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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 06/08/2026 has been entered.
Response to Amendment
The Amendment filed 06/08/2026 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 Claims 27-28, 30-32, 34, 37-38, 42, 48, 50, 55, and 57 are previously withdrawn.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
The objection to the claims has been withdrawn in view of Applicant's amendment.
The rejection of claims 1-4, 7-8, and 18 under 35 U.S.C. 103 as being unpatentable over Suh in view of Holden is withdrawn and replaced with the new ground(s) of rejection based upon a different combination of prior art references and rationale.
Response to Arguments
Applicant's arguments, see pages 9-13, filed 06/08/2026, with respect to the rejections of claims 1-4, 7-8, and 18 under 35 U.S.C. 103, have been fully considered but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument. Nevertheless, the Examiner addresses the arguments below.
Applicant argues (pp. 11-12) that Suh does not teach subjecting a TMPP labeled peptide to a first stage of tandem mass spectrometry followed by a second stage of mass spectrometry, asserting that Suh’s initial MS scan is merely a survey scan rather than a tandem MS experiment.
Applicant’s argument is moot.
The present rejection does not rely on Suh alone to teach the claimed first stage electron-induced dissociation tandem mass spectrometry or the second stage of mass spectrometry. Rather Suh, is relied upon for the clipping-site workflow and first stage tandem mass spectrometry in claims 3 and 18. Holden teaches the first stage ETD (EID) tandem MS, Schulzknappe teaches reporter ion triggering acquisition, and Froelich/Adamczyk teaches performing the second stage of mass spectrometry.
Applicant argues (pp. 11-12) that Holden employs ETD only within hybrid fragmentation methods, such as ETUVPD, EThcD, and ETcaD, and does not disclose generating a first ETD spectrum, identifying a TMPP-labeled peptide by detecting a TMPP reporter ion, or using a TMPP reporter ion to trigger a subsequent fragmentation event. Applicant further contends that separating ETD from Holden’s hybrid methods would defeat the purpose of the reference.
The Examiner respectfully disagrees.
In response to applicant's argument that Holden teaches ETD only as part of hybrid fragmentation and separating ETD from Holden’s hybrid methods would defeat the purpose of the reference, the test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981).
The Examiner does not rely on Holden to teach detecting a TMPP reporter ion to trigger a subsequent stage of mass spectrometry, nor does the Examiner rely on Holden’s hybrid ETuvPD, EThcD, or ETcaD methods as the claimed triggering mechanism. Rather, Holden is relied upon solely for teaching ETD tandem mass spectrometry of TPMPP tagged peptides as a substitution for MALDI-TOF-TOF and nESI-MS/MS taught by Zou (claims 1-2) and Suh (claims 3 and 18), respectively. Holden is also relied upon to teach that fragmentation of TMPP tagged peptides produces the characteristic TMPP+ reporter ion having an m/z of 573 (p. 55). Holden expressly teaches performing ETD as an MS/MS event and further teaches that subsequent hybrid MS/MS experiments were performed via ETD, demonstrating that ETD is a known tandem MS fragmentation technique for TMPP tagged peptides (p. 15, last para., l. 2; p. 16). The Examiner is not extracting ETD from Holden in a manner that defeats the purpose of the reference, but rather substituting one known tandem MS fragmentation technique for another to obtain the recognized benefits of ETD for the peptide sequence characterization.
The claimed reporter ion triggering acquisition is taught by Schulzknappe, while the second stage of MS for obtaining additional sequence information is taught by Froelich/Adamczyk. Accordingly, the rejection relies on the combined teachings of the references, and not on Holden alone, to satisfy the claimed limitations.
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 1-2, 4, and 8 are rejected under 35 U.S.C. 103 as being unpatentable over Zou et al. (CN 104483374 A, see attached English translation), in view of Holden (“Hybrid Activation for Intact Protein and Peptide Characterization”) and Froelich et al. (“Chemical Derivatization and Multistage Tandem Mass Spectrometry for Protein Structural Characterization”) with Adamczyk et al., incorporated by reference (“Charge Derivatization of Peptides to Simplify their Sequencing with an Ion Trap Mass Spectrometer”), as evidenced by Lu et al. (“Sulfonium Ion Derivatization, Isobaric Stable Isotope Labeling and Data Dependent CID- and ETD-MS/MS for Enhanced Phosphopeptide uantitation, Identification and Phosphorylation Site Characterization).
