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 .
Response to Amendment
The Amendment filed 10/22/2025 has been entered. Claims 1-4, 7-8, 18, 27-28, 30-32, 34, 37-38, 42, 48, 50, 55, and 57 are pending. Claims 27-28, 30-32, 34, 37-38, 42, 48, 50, 55, and 57 are previously withdrawn. Claims 1-4, 7-8, and 18 are examined herein.
Status of Objections and Rejections
All objections and rejections from the previous office action are withdrawn in view of Applicant's amendment.
New grounds of claim objections are necessitated by the amendments.
New grounds of rejection under 35 U.S.C. 103 are necessitated by the amendments.
Response to Arguments
Applicant' s arguments, see pages 11-14, filed 10/22/25, with respect to the rejection of claims 1-4, 7-8 and 18 under 35 USC 101 have been fully considered and are persuasive. The rejection of claims 1-4, 7-8 and 18 under 35 USC 101 has been withdrawn.
Applicant's arguments, see pages 14-18, filed 10/22/2025, with respect to the rejections of claims 1-4, 7-8, and 18 under 35 U.S.C. 103, have been fully considered but they are not persuasive.
Applicant argues (pp. 17-18) that Holden teaches the use of EID to maintain labile post-translational modifications (PTMs), whereas the present invention relies on cleaving the TMPP reporter ion from the polypeptide for detection. According to Applicant, Holden’s goal (preserving modifications) is contrary to the claimed method, which depends on releasing a reporter ion. Therefore, Applicant contends that one of ordinary skill in the art would not have been motivated to modify Suh using Holden’s ionization method because it would run counter to the alleged inventive principle. Applicant further argues (pp. 17-18) that the claimed method does more than simply detect a TMPP-labeled peptide, it uses the released TMPP reporter ion to trigger additional fragmentation in a focused manner. In contrast, Suh allegedly fragments the most abundant ions indiscriminately. Applicant asserts that using reporter-ion-triggered fragmentation improves sensitivity and resolution, and therefore the combination of Suh and Holden would not have been obvious.
The Examiner respectfully disagrees.
Applicant’s reference to “EID” appears to be a typographical error, as the claims are directed to ETD, and the arguments have been considered as referring to ETD accordingly. Holden’s disclosure of ETD for maintaining labile PTMs does not teach away from fragmentation or reporter ion generation; rather, it teaches fragmentation techniques that can be used to obtain structural information while preserving useful modifications. The fact that Holden discusses maintaining PTMs does not preclude the generation of detection of fragment ions, including reporter ions. A reference does not teach away merely because it highlights one advantage of a technique (See MPEP 2145(X)(D)(1)). ETD remains a known fragmentation method capable of producing diagnostic ions. Further, the combination of Suh and Holden does not require adopting Holden’s technique for the same stated purpose as the instant application. The proper inquiry is whether a person of ordinary skill would have been motivated to use known fragmentation techniques (such as ETD) in the context of TMPP labeled peptide analysis to obtain improved structural information. Holden teaches hybrid fragmentation using ETD methods to enhance peptide sequence characterization impacted from the location of a specific amino acid (page 37, ll. 1-4). Holden further explains that the use of an ETD has the ability to generate radical precursors that can be used alongside other fragmentation methods for the added benefit of “sequencing, localization of modifications, etc.” (page 2, line 10). Applying such known fragmentation methods to the TMPP-labeled peptides of Suh would have been a predictable use of prior art elements according to their established functions, yielding the predictable result of improved ion separation and sequence identification (See MPEP 2143(I)(A)). With respect to triggering additional fragmentation based on detection of a reporter ion, Suh teaches that “The TMPP labeling method allows peptide detection and subsequence tandem MS analysis of peptides” (p. 1403, col. 2, para. 2, ll. 1-2) and that “MS data were queried for the characteristic TMPP-associated mass shifts of 572 Da” (p. 1400, col. 1, para. 1, ll. 6-7) with database searches modified to account for “N-terminal modification with the TMPP tag” (p. 1396, col. 1, para. 3, ll. 11-12. Thus Suh identifies TMPP-modified peptides and subjects selected precursor ions to MS/MS analysis for sequencing and localization, as shown in Table 3 and Fig. 5. The difference between selecting precursor ions based on intensity and selecting them based on detection of a diagnostic reporter ion is merely a variation in known MS acquisition parameters, which were routinely adjustable in tandem mass spectrometry, and would have represented a predictable optimization rather than a patentable distinction (See MPEP 2144.05).
Accordingly, for the reasons set forth above, claims 1-4, 7-8, and 18 remain rejected under 35 U.S.C. 103.
Claim Objections
Claim 2 is objected to because of the following informalities: line 2 recites “of claim 48,” however, claim 48 is withdrawn. Applicant may correct this by substituting “at least one reporter ion” in line 2 with “at least one TMPP reporter ion”. Consequently, lines 11-12 should read “the TMPP reporter ion”.
Claim 3 is objected to because of the following informalities: lines 4-5 recite “of claim 48,” however, claim 48 is withdrawn. Applicant may correct this by substituting “at least one reporter ion” in line 4 with “at least one TMPP reporter ion”. Consequently, lines 12-13 should read “the TMPP reporter ion”.
Claim 18 is objected to because of the following informalities: lines 4-5 recite “of claim 48,” however, claim 48 is withdrawn. Applicant may correct this by substituting “at least one reporter ion” in line 4 with “at least one TMPP reporter ion”. Consequently, line 13 should read “the TMPP reporter ion”.
