Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant's election with traverse of Group I (claims 1-28) in the reply filed on 11/24/25 is acknowledged. The traversal is on the ground(s) that there would not be a substantial burden on the Office to examine I and II together, in view of the close relationships of the inventions to each other, and the fact that Invention II includes only two claims.
This is not found persuasive because there would be a substantial burden on Examiner to examine the required limitations and scope of those limitations that are different in each group. Examiner notes that if allowable subject matter is found, consideration of any appropriate rejoinder would be made.
The requirement is still deemed proper and is therefore made FINAL.
Claims 29 and 36 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 11/24/25.
Claim Interpretation
The following is a quotation of 35 U.S.C. 112(f):
(f) Element in Claim for a Combination. – An element in a claim for a combination may be expressed as a means or step for performing a specified function without the recital of structure, material, or acts in support thereof, and such claim shall be construed to cover the corresponding structure, material, or acts described in the specification and equivalents thereof.
The following is a quotation of pre-AIA 35 U.S.C. 112, sixth paragraph:
An element in a claim for a combination may be expressed as a means or step for performing a specified function without the recital of structure, material, or acts in support thereof, and such claim shall be construed to cover the corresponding structure, material, or acts described in the specification and equivalents thereof.
The claims in this application are given their broadest reasonable interpretation using the plain meaning of the claim language in light of the specification as it would be understood by one of ordinary skill in the art. The broadest reasonable interpretation of a claim element (also commonly referred to as a claim limitation) is limited by the description in the specification when 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, is invoked.
As explained in MPEP § 2181, subsection I, claim limitations that meet the following three-prong test will be interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph:
(A) the claim limitation uses the term “means” or “step” or a term used as a substitute for “means” that is a generic placeholder (also called a nonce term or a non-structural term having no specific structural meaning) for performing the claimed function;
(B) the term “means” or “step” or the generic placeholder is modified by functional language, typically, but not always linked by the transition word “for” (e.g., “means for”) or another linking word or phrase, such as “configured to” or “so that”; and
(C) the term “means” or “step” or the generic placeholder is not modified by sufficient structure, material, or acts for performing the claimed function.
Use of the word “means” (or “step”) in a claim with functional language creates a rebuttable presumption that the claim limitation is to be treated in accordance with 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph. The presumption that the claim limitation is interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, is rebutted when the claim limitation recites sufficient structure, material, or acts to entirely perform the recited function.
Absence of the word “means” (or “step”) in a claim creates a rebuttable presumption that the claim limitation is not to be treated in accordance with 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph. The presumption that the claim limitation is not interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, is rebutted when the claim limitation recites function without reciting sufficient structure, material or acts to entirely perform the recited function.
Claim limitations in this application that use the word “means” (or “step”) are being interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, except as otherwise indicated in an Office action. Conversely, claim limitations in this application that do not use the word “means” (or “step”) are not being interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, except as otherwise indicated in an Office action.
This application includes one or more claim limitations that do not use the word “means,” but are nonetheless being interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, because the claim limitation(s) uses a generic placeholder that is coupled with functional language without reciting sufficient structure to perform the recited function and the generic placeholder is not preceded by a structural modifier.
Such claim limitation(s) is/are: an electromagnetic mixer in claims 18.
Because this/these claim limitation(s) is/are being interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, it/they is/are being interpreted to cover the corresponding structure described in the specification as performing the claimed function, and equivalents thereof.
An electromagnetic mixer is interpreted to encompass an electromagnetic mixing disclosed by Applicant, such as in paragraph 0017, and its equivalents.
If applicant does not intend to have this/these limitation(s) interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, applicant may: (1) amend the claim limitation(s) to avoid it/them being interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph (e.g., by reciting sufficient structure to perform the claimed function); or (2) present a sufficient showing that the claim limitation(s) recite(s) sufficient structure to perform the claimed function so as to avoid it/them being interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph.
Moreover, Examiner notes that, as to claim 12, while the term “high” is a relative term, the limitation of “a high magnetic response” is understood to be limited to a magnetic response having a Bmax in the range recited in claim 12.
As to claim 13, “a high surface area” is understood to be limited to the surface area recited in claim 13.
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.
Claim(s) 1, 2, 17-20 are rejected under 35 U.S.C. 103 as being unpatentable over CN 103675115 (hereinafter “Zhang”) in view of US 20170233798 (hereinafter “Neely”).
Zhang teaches the following.
The invention relates to natural medicine screening technology field, the invention claims a marine natural active substance bead high-content quick screening method, is based on bead bond and high connotation target protein integrated active substance screening and evaluation method, the arrangement of the invention are: the hardware system is a permanent magnet, a magnetic bead, a target software system responsible for hygroplasm data collection. Compared with the screening method and the traditional ultraviolet screening method of the invention, obviously improves the screening efficiency, shorten the filtering time, and meanwhile able to inspect the multiple biological activities, discovery of a synergistic effect between the compound. screening method of the invention is quick and accurate, large information quantity and good repeatability, which can be used for large-scale screening natural product library or a combinatorial chemical library. See abstract.