Regarding claim 1, Zou teaches a method of characterizing a N-tris(2,4,6- trimethoxyphenyl)phosphonium acetyl (TMPP) labeled peptide in a sample (“This method has good specificity and can selectively label TMPP-Ac at the N-terminus of proteins…When TMPP-Ac labeled peptides are analyzed by mass spectrometry, a characteristic peak of 573.2 is generated, and the spectrum is simple, which is convenient for de novo sequencing,” wherein Examples 1-2 carry out this method on human hemoglobin samples; [0039]-[0040]; See paragraphs [0051]-[0073] for Examples 1-2), comprising:
(i) subjecting the sample to a first stage of tandem mass spectrometry to obtain a first mass spectrum of the TMPP labeled peptide (Figure 3 shows the MALDI-TOF-TOF tandem mass spectra of the N-terminal peptide of human hemoglobin after guanidinolation and TMPP-Ac modification and digestion with staphylococcal protease; [0073]);
(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 (In all the secondary plots, only the peptides with m/z of 1267.50 and 1396.54 Da produced a TMPP-Ac cleavage peak at m/z 573.189. This peak was produced after the N-terminal peptide of the human hemoglobin β chain was modified by TMPP-Ac, which can be used to distinguish the N-terminal peptide of a protein from the intermediate peptide; [0073]);
(iv) characterizing the TMPP labeled peptide by analyzing the first mass spectrum to obtain the amino acid sequence of the TMPP labeled peptide (The sequences can be obtained by searching databases or by de novo sequencing. The sequences are VHLTPEE, C<sup> + </sup> 1396.54 Da (Figure 3a) and VHLTPE, C<sup> + </sup> 1267.50 Da (Figure 3b); [0073]).
Zou fails to teach:
(i) subjecting the sample to a first stage of electron-induced dissociation tandem mass spectrometry to obtain a first mass spectrum of the TMPP labeled peptide (Emphasis added),
(iii) subjecting the identified TMPP labeled peptide to a second stage of mass spectrometry to thereby generate a second mass spectrum of the TMPP labeled peptide; 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 (Emphasis added).
Holden teaches (i) subjecting a sample to a first stage of electron-induced dissociation tandem mass spectrometry to obtain a first mass spectrum of the TMPP labeled peptide (Holden discloses that “all subsequent hybrid MS/MS experiments were performed via both ETD and UVPD” and “an MS/MS event for ETD in the LIT with a 48 msec analytical scan to determine the ions created by ETD,” and then analyzes TMPP-tagged RGAFSTFGAS peptide using ETcaD and EThcD, producing the characteristic TMPP+ reporter ion (m/z 573), wherein “Examples of…ETuvPD mass spectra for the resulting TMPP+- RGAFSTFGAS peptide are shown in Figure 2.10, and the…ETcaD, and EThcD results are summarized in Figure 2.6 and Table 2.1,” (p. 15, last para., l. 2; p. 16, l. 2-3; p. 55)(Paragraph [0011] of the instant publication (US 20220390421 A1) explains that ETD is a type of electron-induced dissociation).
Holden is considered to be analogous to the claimed invention because it is in the same field of endeavor for the characterization of TMPP-tagged peptides using mass spectrometry. Zou utilizes MALDI-TOF/TOF analysis and Holden teaches ETD-MS/MS as an alternative fragmentation technique for obtaining informative peptide sequence data, recognizing that “affixing a fixed-charge tag to the N-terminus of a peptide has also been reported to improve sequence coverage obtained by electron transfer dissociation (ETD)” (p. 16, ll. 2-3). A supporting reference, Lu (“Sulfonium Ion Derivatization, Isobaric Stable Isotope Labeling and Data Dependent CID- and ETD-MS/MS for Enhanced Phosphopeptide Quantitation, Identification and Phosphorylation Site Characterization) also uses EID tandem MS followed to characterize peptides tagged with a fixed-charge label (Abstract). Lu explains that compared to CID-MS/MS, “ETD has an improved ability to localize phosphate groups…as no losses involving the phosphate side chain or phosphate group ‘scrambling’ are observed” (p. 578, col. ll. 43-46). Therefore, 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 substituted the MALDI-TOF/TOF experiment taught by Zou with the ETD-MS/MS experiment taught by Holden because it would produce improved sequence coverage while characterizing the same TMPP-labeled peptides, and this involves simple substitution of one known fragmentation method for another to obtain predictable (See MPEP 2143(I)(B)).