Appropriate correction is required.
Claim Rejections - 35 USC § 103
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 1-4,7-8,18 are rejected under 35 U.S.C. 103 as being unpatentable over Suh et al. (“Using chemical derivatization and mass spectrometric analysis to characterize the post-translationally modified Staphylococcus aureus surface protein G,”; 2010) in view of Holden (“Hybrid Activation for Intact Protein and Peptide Characterization,”; 2015).
Regarding claim 1, Suh teaches a method of characterizing a N-tris(2,4,6- trimethoxyphenyl)phosphonium acetyl (TMPP) labeled peptide in a sample (Comparing TMPP-derivatized SasG and underivatized SasG as a control, peptide analysis of in-gel digests with Trypsin and Glu-C was carried out, and MS data were queried for the characteristic TMPP-associated mass shifts of 572 Da; page 1400, column 1, lines 4-7; See results in Table 3 and Fig. 5)(Under broadest reasonable interpretation the examiner understands ‘characterizing’ to be any new information gathered as a result of analyzing the first mass spectrum and the second mass spectrum for each of the one or more TMPP labeled peptides, wherein the sequences and clipping sites of Table 3 and Fig. 5 satisfy this limitation), comprising:
(i) subjecting the sample to a first stage of tandem mass spectrometry (Fig. 5(b) shows “Comparison of spectra for the SasG peptide 124EVQPK128 from MALDI-TOF MS experiments with and without TMPP labeling,” wherein MALDI-TOF is defined as tandem mass spectrometry since “MALDI target plates were applied to analysis using a MALDI-TOF/TOF mass spectrometer,” which produces “MS/MS fragmentation”; Fig. 5; page 1396, paragraph 1, lines 1-2, 5-6)(“The TMPP labeling method allows peptide detection and subsequence tandem MS analysis of peptides; page 1403, column 2, paragraph 2, lines 1-5) to obtain a first mass spectrum of the TMPP labeled peptide (The first mass spectrum of the TMPP labeled peptide contains “The 10 most intensive ions from the MS scan,” and the second mass spectrum is a result of when these ions “were selected for MS/MS fragmentation,” as shown in Fig. 5(B), wherein “Spectra represented averaged signals of 1000 laser shots for MS analysis and 3000 shots for MS/MS analysis”; page 1396, paragraph 1, lines 5-7)(Since the second fragmented spectrum of Fig. 5(B) shows a positive identification of TMPP, then the first mass spectrum naturally contained ions of the TMPP labeled peptide).
(iii) subjecting the identified TMPP labeled peptide to a second stage of mass spectrometry (The 10 most intensive ions from the MS scan were selected for MS/MS fragmentation; page 1396, paragraph 1, lines 5-6) to thereby generate a second mass spectrum of the TMPP labeled peptide (“Fig. 5…(B) the same m/z segment for trypsin-digested TMPP-derivatized SasG,” wherein “Spectra represented averaged signals of 3000 shots for MS/MS analysis”; Fig. 5; page 1396, paragraph 1, lines 6-7); and
(iv) characterizing the TMPP labeled peptide by analyzing the first mass spectrum and the second mass spectrum to obtain the amino acid sequence of the TMPP labeled peptide (MS and MS/MS data obtained from the MALDI and LC-nESI-MS/MS experiments were searched against the latest releases of the Swiss-Prot and non-redundant NCBI protein databases…to…display the identified peptides with their ranks, the MS/MS scores and a protein sequence coverage summary… For the identification of N-terminal peptides… accounting for N-terminal modification with the TMPP tag. The mass of 572.18 Da was considered as a variable modification; page 1396, paragraph 3) (“As shown in Table 3, additional N-terminal peptides indicative of post-translational protein processing steps were identified, and the characteristic 572 Da mass shift in TMPP-modified peptides was observed,” which lists amino acid position (clipping site) and sequence; page 1402, column 2, lines 1-4).
Suh is silent to teaching that the first mass spectrum is generated by electron transfer dissociation (ETD) and (ii) identifying the TMPP labeled peptide by detecting or separating a TMPP reporter ion in the first mass spectrum of the TMPP labeled peptide, wherein the TMPP reporter ion has a nominal mass-to-charge (m/z) of about 533, about 590, or about 573.
Suh does teach the use of MALDI-TOF MS/MS and LC-nESI-MS/MS when creating first and second mass spectra, one spectra on MS and the other on MS/MS (Fig. 5 and Table 3). Suh also discloses identifying TMPP-derivatized peptides by observing a characteristic mass shift of approximately 572 Da corresponding to incorporation of the TMPP tag (p. 1400, col. 1, ll. 4-7). This disclosure pertains to the increased mass of the modified peptide rather than to a detached reporter ion having a specific nominal m/z value. Suh does not describe generation or detection of a free TMPP+ reporter ion at m/z 573, nor does it disclose reporter ions at m/z ratios at about 533 or about 590. Accordingly, the specific nominal m/z values recited are not expressly taught by Suh alone.
