatentable over Dou et al. (2019 Anal Chem 91: 13119-13127, published September 11, 2019; previously cited). The teachings of Dou et al. over at least instant claims 36, 38, 43-44 are noted above.
Regarding instant claim 39, Dou et al. disclose fractionating the mixed sample by nanoflow LC in buffer (at least p. 13121). “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). MPEP 2144.05. In this instance, it would have been obvious to determine optimum pH ranges, including a high pH, for the conditions used to separate the peptides by nanoflow LC.
Regarding instant claims 40-42, Dou et al. disclose MS-based proteomic analyses are capable of quantifying thousands of proteins in an unbiased manner (p. 13119). Dou et al. disclose efforts to improve sample recovery include the use of low-binding tubes and MS-compatible surfactants (p. 13119). Dou et al. has already disclosed the surfactant DDM (p. 13120). Dou et al. disclose the separated fractions are collected into containers with buffer solutions (at least p. 13119, also Fig. 1). Therefore, it would have been obvious that the fraction is collected at low volume in a dilution buffer (instant claim 40), where the container is of a material that is low-binding, including common lab container materials such as polypropylene and polyethylene (instant claim 41), and the buffer comprises a MS-compatible surfactant such as n-dodecyl β-D-maltoside (instant claim 42).
Regarding instant claim 37, Dou et al. disclose setting an AGC target of 1 x 106, injection times including 246 ms, and adjusting the AGC levels and injection times (at least p. 13121). Dou et al. also disclose the single cell quantification accuracy can be improved by incorporating real time search MS3 (p. 13126). “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). MPEP 2144.05. In this instance, it would be obvious to arrive at the recited automatic gain control levels for M3 analysis of > 5 x 105 and injection times of > 250 ms by routine experimentation and adjust the automatic gain control levels and injection times based also by routine experimentation and/or optimization.
Reply: Applicants’ amendments/remarks have been considered but they are not persuasive. The reasons for maintaining Dou et al. are the same as noted above.
Claims 36-39, 43-44 are rejected under 35 U.S.C. 103 as being unpatentable over Zhu et al. (2018 Angew Chem 130: 12550-12554; IDS 06.07.22, previously cited) in view of McAlister et al. (2012 Anal Chem 84: 7469-7478; previously cited) and Yi et al. (2019 Anal Chem 91: 5794-5801; IDS 06.07.22, previously cited). Zhu et al. disclose proteomic analysis of single mammalian cells enabled by microfluidic nanodroplet sample preparation and ultrasensitive nanoLC-MS (p. 12550). Zhu et al. disclose NanoPOTS chips containing an array of nanowells with diameters of 1 mm are loaded with reagents and single cell samples (p. 12553). Zhu et al. disclose cells are lysed and proteins are extracted with DDM and DDT; proteins are alkylated with IAA and digested with Lys-C and Trypsin (p. 12553). Zhu et al. disclose the digested samples containing peptides are cleaned with a SPE column and separated on a nanoLC column with a buffer gradient where all data are acquired by a mass spectrometer (p. 12553). Zhu et al. disclose that to enable large-scale study of single cells, the analysis throughput can be increased by using sample multiplexing based on isobaric labeling, citing reference 29 to McAlister et al. (p. 12553). Zhu et al. do not explicitly teach a booster well and/or labeling the peptides with TMT labels.
McAlister et al. disclose isobaric chemical tags TMT provides an avenue for mass spectrometry-based proteome quantitation experiments (at least p. 7469). McAlister et al. disclose a peptide mixture obtained after cell lysis, protein extraction, and digestion, are loaded into wells and labeled with TMT reagents (at least p. 7470-7471). The peptide mixture labeled with TMT reagent are separated and analyzed by LC-MS/MS (at least p. 7471).
Yi et al. disclose that it is appealing to take advantage of the multiplexing nature of isobaric labeling approaches (e.g., TMT) to achieve higher sensitivity and proteome coverage with limited individual amounts (at least p. 5799). Yi et al. disclose the small quantity of study samples and the much larger amount of boosting sample are individual labeled with TMT tags (at least p. 5795, also Fig. 1). Yi et al. disclose protein extraction and digestion of cells suspended in lysis buffer, where after digestion the digested peptides are placed into new tubes and mixed with TMT reagents (at least p. 5796). Yi et al. disclose mixing all the peptides labeled with different TMT tags into the same tube, including labeled BSA peptides, fractionating the peptides, and then subjecting the peptides to LC-MS/MS analysis (at least p. 5796-5797). Yi et al. disclose an advantageous utilization of this concept is to use samples, with similar proteome contents to the other study samples, in a much larger quantity as a “boosting” of the sample in one of the TMT channels; so that the proteins contributing to the other lower-input study samples will have a better chance of being identified and thus quantified (at least p. 5799). For instance, Yi et al. disclose Budnik et al. used a carrier TMT channel with much higher protein input than the single-cell samples in the other channels to increase the number of detectable proteins from the single-cell samples (p. 5799).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention combine the references and arrive at the claimed method of performing proteomic analysis comprising the steps of: placing a single cell sample into a nanowell disposed within a chip, the nanowell having a diameter < 2 mm; placing a peptide mixture into another well having a diameter < 2 mm but greater than the nanowell diameter; lysing the cell sample; extracting proteins from the cell sample; digesting the proteins into peptides; labeling the peptides and the peptide mixture using TMT labels; mixing the labeled peptides from the wells together; separating the mixed sample; and acquiring data from the separated sample using a mass spectrometer (instant claim 36). The motivation to do so is given by the prior art. Zhu et al. disclose a method of performing proteomic analysis comprising single-cell samples by the NanoPOTS approach. Zhu et al. differ from the claimed method by not explicitly teaching a booster well and/or labeling the peptides with TMT labels. However, Zhu et al. disclose that to enable large-scale study of single cells, the analysis throughput can be increased by using sample multiplexing based on isobaric labeling with TMT reagents (citing McAlister et al.). Yi et al. disclose a “boosting” sample containing much larger amounts of peptide mass labeled with TMT achieves higher sensitivity and proteome coverage with mass-limited samples. Therefore, one of ordinary skill would have reasonable motivation to incorporate the TMT isobaric labeling method of Yi et al. with the nanoPOTS approach of Zhu et al. to arrive at the claimed method of performing proteomic analysis. One of ordinary skill would have a reasonable expectation of success because the prior art disclose combining a carrier TMT channel having a higher protein input with digested peptides from single-cell samples to increase of identifying and/or quantifying the lower-input study sample from the single-cell sample and methods for performing proteomic analysis of single cell samples by the nanoPOTS approach and TMT isobaric labeling were known.