Modified Lou fails to teach (iii) subjecting the identified TMPP labeled peptide to a second stage of mass spectrometry to thereby generate a second mass spectrum of the TMPP labeled peptide; 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 (Emphasis added).
Froelich teaches (iii) subjecting the identified TMPP labeled peptide to a second stage of mass spectrometry to thereby generate a second mass spectrum of the TMPP labeled peptide (Adamczyk et al. have demonstrated that N-terminal b- and a-type ions are formed predominately following CID-MS3 analysis of the TMPP-Ac containing b-type ions produced in the -first stage of tandem mass spectrometry analysis [77]; Froelich, pp. 88-89); 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 (Emphasis added)(See sequencing data in table 1 and Figs. 1-6 of Adamczyk)(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).
Froelich is considered to be analogous to the claimed invention because it is in the same field of endeavor for the characterization of TMPP-tagged peptides using mass spectrometry. Zou recognizes that a known method for sequencing peptides is “performed using various chemical labels at the N-terminus, followed by…multi-stage mass spectrometry (MSn)” ([0007]), and Froelich teaches that such a method “can be particularly useful for the analysis of larger peptides, for which incomplete sequence information is obtained frequently following CID-MS/MS alone” (p. 89, ll. 3-5). Therefore, 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 the data acquisition method taught by Zou in view of Holden by subjecting the identified TMPP labeled peptide to a second stage of mass spectrometry as taught by Froelich because it would be “useful for obtaining the complete sequence information of the peptides” (Adamczyk; Abstract), and this involves combining prior art elements according to known methods to yield predictable results (See MPEP 2143(I)(A)).
Regarding claim 2, Zou teaches a method of characterizing a polypeptide, comprising:
(i) labeling the polypeptide with at least one TMPP reporter ion to obtain a TMPP labeled polypeptide (“add 3 μL of freshly prepared 10 mM (N-succinimideoxocarbonylmethyl) tris(2,4,6-trimethoxyphenyl)phosphine bromide (TMPP-Ac-OSu) acetonitrile solution,” to extracted proteins from step 1.1, wherein “This method has good specificity and can selectively label TMPP-Ac at the N-terminus of proteins”; [0055];[0039]);
(ii) digesting the TMPP labeled polypeptide to generate a mixture comprising one or more unlabeled peptides and one or more TMPP labeled peptides (“Add 50mM NH4NER6HCO3NER7 solution containing 10μL of 20ng/μL trypsin and incubate at 37℃ for 17 hours,” to produce “The enzymatically digested peptides”; [0057])(Paragraph [0073] explains that the tandem MS spectra showed N-terminal peptides (labeled with TMPP-Ac cleavage peak at m/z 573.189) and intermediate peptides (unlabeled));
(iii) subjecting the mixture to liquid chromatography (LC) to generate elutes of the LC (“The enzymatically digested peptides were eluted using conventional methods in the field,” wherein “The N-terminal peptides of the enriched protein were separated by reverse phase liquid chromatography”; [0057]; [0018]);
(iv) subjecting the elutes to a first stage of tandem mass spectrometry to obtain a first mass spectrum of each of the one or more TMPP labeled peptides (Figure 3 shows the MALDI-TOF-TOF tandem mass spectra of the N-terminal peptide of human hemoglobin after guanidinolation and TMPP-Ac modification and digestion with staphylococcal protease; [0073]);
(v) identifying the one or more TMPP labeled peptides by detecting or separating the TMPP reporter ion in the first mass spectrum of each of the one or more TMPP labeled peptides, wherein the TMPP reporter ion has a nominal mass-to-charge (m/z) of about 533, about 590, or about 573 (In all the secondary plots, only the peptides with m/z of 1267.50 and 1396.54 Da produced a TMPP-Ac cleavage peak at m/z 573.189. This peak was produced after the N-terminal peptide of the human hemoglobin β chain was modified by TMPP-Ac, which can be used to distinguish the N-terminal peptide of a protein from the intermediate peptide; [0073];
(vii) characterizing the polypeptide by analyzing the first mass spectrum for each of the one or more TMPP labeled peptides to obtain the amino acid sequence of the polypeptide (The sequences can be obtained by searching databases or by de novo sequencing. The sequences are VHLTPEE, C<sup> + </sup> 1396.54 Da (Figure 3a) and VHLTPE, C<sup> + </sup> 1267.50 Da (Figure 3b); [0073]).