Holden teaches the first mass spectrum is generated by electron transfer dissociation (ETD). (We report a hybrid fragmentation method involving electron transfer dissociation (ETD); page 1, 1.1, line 2; Title; page 4, lines 13-14; See spectrum of Fig. 1.1), and (ii) identifying the TMPP labeled peptide by detecting or separating a TMPP reporter ion in a mass spectrum of the TMPP labeled peptide, wherein the TMPP reporter ion has a nominal mass-to-charge (m/z) of about 533, about 590, or about 573 (The even electron fixed-charge peptide dissociated predominantly by the loss of the charged TMPP tag (resulting in the TMPP+ ion of m/z 573); p. 55, ll. 4-6; (Figure 2.10A)).
Holden is considered to be analogous to the claimed invention because it is in the same field of endeavor for peptide characterization of TMPP-tagged peptides using mass spectrometry (Title). It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Suh to incorporate the teachings of Holden by using ETD fragmentation to obtain the first spectrum and its resulting TMPP+ reporter ion at m/z 573 in order to improve amino acid sequence characterization and cleavage-site localization. Holden explains that the use of an ETD has the ability to generate radical precursors that can be used alongside other fragmentation methods for the added benefit of “sequencing, localization of modifications, etc.” (page 2, line 10). In regards to step (ii), Suh already teaches that TMPP derivatization produces a characteristic mass shift of approximately 572 Da that is specifically queried during MS analysis (p. 1400, col. 1, ll. 4-7). Suh thus establishes that the TMPP tag has a known and predictable mass contribution and serves as a diagnostic feature for identifying labeled peptides. Holden further teaches that fragmentation of TMPP-labeled peptides results in loss of the charged TMPP tag, producing the TMPP+ ion at m/z 573. Given Suh’s recognition of the TMPP tag as a characteristic mass marker and Holden’s express disclosure that fragmentation yields the corresponding charged TMPP+ ion at nominal m/z 573, a person of ordinary skill in the art would have found it predictable and straightforward to detect that diagnostic TMPP+ fragment during tandem MS analysis. Incorporating Holden’s explicit reporter-ion teaching into Suh’s established TMPP mass-shift workflow would have represented the routine use of a known fragmentation product corresponding to a known modification mass, yielding no more than the predictable identification of the same chemical moiety in fragment form (See MPEP 2143(I)(A)).
Regarding claim 2, Suh teaches a method of characterizing a polypeptide (Comparing TMPP-derivatized SasG and underivatized SasG as a control, peptide analysis of in-gel digests with Trypsin and Glu-C was carried out, and MS data were queried for the characteristic TMPP-associated mass shifts of 572 Da; page 1400, column 1, lines 4-7)(Under broadest reasonable interpretation the examiner understands ‘characterizing’ to be any new information gathered as a result of analyzing the first mass spectrum and the second mass spectrum for each of the one or more TMPP labeled peptides, wherein the sequences and clipping sites of Table 3 and Fig. 5 satisfy this limitation), comprising:
(i) labeling the polypeptide with at least one reporter ion of claim 48 [sic], (“The TMPP labeling method permits the discernment of a peptide with a natural N-terminus from an equivalent peptide derived from protease cleavage during MS sample preparation,” wherein the reporter ion of claim 48 is TMPP; page 1400, column 2, paragraph 2, lines 1-3), to obtain a TMPP labeled polypeptide (obtaining one or more TMPP labeled clipped polypeptides is the intended result of labeling the protease cleaved peptide or clipped polypeptide with TMPP)(the italicized clause in the method claim is not given weight when it simply expresses the intended result of a process step positively recited; MPEP 2111.04);
(ii) digesting the TMPP labeled polypeptide (Fig. 6…(C) The corresponding peptide from trypsin-digested TMPP-labeled SasG; page 1402, caption for Fig. 6) to generate a mixture comprising one or more unlabeled peptides and one or more TMPP labeled peptides (The TMPP labeling method allows peptide detection and subsequence tandem MS analysis of peptides…from complex mixture after proteolytic digestion; page 1403, column 2, paragraph 2, lines 1-5; see labeled and unlabeled peptides of Fig. 6);
(iii) subjecting the mixture to liquid chromatography (LC) to generate elutes of the LC (“LC-nESI-MS/MS: LC-nESI-MS/MS analysis was performed…to elute peptides off the trap,” wherein Fig. 6 shows “LC-nESI-MS/MS spectra for the N-terminal peptide 89SEVTSNK∼”; page 1396, paragraph 2, lines 1-7; Fig. 6);
(iv) subjecting the elutes to a first stage of tandem mass spectrometry (Fig. 6…LC-nESI-MS/MS spectra for the N-terminal peptide 89SEVTSNK∼…(C) The corresponding peptide from trypsin-digested TMPP-labeled SasG; page 1402; Fig. 5)(“The TMPP labeling method allows peptide detection and subsequence tandem MS analysis of peptides; page 1403, column 2, paragraph 2, lines 1-5) to obtain a first mass spectrum of each of the one or more TMPP labeled peptides (The first mass spectrum of the TMPP labeled peptide contains “the top five parent ions,” and the second mass spectrum is a result of when these ions were “selected for fragmentation,” as shown in Fig. 6(C), wherein “Spectra were acquired in automated MS/MS mode, with the top five parent ions above a threshold of 10,000 selected for fragmentation.”; page 1396, paragraph 2, lines 9-12)(Since the second fragmented spectrum of Fig. 6(C) shows a positive identification of TMPP, then the first mass spectrum naturally contained ions of the TMPP labeled peptide).;
(vi) subjecting the identified one or more TMPP labeled peptides to a second mass spectrometry to thereby generate a second mass spectrum of the each of the one or more TMPP labeled peptides (Spectra were acquired in automated MS/MS mode, with the top five parent ions above a threshold of 10,000 selected for fragmentation ; page 1396 paragraph 2, last 4 lines)( Fig. 6…LC-nESI-MS/MS spectra for the N-terminal peptide 89SEVTSNK∼…(C) The corresponding peptide from trypsin-digested TMPP-labeled SasG; page 1402; Fig. 6); and
(vii) characterizing the polypeptide by analyzing the first mass spectrum and the second mass spectrum for each of the one or more TMPP labeled peptides (MS and MS/MS data obtained from the MALDI and LC-nESI-MS/MS experiments were searched against the latest releases of the Swiss-Prot and non-redundant NCBI protein databases…to…display the identified peptides with their ranks, the MS/MS scores and a protein sequence coverage summary… For the identification of N-terminal peptides… accounting for N-terminal modification with the TMPP tag. The mass of 572.18 Da was considered as a variable modification; page 1396, paragraph 3)(Under broadest reasonable interpretation the examiner understands ‘characterizing’ to be any new information gathered as a result of analyzing the first mass spectrum and the second mass spectrum for each of the one or more TMPP labeled peptides) to obtain the amino acid sequence of the polypeptide.