Regarding the instant limitation that the diameter of the nanowell is less than the diameter of the “booster well”, as noted above, Yi et al. disclose the “boosting” sample containing much larger amounts of peptide mass labeled with TMT achieves higher sensitivity and proteome coverage with mass-limited samples (p. 5799). Yi et al. further disclose sample loss after TMT labeling can be minimized by the presence of a large amount of sample from the boosting sample (p. 5800). Therefore, it would be obvious that the “booster” well comprising the “boosting” sample has a larger diameter or is larger than the nanowell because the prior art suggests utilizing a large amount of sample for the boosting sample.
Regarding instant claim 37, Yi et al. disclose the boosting to amplify signal with isobaric labeling strategy is compatible with M3 analysis (p. 5800). McAlister et al. disclose analysis with a MS1 spectrum having an AGC 1 x 106 and an injection time 150 ms (p. 7471). McAlister et al. also disclose M2/M3 analysis where M2 analysis has an AGC 2 x 103, injection time 100 ms and M3 analysis having an AGC 1.5 x 105 and injection time 250 ms (p. 7471). “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). MPEP 2144.05. In this instance, it would be obvious to arrive at the recited automatic gain control levels for M3 analysis of > 5 x 105 and injection times of > 250 ms by routine experimentation and adjust the automatic gain control levels and injection times based also by routine experimentation and/or optimization.
Regarding instant claims 38-39, Yi et al. disclose the labeled mixture comprising the digested peptide and peptide mixture is separated or fractionated before LC-MS/MS analysis (p. 5795-5796). Zhu et al. disclose separation on a nanoLC column in buffer before LC-MS/MS analysis (p. 12553). “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). MPEP 2144.05. In this instance, it would have been obvious to determine optimum pH ranges, including a high pH, for the conditions used to separate the peptides by nanoflow LC. Therefore, it would be obvious that the mixed sample comprising labeled peptides from the single cell sample and the labeled peptide mixture can be separated on a nanoflow LC column with buffer as suggested by Zhu et al., where it would have been obvious to determine optimum pH ranges, including a high pH, for the conditions used to separate the peptides by nanoflow LC.
Regarding instant claim 43, Zhu et al. disclose the digested samples containing peptides are cleaned with a SPE column and separated on a nanoLC column with a buffer gradient where all data are acquired by a mass spectrometer (p. 12553). Therefore, it would be obvious that the mixed sample comprising labeled peptides from the single cell sample can be washed as suggested by Zhu et al. prior to combining with the labeled peptide mixture in the boosting well as suggested by Yi et al. and then separated on a nanoflow LC column with buffer as suggested by Zhu et al.
Regarding instant claim 44, Zhu et al. disclose the proteins in the sample are alkylated during protein extraction (p. 12553).
Reply: Applicants’ remarks have been considered but they are not persuasive. The reasons for maintaining Zhu et al. are the same as previously noted and are incorporated herein.
See also the reasons noted on at least p. 10-11 of the June 25, 2024 non-final office action.
Claims 36-44 are rejected under 35 U.S.C. 103 as being unpatentable over Zhu et al. (2018 Angew Chem 130: 12550-12554; IDS 06.07.22, previously cited) in view of McAlister et al. (2012 Anal Chem 84: 7469-7478; previously cited), Yi et al. (2019 Anal Chem 91: 5794-5801; IDS 06.07.22, previously cited), and Kelly et al. (WO 2018085835; IDS 06.07.22, previously cited). The teachings of Zhu et al. in view of McAlister et al. and Yi et al. over at least instant claims 36-39, 43-44 are noted above.
Regarding instant claims 40-42, Kelly et al. disclose preparation of single-cell samples in protein low-binding vials (at least paragraph 000131). Kelly et al. also disclose efforts to improve sample recovery include use of low-binding sample tubes and MS-friendly surfactants (at least paragraph 00060). Zhu et al. has already disclosed the surfactant DDM (p. 12553). Kelly et al. reasonably disclose the separated fractions are collected into containers with buffer solutions (Kelly et al. at least p. 33-34). Therefore, it would have been obvious that the fraction is collected at low volume in a dilution buffer (instant claim 40), where the container is of a material that is low-binding, including common lab container materials such as polypropylene and polyethylene (instant claim 41), and the buffer comprises a MS-compatible surfactant such as n-dodecyl β-D-maltoside (instant claim 42).
Reply: Applicants’ remarks are not persuasive. The reasons for maintaining Zhu et al. are the same as noted above.
See also the reasons noted on at least p. 12 of the June 25, 2024 non-final office action.
No claim is allowed.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any extension fee pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/Marsha Tsay/Primary Examiner, Art Unit 1656
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