Zou fails to teach:
(iv) subjecting the elutes to a first stage of electron-induced dissociation tandem mass spectrometry to obtain a first mass spectrum of each of the one or more TMPP labeled peptides (Emphasis added);
(vi) subjecting the identified one or more TMPP labeled peptides to a second stage of mass spectrometry to thereby generate a second mass spectrum of the each of the one or more TMPP labeled peptides; 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 to obtain the amino acid sequence of the polypeptide (Emphasis added).
Holden teaches (iv) subjecting a sample to a first stage of electron-induced dissociation tandem mass spectrometry to obtain a first mass spectrum of the TMPP labeled peptide (Holden discloses that “all subsequent hybrid MS/MS experiments were performed via both ETD and UVPD” and “an MS/MS event for ETD in the LIT with a 48 msec analytical scan to determine the ions created by ETD,” and then analyzes TMPP-tagged RGAFSTFGAS peptide using ETcaD and EThcD, producing the characteristic TMPP+ reporter ion (m/z 573), wherein “Examples of…ETuvPD mass spectra for the resulting TMPP+- RGAFSTFGAS peptide are shown in Figure 2.10, and the…ETcaD, and EThcD results are summarized in Figure 2.6 and Table 2.1,” (p. 15, last para., l. 2; p. 16, l. 2-3; p. 55)(Paragraph [0011] of the instant publication (US 20220390421 A1) explains that ETD is a type of electron-induced dissociation).
Holden is considered to be analogous to the claimed invention because it is in the same field of endeavor for the characterization of TMPP-tagged peptides using mass spectrometry. Zou utilizes MALDI-TOF/TOF analysis and Holden teaches ETD-MS/MS as an alternative fragmentation technique for obtaining informative peptide sequence data, recognizing that “affixing a fixed-charge tag to the N-terminus of a peptide has also been reported to improve sequence coverage obtained by electron transfer dissociation (ETD)” (p. 16, ll. 2-3). A supporting reference, Lu (“Sulfonium Ion Derivatization, Isobaric Stable Isotope Labeling and Data Dependent CID- and ETD-MS/MS for Enhanced Phosphopeptide Quantitation, Identification and Phosphorylation Site Characterization) also uses EID tandem MS to characterize peptides tagged with a fixed-charge label. Lu explains that compared to CID-MS/MS, “ETD has an improved ability to localize phosphate groups…as no losses involving the phosphate side chain or phosphate group ‘scrambling’ are observed” (p. 578, col. ll. 43-46). Therefore, 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 substituted the MALDI-TOF/TOF experiment taught by Zou with the ETD-MS/MS experiment taught by Holden because it would produce improved sequence coverage while characterizing the same TMPP-labeled peptides, and this involves simple substitution of one known fragmentation method for another to obtain predictable (See MPEP 2143(I)(B)).
Modified Lou fails to teach (vi) subjecting the identified one or more TMPP labeled peptides to a second stage of mass spectrometry to thereby generate a second mass spectrum of the each of the one or more TMPP labeled peptides; 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 to obtain the amino acid sequence of the polypeptide (Emphasis added).