Suh is silent to teaching that the first mass spectrum is generated by electron transfer dissociation (ETD) and (v) identifying the one or more TMPP labeled peptide by detecting or separating a TMPP reporter ion in the first mass spectrum of each of the one or more TMPP labeled peptide, wherein the TMPP reporter ion has a nominal mass-to-charge (m/z) of about 533, about 590, or about 573.
Suh does teach the use of MALDI-TOF MS/MS and LC-nESI-MS/MS when creating first and second mass spectra, one spectra on MS and the other on MS/MS (Fig. 5 and Table 3). Suh also discloses identifying TMPP-derivatized peptides by observing a characteristic mass shift of approximately 572 Da corresponding to incorporation of the TMPP tag (p. 1400, col. 1, ll. 4-7). This disclosure pertains to the increased mass of the modified peptide rather than to a detached reporter ion having a specific nominal m/z value. Suh does not describe generation or detection of a free TMPP+ reporter ion at m/z 573, nor does it disclose reporter ions at m/z ratios at about 533 or about 590. Accordingly, the specific nominal m/z values recited are not expressly taught by Suh alone.
Holden teaches the first mass spectrum is generated by electron transfer dissociation (ETD). (We report a hybrid fragmentation method involving electron transfer dissociation (ETD); page 1, 1.1, line 2; Title; page 4, lines 13-14; See spectrum of Fig. 1.1), and (ii) identifying the one or more TMPP labeled peptide by detecting or separating a TMPP reporter ion in a mass spectrum of each of the one or more TMPP labeled peptide, wherein the TMPP reporter ion has a nominal mass-to-charge (m/z) of about 533, about 590, or about 573 (The even electron fixed-charge peptide dissociated predominantly by the loss of the charged TMPP tag (resulting in the TMPP+ ion of m/z 573); p. 55, ll. 4-6; (Figure 2.10A)).
Holden is considered to be analogous to the claimed invention because it is in the same field of endeavor for peptide characterization of TMPP-tagged peptides using mass spectrometry (Title). It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Suh to incorporate the teachings of Holden by using ETD fragmentation to obtain the first spectrum and its resulting TMPP+ reporter ion at m/z 573 in order to improve amino acid sequence characterization and cleavage-site localization. Holden explains that the use of an ETD has the ability to generate radical precursors that can be used alongside other fragmentation methods for the added benefit of “sequencing, localization of modifications, etc.” (page 2, line 10). In regards to step (ii), Suh already teaches that TMPP derivatization produces a characteristic mass shift of approximately 572 Da that is specifically queried during MS analysis (p. 1400, col. 1, ll. 4-7). Suh thus establishes that the TMPP tag has a known and predictable mass contribution and serves as a diagnostic feature for identifying labeled peptides. Holden further teaches that fragmentation of TMPP-labeled peptides results in loss of the charged TMPP tag, producing the TMPP+ ion at m/z 573. Given Suh’s recognition of the TMPP tag as a characteristic mass marker and Holden’s express disclosure that fragmentation yields the corresponding charged TMPP+ ion at nominal m/z 573, a person of ordinary skill in the art would have found it predictable and straightforward to detect that diagnostic TMPP+ fragment during tandem MS analysis. Incorporating Holden’s explicit reporter-ion teaching into Suh’s established TMPP mass-shift workflow would have represented the routine use of a known fragmentation product corresponding to a known modification mass, yielding no more than the predictable identification of the same chemical moiety in fragment form (See MPEP 2143(I)(A)).