The invention is directed to screening a target, comprising the following steps (para. 0007):
A, using magnetic bead as carrier, functionalized to bind to a target (para. 0008)
B, incubating the magnetic bead with a sample (para. 0009)
C, removing the supernatant, washing the magnetic beads by denaturing solvent to denature the enzyme, and collecting the eluent (para. 0010);
identifying a substance in the eluent through liquid phase chromatography-mass spectrometry and nuclear magnetic resonance technology authentication analyzing, obtaining target protein inhibitor, wherein the liquid chromatography-mass spectrometry (hygroplasm) parameter is ESI positive and negative ion mode (para. 0011);
said target protein is a DNA topoisomerase, alpha-glucosidase or triglyceride enzyme and so on (para. 0012)
in said step A is specifically as follows: the magnetic bead washing several times, adding 1 - (3 amino propyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) activated magnetic bead surface, adding dissolved DNA topoisomerase, alpha-glucosidase and triglycerides such as drug-related target protein, after incubating for a period of time, the enzyme is bonded to the magnetic bead surface, cleaning for several times (para. 0013).
The sample comprises a marine organism (para. 0014).
The screening method of the invention includes providing
a concentration of 50 mg/mL with 25mM MES (pH 6) is 50 mg/mL N-hydroxy succinimide (NHS) solution. on the magnetic beads after cleaning, adding 50 microlitres of EDC solution and 50 microlitres of NHS solution, uniformly mixing, slowly inclining rotating at room temperature hatching for 30 min. after incubation, putting the centrifugal tube absorbing 4 min on the magnet, removing the supernatant, using 300 mu L 25mM MES (pH6) washing for two times (para. 0019);
second step: on the activated magnetic bead, respectively adding 60 mu L dissolved in 25mM MES solution (pH DNA topoisomerase 6) 1, alpha-glucosidase and triglyceride, and respectively adding 40 mu L 25mM MES solution (pH 6) to a final volume of lOOyL, oscillating and mixing uniformly at room temperature incubation overnight, after incubation, putting the centrifugal tube absorbing 4 min on the magnet, removing the supernatant; using 300 mu L PBS coated bead 4, covers proteins such as bovine serum albumin (BSA) or skim milk powder concentration can reach 0.1-0.5 %, the coated beads dispersed in the required concentration of PBS and Tris buffer solution again (para. 0021);
the third step: using the transmission electronic microscope to blank beads and bonded bead, characterizing the properties [para. 0022];
a fourth step: weighing marine natural product extract, adding a small amount of dimethyl sulfoxide (DMSO) to dissolve, then adding
0.1 mL phosphate buffer saline (PBS) solution (pH 6.8, 10mM) ultrasonic IOmin to dissolve, absorbing supernatant liquid mass analysis to obtain HP(para. 0023).LC and molecular quantity information
fifth step: fourth step marine natural product to obtain supernatant 40 μ L, respectively adding to 100 mu L, DNA topoisomerase 1 alpha-glucosidase and triglyceride enzyme beads dispersed in acetic acid solution and blank beads dispersed in the PBS buffer solution, placing on the oscillator mixing uniformly, incubated for I hour, using PBS buffer solution to wash for 3 times, each time of cleaning solution in liquid phase analysis (para. 0024);
a sixth step of removing the cleaning liquid, each connected in the container adding 10% acetonitrile-water (mL) to wash eluent to liquid-mass spectrometry analysis (para. 0025);
seventh step: through comparing the fourth step and the sixth step liquid obtained by mass analysis spectrum can be obtained with DNA topoisomerase 1, alpha-glucosidase and bead triglyceride binding of marine natural product retention time and molecular quantity information (para. 0026).
An instrument is disclosed for high performance liquid chromatography tandem dbrs LCQ Deca XPplus ion trap mass spectrometry, chromatographic column: Agilent Zorbax SB-C18 column (4.6X 250 mm, 5 microns) ;Agilent 1200 preparation liquid phase (XDB-C18column, 30X, 250 mm, 5 microns); constant temperature oscillation incubator. ELx800 microplate reader. NMR (Bruker AvanceIII 500M NMR). (Paras. 0044-0045).
Embodiment 1: key based on magnetic beads and DNA topoisomerase, high-content screening method of alpha-glucosidase and triglyceride (para. 0046) on the activated magnetic beads, respectively adding 60 mu L dissolved in 25mM MES solution (pH 6) of DNA topoisomerase Ka-glucosidase and triglyceride, and respectively adding 40 mu L 25mM MES solution (pH 6) to a final volume of 100 [mu] L, oscillating and mixing uniformly at room temperature incubation overnight, after incubation, the tube is placed on the magnet 4 min and removing the supernatant. using 300 mu L PBS coated bead 4, covers proteins such as BSA or skim milk powder concentration can reach 0.1-0.5 %, the coated beads dispersed in the required concentration of PBS and Tris buffer solution again (para. 0048).