Froelich teaches (vi) subjecting the identified TMPP labeled peptide to a second stage of mass spectrometry to thereby generate a second mass spectrum of the TMPP labeled peptide (Adamczyk et al. have demonstrated that N-terminal b- and a-type ions are formed predominately following CID-MS3 analysis of the TMPP-Ac containing b-type ions produced in the -first stage of tandem mass spectrometry analysis [77]; Froelich, pp. 88-89); and
(vii) 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 (Emphasis added)(See sequencing data in table 1 and Figs. 1-6 of Adamczyk)(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).
Froelich is considered to be analogous to the claimed invention because it is in the same field of endeavor for the characterization of TMPP-tagged peptides using mass spectrometry. Zou recognizes that a known method for sequencing peptides is “performed using various chemical labels at the N-terminus, followed by…multi-stage mass spectrometry (MSn)” ([0007]), and Froelich teaches that such a method “can be particularly useful for the analysis of larger peptides, for which incomplete sequence information is obtained frequently following CID-MS/MS alone” (p. 89, ll. 3-5). Therefore, 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 the data acquisition method taught by Zou in view of Holden by subjecting the identified TMPP labeled peptide to a second stage of mass spectrometry as taught by Froelich because it would be “useful for obtaining the complete sequence information of the peptides” (Adamczyk; Abstract), and this involves combining prior art elements according to known methods to yield predictable results (See MPEP 2143(I)(A)).
Regarding claim 4, The method of claim 1, wherein the electron-induced dissociation is electron transfer dissociation (ETD) (“all subsequent hybrid MS/MS experiments were performed via both ETD and UVPD,” wherein “an MS/MS event for ETD in the LIT with a 48 msec analytical scan to determine the ions created by ETD”; Holden, p. 15, last para., l. 2; p. 16, ll. 2-3) or electron capture dissociation (ECD).
Regarding claim 8,The method of claim 1, wherein the second mass spectrometry comprises collision-induced dissociation (CID) (CID-MS3 analysis; Froelich, p. 89, l. 1), higher-energy collisional dissociation (HCD), or ultraviolet photodissociation (UVPD).
Claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Zou et al. (CN 104483374 A, see attached English translation), in view of Holden (“Hybrid Activation for Intact Protein and Peptide Characterization”) and Froelich et al. (“Chemical Derivatization and Multistage Tandem Mass Spectrometry for Protein Structural Characterization”) with Adamczyk et al., incorporated by reference (“Charge Derivatization of Peptides to Simplify their Sequencing with an Ion Trap Mass Spectrometer”) as evidenced by Lu et al. (“Sulfonium Ion Derivatization, Isobaric Stable Isotope Labeling and Data Dependent CID- and ETD-MS/MS for Enhanced Phosphopeptide uantitation, Identification and Phosphorylation Site Characterization), as applied to claim 1 above, and in further view of Schulzknappe et al. (US 20150241443 A1).
Regarding claim 7, The method of claim 1.
Modified Zou fails to teach the TMPP reporter ion triggers the second mass spectrometry.
Schulzknappe teaches a TMPP reporter ion triggers the second mass spectrometry (In a preferred embodiment, the presence of a reporter ion from the trigger indicates that an analyte of interest is eluting from an LC column during LC-MS. This would "trigger" the execution of a pre-defined MS/MS experiment; [0201]).
Schulzknappe is considered to be analogous to the claimed invention because it is in the same field of endeavor for the characterization of labeled peptides using mass spectrometry. Adamczyk deliberately targets sequential fragmentation of TMPP-tagged peptides in the sample, stating that “MSn of the charge-derivatized peptides provides a unique advantage over their underivatized counterparts, because all of the product ions from the first stage CID have a retained, fixed charge on the N-terminus, which in turn will direct the fragmentation of the next stage CID, producing exclusively N-terminal ‘granddaughter’ ions. Therefore, 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 the data acquisition method taught by Zou in view of Holden and Froelich by triggering the second mass spectrometry when the reporter ion is detected because automating the process would improve analytical throughput and eliminate unnecessary manual or indiscriminate MS/MS acquisitions (See MPEP 2144.04(III)).