Regarding claim 3, Suh teaches a method of identifying a clipping site on a protein (“The Staphylococcus aureus surface protein G (SasG)...The protein was also derivatized with...TMPPAc-OSu) to assess the presence of additional N-terminal sites of mature SasG,” wherein, “TMPP derivatization was employed to elucidate whether this protein is proteolytically clipped beyond its in silico predicted N-terminus. Key to the identification of N-terminal peptides is a comparison of mass spectra prior to and after derivatization with the TMPP reagent”; Abstract; page 1395, column 1, paragraph 2, lines 8-12) comprising:
(i) obtaining a sample containing one or more clipped polypeptides of the protein (peptide derived from protease cleavage during MS sample preparation; page 1400, column 2, paragraph 2, lines 2-3);
(ii) labeling the one or more clipped polypeptides with at least one reporter ion of claim 48 [sic], (“The TMPP labeling method permits the discernment of a peptide with a natural N-terminus from an equivalent peptide derived from protease cleavage during MS sample preparation,” wherein the reporter ion of claim 48 is TMPP; page 1400, column 2, paragraph 2, lines 1-3), to obtain a TMPP labeled polypeptide (obtaining one or more TMPP labeled clipped polypeptides is the intended result of labeling the protease cleaved peptide or clipped polypeptide with TMPP)(the italicized clause in the method claim is not given weight when it simply expresses the intended result of a process step positively recited; MPEP 2111.04);
(iii) digesting the one or more TMPP labeled clipped polypeptides (Fig. 6…(C) The corresponding peptide from trypsin-digested TMPP-labeled SasG; page 1402, caption for Fig. 6) to generate a mixture comprising one or more unlabeled peptides and one or more TMPP labeled peptides (The TMPP labeling method allows peptide detection and subsequence tandem MS analysis of peptides…from complex mixture after proteolytic digestion; page 1403, column 2, paragraph 2, lines 1-5; see labeled and unlabeled peptides of Fig. 6);
(iv) subjecting the mixture to liquid chromatography (LC) to generate elutes of the LC (“LC-nESI-MS/MS: LC-nESI-MS/MS analysis was performed…to elute peptides off the trap,” wherein Fig. 6 shows “LC-nESI-MS/MS spectra for the N-terminal peptide 89SEVTSNK∼”; page 1396, paragraph 2, lines 1-7; Fig. 6);
(v) subjecting the elutes to a first stage of tandem mass spectrometry to obtain a first mass spectrum of each of the TMPP labeled peptides (Fig. 6…LC-nESI-MS/MS spectra for the N-terminal peptide 89SEVTSNK∼…(C) The corresponding peptide from trypsin-digested TMPP-labeled SasG; page 1402; Fig. 5)(“The TMPP labeling method allows peptide detection and subsequence tandem MS analysis of peptides; page 1403, column 2, paragraph 2, lines 1-5) to obtain a first mass spectrum of each of the one or more TMPP labeled peptides (The first mass spectrum of the TMPP labeled peptide contains “the top five parent ions,” and the second mass spectrum is a result of when these ions were “selected for fragmentation,” as shown in Fig. 6(C), wherein “Spectra were acquired in automated MS/MS mode, with the top five parent ions above a threshold of 10,000 selected for fragmentation.”; page 1396, paragraph 2, lines 9-12)(Since the second fragmented spectrum of Fig. 6(C) shows a positive identification of TMPP, then the first mass spectrum naturally contained ions of the TMPP labeled peptide);
(vii) subjecting each of the identified TMPP labeled peptides to a second mass spectrometry to thereby generate a second mass spectrum for each of the TMPP labeled peptides (Spectra were acquired in automated MS/MS mode, with the top five parent ions above a threshold of 10,000 selected for fragmentation ; page 1396 paragraph 2, last 4 lines)( Fig. 6…LC-nESI-MS/MS spectra for the N-terminal peptide 89SEVTSNK∼…(C) The corresponding peptide from trypsin-digested TMPP-labeled SasG; page 1402; Fig. 6);and
(viii) obtaining the sequence of the protein and identifying the clipping site on the protein by analyzing the first mass spectrum and the second mass spectrum for each of the TMPPI labeled peptides (The peptide TMPP-Ac-89SEVTSNK95 at m/z 668.96 (2+) was identified from tryptic digests of TMPP-labeled SasG by LCMS/MS (Fig. 6C); page 1402, column 1, lines 5-7)(MS and MS/MS data obtained from the MALDI and LC-nESI-MS/MS experiments were searched against the latest releases of the Swiss-Prot and non-redundant NCBI protein databases…to…display the identified peptides with their ranks, the MS/MS scores and a protein sequence coverage summary… For the identification of N-terminal peptides… accounting for N-terminal modification with the TMPP tag. The mass of 572.18 Da was considered as a variable modification; page 1396, paragraph 3)(Under broadest reasonable interpretation the examiner understands ‘characterizing’ to be any new information gathered as a result of analyzing the first mass spectrum and the second mass spectrum for each of the one or more TMPP labeled peptides).
Suh is silent to teaching that the first mass spectrum is generated by electron transfer dissociation (ETD) and (vi) identifying each of the TMPP labeled peptide by detecting or separating a TMPP reporter ion in the first mass spectrum for each of the TMPP labeled peptide, wherein the TMPP reporter ion has a nominal mass-to-charge (m/z) of about 533, about 590, or about 573.
Suh does teach the use of MALDI-TOF MS/MS and LC-nESI-MS/MS when creating first and second mass spectra, one spectra on MS and the other on MS/MS (Fig. 5 and Table 3). Suh also discloses identifying TMPP-derivatized peptides by observing a characteristic mass shift of approximately 572 Da corresponding to incorporation of the TMPP tag (p. 1400, col. 1, ll. 4-7). This disclosure pertains to the increased mass of the modified peptide rather than to a detached reporter ion having a specific nominal m/z value. Suh does not describe generation or detection of a free TMPP+ reporter ion at m/z 573, nor does it disclose reporter ions at m/z ratios at about 533 or about 590. Accordingly, the specific nominal m/z values recited are not expressly taught by Suh alone.