Claims 3 and 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”), in view of Holden (“Hybrid Activation for Intact Protein and Peptide Characterization”) and Froelich et al. (“Chemical Derivatization and Multistage Tandem Mass Spectrometry for Protein Structural Characterization”) with Adamczyk et al., incorporated by reference (“Charge Derivatization of Peptides to Simplify their Sequencing with an Ion Trap Mass Spectrometer”) as evidenced by Lu et al. (“Sulfonium Ion Derivatization, Isobaric Stable Isotope Labeling and Data Dependent CID- and ETD-MS/MS for Enhanced Phosphopeptide uantitation, Identification and Phosphorylation Site Characterization).
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 TMPP reporter ion, (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; page 1400, column 2, paragraph 2, lines 1-3), to thereby obtain one or more TMPP labeled clipped polypeptides (obtaining one or more TMPP labeled clipped polypeptides is the intended result of labeling the protease cleaved peptide or clipped polypeptide with TMPP);
(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 (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);
(viii) obtaining the sequence of the protein and identifying the clipping site on the protein by analyzing the first 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 fails to teach:
(v) subjecting the elutes to a first stage of electron-induced dissociation tandem mass spectrometry (Emphasis added);
(vi) identifying each of the TMPP labeled peptides by detecting or separating the TMPP reporter ion in the first 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;
(vii) subjecting each of the identified TMPP labeled peptides to a second stage of mass spectrometry to thereby generate a second mass spectrum for each of the TMPP labeled peptides; and
(viii) analyzing the second mass spectrum for each of the TMPP labeled peptides.
Holden teaches (v) subjecting a sample to a first stage of electron-induced dissociation tandem mass spectrometry to obtain a first mass spectrum of the TMPP labeled peptide (Holden discloses that “all subsequent hybrid MS/MS experiments were performed via both ETD and UVPD” and “an MS/MS event for ETD in the LIT with a 48 msec analytical scan to determine the ions created by ETD,” and then analyzes TMPP-tagged RGAFSTFGAS peptide using ETcaD and EThcD, producing the characteristic TMPP+ reporter ion (m/z 573), wherein “Examples of…ETuvPD mass spectra for the resulting TMPP+- RGAFSTFGAS peptide are shown in Figure 2.10, and the…ETcaD, and EThcD results are summarized in Figure 2.6 and Table 2.1,” (p. 15, last para., l. 2; p. 16, l. 2-3; p. 55)(Paragraph [0011] of the instant publication (US 20220390421 A1) explains that ETD is a type of electron-induced dissociation); and
(vi) identifying each of the TMPP labeled peptides by detecting or separating the TMPP reporter ion in the first 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 (A second N-terminal fixed-charge peptide was generated by attachment of the TMPP tag to RGAFSTFGAS…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); See Figs. 2.6, 2.10 and Table 2.1)
Holden is considered to be analogous to the claimed invention because it is in the same field of endeavor for the characterization of TMPP-tagged peptides using mass spectrometry. Suh utilizes nESI-MS/MS analysis and Holden teaches ETD-MS/MS as an alternative fragmentation technique for obtaining informative peptide sequence data, recognizing that “affixing a fixed-charge tag to the N-terminus of a peptide has also been reported to improve sequence coverage obtained by electron transfer dissociation (ETD)” (p. 16, ll. 2-3). A supporting reference, Lu (“Sulfonium Ion Derivatization, Isobaric Stable Isotope Labeling and Data Dependent CID- and ETD-MS/MS for Enhanced Phosphopeptide Quantitation, Identification and Phosphorylation Site Characterization) also uses EID tandem MS to characterize peptides tagged with a fixed-charge label. Lu explains that compared to CID-MS/MS, “ETD has an improved ability to localize phosphate groups…as no losses involving the phosphate side chain or phosphate group ‘scrambling’ are observed” (p. 578, col. ll. 43-46). Therefore, 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 substituted the nESI-MS/MS experiment taught by Suh with the ETD-MS/MS experiment taught by Holden because it would produce improved sequence coverage while characterizing the same TMPP-labeled peptides, and this involves simple substitution of one known fragmentation method for another to obtain predictable (See MPEP 2143(I)(B)).
Suh fails to teach:
(vii) subjecting each of the identified TMPP labeled peptides to a second stage of mass spectrometry to thereby generate a second mass spectrum for each of the TMPP labeled peptides; and
(viii) analyzing the second mass spectrum for each of the TMPP labeled peptides.
Froelich teaches (iii) subjecting the identified TMPP labeled peptide to a second stage of mass spectrometry to thereby generate a second mass spectrum of the TMPP labeled peptide (Adamczyk et al. have demonstrated that N-terminal b- and a-type ions are formed predominately following CID-MS3 analysis of the TMPP-Ac containing b-type ions produced in the -first stage of tandem mass spectrometry analysis [77]; Froelich, pp. 88-89); and
(iv) analyzing the second mass spectrum of the TMPP labeled peptide (Emphasis added)(See sequencing data in table 1 and Figs. 1-6 of Adamczyk)(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).
Froelich is considered to be analogous to the claimed invention because it is in the same field of endeavor for the characterization of TMPP-tagged peptides using mass spectrometry. Zou recognizes that a known method for sequencing peptides is “performed using various chemical labels at the N-terminus, followed by…multi-stage mass spectrometry (MSn)” ([0007]), and Froelich teaches that such a method “can be particularly useful for the analysis of larger peptides, for which incomplete sequence information is obtained frequently following CID-MS/MS alone” (p. 89, ll. 3-5). Therefore, 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 the data acquisition method taught by Suh in view of Holden by subjecting the identified TMPP labeled peptide to a second stage of mass spectrometry as taught by Froelich because it would be “useful for obtaining the complete sequence information of the peptides” (Adamczyk; Abstract), and this involves combining prior art elements according to known methods to yield predictable results (See MPEP 2143(I)(A)).
Regarding claim 18, 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 TMPP reporter ion, (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; page 1400, column 2, paragraph 2, lines 1-3), to thereby obtain one or more TMPP labeled clipped polypeptides (obtaining one or more TMPP labeled clipped polypeptides is the intended result of labeling the protease cleaved peptide or clipped polypeptide with TMPP);
(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 (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);
(viii) obtaining the sequence of the protein and identifying the clipping site on the protein by analyzing the first 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 fails to teach:
(v) subjecting the elutes to a first stage of electron-induced dissociation tandem mass spectrometry (Emphasis added);
(vi) detecting or separating the 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;
(vii) upon detection or separation of the TMPP reporter ion, subjecting each of the TMPP labeled peptides to a second stage of mass spectrometry, comprising a collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), or ultraviolet photodissociation (UVPD), to thereby generate a CID, HCD, or UVPD mass spectrum for each of the TMPP labeled peptides, respectively; and
(viii) analyzing the CID, HCD, or UVPD mass spectrum for each of the TMPP labeled peptides.