Holden teaches the first mass spectrum is generated by electron transfer dissociation (ETD). (We report a hybrid fragmentation method involving electron transfer dissociation (ETD); page 1, 1.1, line 2; Title; page 4, lines 13-14; See spectrum of Fig. 1.1), and (v) identifying each of the TMPP labeled peptide by detecting or separating a TMPP reporter ion in a mass spectrum for each of the TMPP labeled peptide, wherein the TMPP reporter ion has a nominal mass-to-charge (m/z) of about 533, about 590, or about 573 (The even electron fixed-charge peptide dissociated predominantly by the loss of the charged TMPP tag (resulting in the TMPP+ ion of m/z 573); p. 55, ll. 4-6; (Figure 2.10A)).
Holden is considered to be analogous to the claimed invention because it is in the same field of endeavor for peptide characterization of TMPP-tagged peptides using mass spectrometry (Title). It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Suh to incorporate the teachings of Holden by using ETD fragmentation to obtain the first spectrum and its resulting TMPP+ reporter ion at m/z 573 in order to improve amino acid sequence characterization and cleavage-site localization. Holden explains that the use of an ETD has the ability to generate radical precursors that can be used alongside other fragmentation methods for the added benefit of “sequencing, localization of modifications, etc.” (page 2, line 10). In regards to step (ii), Suh already teaches that TMPP derivatization produces a characteristic mass shift of approximately 572 Da that is specifically queried during MS analysis (p. 1400, col. 1, ll. 4-7). Suh thus establishes that the TMPP tag has a known and predictable mass contribution and serves as a diagnostic feature for identifying labeled peptides. Holden further teaches that fragmentation of TMPP-labeled peptides results in loss of the charged TMPP tag, producing the TMPP+ ion at m/z 573. Given Suh’s recognition of the TMPP tag as a characteristic mass marker and Holden’s express disclosure that fragmentation yields the corresponding charged TMPP+ ion at nominal m/z 573, a person of ordinary skill in the art would have found it predictable and straightforward to detect that diagnostic TMPP+ fragment during tandem MS analysis. Incorporating Holden’s explicit reporter-ion teaching into Suh’s established TMPP mass-shift workflow would have represented the routine use of a known fragmentation product corresponding to a known modification mass, yielding no more than the predictable identification of the same chemical moiety in fragment form (See MPEP 2143(I)(A)).
Regarding claim 4, Modified Suh teaches the method of claim 1, wherein the electron-induced dissociation is electron transfer dissociation (ETD) or electron capture dissociation (ECD) (We report a hybrid fragmentation method involving electron transfer dissociation (ETD); Holden, page 1, 1.1, line 2).
Regarding claim 7, Modified Suh teaches the method of claim 1, wherein the TMPP reporter ion triggers the second mass spectrometry (“The 10 most intensive ions from the MS scan were selected for MS/MS fragmentation,” and “Spectra were acquired in automated MS/MS mode, with the top five parent ions above a threshold of 10,000 selected for fragmentation,” wherein the parent ions are naturally formed in the first MS run before fragmentation and since the TMPP-Ac-124EVQPK128 reporter ion was detected in the second mass spectrometry run (Fig. 5), the parent ion of TMPP-Ac-124EVQPK128 triggered the second mass spectrometry; Suh, page 1396, paragraph 2, lines 9-11).
Regarding claim 8, Modified Suh teaches the method of claim 1.
Suh is silent to teaching that the second mass spectrometry comprises collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), or ultraviolet photodissociation (UVPD).
Suh does teach the use of MALDI-TOF MS/MS and LC-nESI-MS/MS when creating first and second spectra, one spectra on MS and the other on MS/MS (Figs. 5-6 and Table 3)
Holden teaches the second mass spectrometry comprises collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), or ultraviolet photodissociation (UVPD). (“We report a hybrid fragmentation method involving electron transfer dissociation (ETD) combined with ultraviolet photodissociation (UVPD),” for “peptide characterization,” wherein “ETD can precede or follow UVPD in the HCD cell or both activation processes can be undertaken simultaneously,” and therefore can obtain an ETD first mass spectrum and a second UVPD mass spectrum; page 1, 1.1, line 2; Title; page 4, lines 13-14; See spectrum of Fig. 1.1).
Holden is considered to be analogous to the claimed invention because it is in the same field of endeavor for TMPP-tagged peptides and analysis of cleavage sites using mass spectrometry. It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Suh to incorporate the teachings of Holden by using a hybridized ETD-UVPD method when evaluating spectra for a more “for accurate amino acid sequence characterization and localization of post-translational modifications” (Holden, page iv lines 2-3) . Holden explains that “ETHCD, was shown to provide an informative array of predominantly b-, c-, y-, and z type ions,” which results in an increase in information for the exact locations of cleavage sites (page 2, lines 17-18). However, “the most diverse array of fragment ions was obtained from the ETUVPD hybrid method, an outcome that proved particularly beneficial for specific localization of modifications for which fragmentation was suppressed for other activation methods” (Holden, page 3, lines 2-4). With a wider array of this ionic fragmentation information, analysis of cleavage or clipping sites using “hybrid fragmentation of intact proteins is feasible even for complex mixtures” (Holden, page 3, paragraph 2, lines 2-4). The use of ETD alone has the “ability to maintain labile post translational modifications (PTMs) while indiscriminately fragmenting the polypeptide backbone,” which is useful when applying the TMPP label taught by both Holden and Suh for analysis. However, when UVPD instrumentation further fragments the ions produced from the ETD, “the location of very basic sites (like Arg) at the C versus N-terminus” are highlighted even more effectively than ETD or UVPD alone (Holden, page 3, lines 5-8). It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Suh to incorporate the teachings of Holden so as to maximize all possible candidates for clipping/cleavage sites and better understand the functional limitations of specific clipping sites when evaluating vaccine and diagnostic candidates (See Abstract and Introduction of Suh).