Holden teaches (v) subjecting a sample to a first stage of electron-induced dissociation tandem mass spectrometry to obtain a first mass spectrum of the TMPP labeled peptide (Holden discloses that “all subsequent hybrid MS/MS experiments were performed via both ETD and UVPD” and “an MS/MS event for ETD in the LIT with a 48 msec analytical scan to determine the ions created by ETD,” and then analyzes TMPP-tagged RGAFSTFGAS peptide using ETcaD and EThcD, producing the characteristic TMPP+ reporter ion (m/z 573), wherein “Examples of…ETuvPD mass spectra for the resulting TMPP+- RGAFSTFGAS peptide are shown in Figure 2.10, and the…ETcaD, and EThcD results are summarized in Figure 2.6 and Table 2.1,” (p. 15, last para., l. 2; p. 16, l. 2-3; p. 55)(Paragraph [0011] of the instant publication (US 20220390421 A1) explains that ETD is a type of electron-induced dissociation); and
(vi) identifying each of the TMPP labeled peptides by detecting or separating the TMPP reporter ion in the first 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 (A second N-terminal fixed-charge peptide was generated by attachment of the TMPP tag to RGAFSTFGAS…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); See Figs. 2.6, 2.10 and Table 2.1)
Holden is considered to be analogous to the claimed invention because it is in the same field of endeavor for the characterization of TMPP-tagged peptides using mass spectrometry. Suh utilizes nESI-MS/MS analysis and Holden teaches ETD-MS/MS as an alternative fragmentation technique for obtaining informative peptide sequence data, recognizing that “affixing a fixed-charge tag to the N-terminus of a peptide has also been reported to improve sequence coverage obtained by electron transfer dissociation (ETD)” (p. 16, ll. 2-3). A supporting reference, Lu (“Sulfonium Ion Derivatization, Isobaric Stable Isotope Labeling and Data Dependent CID- and ETD-MS/MS for Enhanced Phosphopeptide Quantitation, Identification and Phosphorylation Site Characterization) also uses EID tandem MS to characterize peptides tagged with a fixed-charge label. Lu explains that compared to CID-MS/MS, “ETD has an improved ability to localize phosphate groups…as no losses involving the phosphate side chain or phosphate group ‘scrambling’ are observed” (p. 578, col. ll. 43-46). Therefore, 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 substituted the nESI-MS/MS experiment taught by Suh with the ETD-MS/MS experiment taught by Holden because it would produce improved sequence coverage while characterizing the same TMPP-labeled peptides, and this involves simple substitution of one known fragmentation method for another to obtain predictable (See MPEP 2143(I)(B)).
Suh fails to teach:
(vii) upon detection or separation of the TMPP reporter ion, subjecting each of the TMPP labeled peptides to a second stage of mass spectrometry, comprising a collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), or ultraviolet photodissociation (UVPD), to thereby generate a CID, HCD, or UVPD mass spectrum for each of the TMPP labeled peptides, respectively; and
(viii) analyzing the CID, HCD, or UVPD mass spectrum for each of the TMPP labeled peptides.
Froelich teaches (vii) upon detection or separation of the TMPP reporter ion, subjecting each of the TMPP labeled peptides to a second stage of mass spectrometry, comprising a collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), or ultraviolet photodissociation (UVPD), to thereby generate a CID, HCD, or UVPD mass spectrum for each of the TMPP labeled peptides, respectively (Adamczyk et al. have demonstrated that N-terminal b- and a-type ions are formed predominately following CID-MS3 analysis of the TMPP-Ac containing b-type ions produced in the -first stage of tandem mass spectrometry analysis [77]; Froelich, pp. 88-89; See sequencing data in table 1 and Figs. 1-6 of Adamczyk) and
(viii) analyzing the CID, HCD, or UVPD mass spectrum for each of the TMPP labeled peptides (See sequencing data in table 1 and Figs. 1-6 of Adamczyk).
Froelich is considered to be analogous to the claimed invention because it is in the same field of endeavor for the characterization of TMPP-tagged peptides using mass spectrometry. Zou recognizes that a known method for sequencing peptides is “performed using various chemical labels at the N-terminus, followed by…multi-stage mass spectrometry (MSn)” ([0007]), and Froelich teaches that such a method “can be particularly useful for the analysis of larger peptides, for which incomplete sequence information is obtained frequently following CID-MS/MS alone” (p. 89, ll. 3-5). Therefore, 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 the data acquisition method taught by Suh in view of Holden by subjecting the identified TMPP labeled peptide to a second stage of mass spectrometry as taught by Froelich because it would be “useful for obtaining the complete sequence information of the peptides” (Adamczyk; Abstract), and this involves combining prior art elements according to known methods to yield predictable results (See MPEP 2143(I)(A)).
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure:
Lu et al., 2011 (instant PTO-892) teaches the advantages of using ETD-MS/MS over CID-MS/MS when characterizing labeled peptides. Lu also teaches automatically initiating (triggering) CID-MS/MS, -MS3, and/or ETDMS/ based on input criteria.
No claims are allowed.
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/V.S./Examiner, Art Unit 1758
/MARIS R KESSEL/Supervisory Patent Examiner, Art Unit 1758
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