Regarding claim 18, Suh teaches a method of identifying a clipping site on a protein (“The Staphylococcus aureus surface protein G (SasG)...The protein was also derivatized with...TMPPAc-OSu) to assess the presence of additional N-terminal sites of mature SasG,” wherein, “TMPP derivatization was employed to elucidate whether this protein is proteolytically clipped beyond its in silico predicted N-terminus. Key to the identification of N-terminal peptides is a comparison of mass spectra prior to and after derivatization with the TMPP reagent”; Abstract; page 1395, column 1, paragraph 2, lines 8-12)(As shown in Table 3, additional N-terminal peptides indicative of post-translational protein processing steps were identified; page 142, column 2, paragraph 1, lines 1-2), comprising:
(i) obtaining a sample containing one or more clipped polypeptides of the protein (peptide derived from protease cleavage during MS sample preparation; page 1400, column 2, paragraph 2, lines 2-3);
(ii) labeling the one or more clipped polypeptides with at least one reporter ion of claim 48 [sic] (“The TMPP labeling method permits the discernment of a peptide with a natural N-terminus from an equivalent peptide derived from protease cleavage during MS sample preparation,” wherein the reporter ion of claim 48 is TMPP; page 1400, column 2, paragraph 2, lines 1-3), to obtain a TMPP labeled polypeptide.
(iii) digesting the one or more TMPP labeled clipped polypeptides (Fig. 6…(C) The corresponding peptide from trypsin-digested TMPP-labeled SasG; page 1402, caption for Fig. 6) to generate a mixture comprising one or more unlabeled peptides and one or more TMPP labeled peptides (The TMPP labeling method allows peptide detection and subsequence tandem MS analysis of peptides…from complex mixture after proteolytic digestion; page 1403, column 2, paragraph 2, lines 1-5; see labeled and unlabeled peptides of Fig. 6);
(iv) subjecting the mixture to liquid chromatography (LC) to generate elutes of the LC (“LC-nESI-MS/MS: LC-nESI-MS/MS analysis was performed…to elute peptides off the trap,” wherein Fig. 6 shows “LC-nESI-MS/MS spectra for the N-terminal peptide 89SEVTSNK∼”; page 1396, paragraph 2, lines 1-7; Fig. 6);
(v) subjecting the elutes to a first stage of tandem mass spectrometry (Fig. 6…LC-nESI-MS/MS spectra for the N-terminal peptide 89SEVTSNK∼…(C) The corresponding peptide from trypsin-digested TMPP-labeled SasG; page 1402; Fig. 5)(“The TMPP labeling method allows peptide detection and subsequence tandem MS analysis of peptides; page 1403, column 2, paragraph 2, lines 1-5) to obtain a first mass spectrum of each of the one or more TMPP labeled peptides (The first mass spectrum of the TMPP labeled peptide contains “the top five parent ions,” and the second mass spectrum is a result of when these ions were “selected for fragmentation,” as shown in Fig. 6(C), wherein “Spectra were acquired in automated MS/MS mode, with the top five parent ions above a threshold of 10,000 selected for fragmentation.”; page 1396, paragraph 2, lines 9-12)(Since the second fragmented spectrum of Fig. 6(C) shows a positive identification of TMPP, then the first mass spectrum naturally contained ions of the TMPP labeled peptide);
(vii) upon detection or separation of the TMPP reporter ion, subjecting each of the TMPP labeled peptides to a second mass spectrometry to thereby generate a second mass spectrum for each of the TMPP labeled peptides, respectively (Spectra were acquired in automated MS/MS mode, with the top five parent ions above a threshold of 10,000 selected for fragmentation ; page 1396 paragraph 2, last 4 lines)( Fig. 6…LC-nESI-MS/MS spectra for the N-terminal peptide 89SEVTSNK∼…(C) The corresponding peptide from trypsin-digested TMPP-labeled SasG; page 1402; Fig. 6); and
(viii) obtaining the sequence of the protein and identifying the clipping site on the protein by analyzing the mass spectrum for each of the TMPP labeled peptides (The peptide TMPP-Ac-89SEVTSNK95 at m/z 668.96 (2+) was identified from tryptic digests of TMPP-labeled SasG by LCMS/MS (Fig. 6C); page 1402, column 1, lines 5-7)(MS and MS/MS data obtained from the MALDI and LC-nESI-MS/MS experiments were searched against the latest releases of the Swiss-Prot and non-redundant NCBI protein databases…to…display the identified peptides with their ranks, the MS/MS scores and a protein sequence coverage summary… For the identification of N-terminal peptides… accounting for N-terminal modification with the TMPP tag. The mass of 572.18 Da was considered as a variable modification; page 1396, paragraph 3)(Under broadest reasonable interpretation the examiner understands ‘characterizing’ to be any new information gathered as a result of analyzing the first mass spectrum and the second mass spectrum for each of the one or more TMPP labeled peptides).
Suh is silent to teaching that the first mass spectrum is generated by electron transfer dissociation (ETD) and the second mass spectrum is generated by collision induced dissociation (CID), higher-energy collisional dissociation (HCD), or ultraviolet photodissociation (UVPD). Suh is also silent to teaching (vi) detecting or separating a TMPP reporter ion in the ETD mass spectrum for each of the TMPP labeled peptides, wherein the TMPP reporter ion has a nominal mass-to-charge (m/z) of about 533, about 590, or about 573.
Suh does teach the use of MALDI-TOF MS/MS and LC-nESI-MS/MS when creating first and second spectra, one spectra on MS and the other on MS/MS (Figs. 5-6 and Table 3). Suh also discloses identifying TMPP-derivatized peptides by observing a characteristic mass shift of approximately 572 Da corresponding to incorporation of the TMPP tag (p. 1400, col. 1, ll. 4-7). This disclosure pertains to the increased mass of the modified peptide rather than to a detached reporter ion having a specific nominal m/z value. Suh does not describe generation or detection of a free TMPP+ reporter ion at m/z 573, nor does it disclose reporter ions at m/z ratios at about 533 or about 590. Accordingly, the specific nominal m/z values recited are not expressly taught by Suh alone.
Holden teaches the first mass spectrum is generated by electron transfer dissociation (ETD) and the second mass spectrum is generated by ultraviolet photodissociation (UVPD). (“We report a hybrid fragmentation method involving electron transfer dissociation (ETD) combined with ultraviolet photodissociation (UVPD),” for “peptide characterization,” wherein “ETD can precede or follow UVPD in the HCD cell or both activation processes can be undertaken simultaneously”; page 1, 1.1, line 2; Title; page 4, lines 13-14; See spectra of Fig. 1), and (vi) detecting or separating a TMPP reporter ion in the ETD mass spectrum for each of the TMPP labeled peptides, wherein the TMPP reporter ion has a nominal mass-to-charge (m/z) of about 533, about 590, or about 573 (The even electron fixed-charge peptide dissociated predominantly by the loss of the charged TMPP tag (resulting in the TMPP+ ion of m/z 573); p. 55, ll. 4-6; (Figure 2.10A)).
Holden is considered to be analogous to the claimed invention because it is in the same field of endeavor for TMPP-tagged peptides and analysis of cleavage sites using mass spectrometry. It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Suh to incorporate the teachings of Holden by using a hybridized ETD-UVPD method when evaluating spectra and its resulting TMPP+ reporter ion at m/z 573 in order to improve amino acid sequence characterization and cleavage-site localization . Holden explains that “ETHCD, was shown to provide an informative array of predominantly b-, c-, y-, and z type ions,” which results in an increase in information for the exact locations of cleavage sites (page 2, lines 17-18). However, “the most diverse array of fragment ions was obtained from the ETUVPD hybrid method, an outcome that proved particularly beneficial for specific localization of modifications for which fragmentation was suppressed for other activation methods” (Holden, page 3, lines 2-4). With a wider array of this ionic fragmentation information, analysis of cleavage or clipping sites using “hybrid fragmentation of intact proteins is feasible even for complex mixtures” (Holden, page 3, paragraph 2, lines 2-4). The use of ETD alone has the “ability to maintain labile post translational modifications (PTMs) while indiscriminately fragmenting the polypeptide backbone,” which is useful when applying the TMPP label taught by both Holden and Suh for analysis. However, when UVPD instrumentation further fragments the ions produced from the ETD, “the location of very basic sites (like Arg) at the C versus N-terminus” are highlighted even more effectively than ETD or UVPD alone (Holden, page 3, lines 5-8). It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Suh to incorporate the teachings of Holden so as to maximize all possible candidates for clipping/cleavage sites and better understand the functional limitations of specific clipping sites when evaluating vaccine and diagnostic candidates (See Abstract and Introduction of Suh). In regards to step (vi), Suh already teaches that TMPP derivatization produces a characteristic mass shift of approximately 572 Da that is specifically queried during MS analysis (p. 1400, col. 1, ll. 4-7). Suh thus establishes that the TMPP tag has a known and predictable mass contribution and serves as a diagnostic feature for identifying labeled peptides. Holden further teaches that fragmentation of TMPP-labeled peptides results in loss of the charged TMPP tag, producing the TMPP+ ion at m/z 573. Given Suh’s recognition of the TMPP tag as a characteristic mass marker and Holden’s express disclosure that fragmentation yields the corresponding charged TMPP+ ion at nominal m/z 573, a person of ordinary skill in the art would have found it predictable and straightforward to detect that diagnostic TMPP+ fragment during tandem MS analysis. Incorporating Holden’s explicit reporter-ion teaching into Suh’s established TMPP mass-shift workflow would have represented the routine use of a known fragmentation product corresponding to a known modification mass, yielding no more than the predictable identification of the same chemical moiety in fragment form (See MPEP 2143(I)(A)).
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). 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 nonprovisional extension fee (37 CFR 1.17(a)) 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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/V.S./Examiner, Art Unit 1758
/MARIS R KESSEL/Supervisory Patent Examiner, Art Unit 1758