Using a transmission electron microscope to the blank bead to bead and bonding property characterization, can be seen from FIG. 1, clear of blank beads (A) under the transmission electron microscope, and combines the bead of the triglyceride (B) surface is very fuzzy, which means it has a protein adhesion on the magnetic bead surface (para. 0049).
Thus, as to Applicant’s claim 1, Zhang teaches:
a method of preparing a protein analyte for characterization [see abstract, paras. 0007, 0011, 0023, 0025-0026]
comprising pretreating a protein analyte [abstract, paras. 0010 and 0012],
in the presence of first magnetic beads [see abstract, paras. 0046]
and enzymatically digesting the pretreated protein analyte to provide a plurality of peptides [see paras. 0010].
However, Zhang does not teach that the pretreatment or enzymatically digesting is done on magnetic beads in a changing magnetic field.
Zhang however does teach oscillating an mixing uniformly the activated magnetic beads the reagents used prior to analysis under mass spectrometry (see paras. 0021, 0045, 0048).
Moreover, Neely discloses the following.
Embodiments of the invention include devices, systems, and/or methods for detecting and/or measuring the concentration of one or more analytes in a sample (e.g., a protein, a peptide, an enzyme, a polypeptide, an amino acid... The analytes may include identification of cells or specific cell types. The analyte(s) may include one or more biologically active substances and/or metabolite(s), marker(s), and/or other indicator(s) of biologically active substances. A biologically active substance may be described as a single entity or a combination of entities…Para. 0270.
Medical tests (e.g., blood tests, urine tests, and/or other human or animal tissue tests) that may be performed using various embodiments of the invention described herein include, for example, general chemistry tests (e.g., analytes include albumin, blood urea nitrogen, calcium, creatinine, magnesium, phosphorus, total protein, and/or uric acid); electrolyte tests (e.g., analytes include sodium, potassium, chloride, and/or carbon dioxide); diabetes….; tests for protein—CSF (e.g., analytes include immunoglobulin G and/or urinary/cerebrospinal fluid protein); toxicology tests… Para. 0286.
The systems and methods of the invention can also be used to monitor and diagnose neurological disease, such as dementia (a loss of cognitive ability in a previously-unimpaired person) and other forms of cognitive impairment. Without careful assessment of history, the short-term syndrome of delirium (often lasting days to weeks) can easily be confused with dementia, because they have all symptoms in common, save duration, and the fact that delirium is often associated with over-activity of the sympathetic nervous system. Some mental illnesses, including depression and psychosis, may also produce symptoms that must be differentiated from both delirium and dementia. Routine blood tests are also usually performed to rule out treatable causes. These tests include vitamin B12, folic acid, thyroid-stimulating hormone (TSH), C-reactive protein, full blood count, electrolytes, calcium, renal function, and liver enzymes…The method of the invention can be a multiplexed, no sample preparation, single detection method, automated system to determine the drug level, the toxicity or adverse effect determinant, and the potential biomarker of the progression of the disease. For example, a cartridge having portals or wells containing 1) magnetic particles having protein biomarker specific antibodies decorated on their surface, 2) magnetic particles having specific antibodies on their surface, and 3) magnetic particles having nucleic acid specific probes to identify protein expression levels could be employed to rapidly determine and provide clinical management values for a given dementia subject. Para. 0294.
The systems and methods of the invention can also be used to monitor and diagnose heart disease in a subject, such as a myocardial infarction. Cardiac markers or cardiac enzymes are proteins that leak out of injured myocardial cells and are used to assess cardiac injury. Cardiac markers include, without limitation, the enzymes SGOT, LDH, the MB subtype of the enzyme creatine kinase, and cardiac troponins (T and I). … The systems and methods of the invention can be used to provide a multiplexed, no sample preparation, single detection method, automated system to determine the drug level, the toxicity or adverse effect determinants, and the potential biomarker of the progression of the disease. For example, a cartridge having portals or wells containing 1) magnetic particles having anti-troponin I or troponin T specific antibodies decorated on their surface, 2) magnetic particles having toxicity biomarker specific antibodies on their surface, and 3) magnetic particles having specific probes to identify disease markers of progression could be employed to rapidly determine and provide clinical management values for a given myocardial infarction patient. Para. 0312.
The proteins described above would be analyzed in the hepatic function panel using the systems and methods of the invention. Para. 0314.
The cycling MAA approach described herein can accelerate the kinetics of magnetic particle-analyte clustering by (i) reducing the spatial entropy of the binding interaction step by maintaining local concentration of the magnetic particles, (ii) introducing localized mixing by magnet mediated transportation of the pellet from position to position, (iii) reducing shearing of the specific-bound clusters by reducing the need for more energetic dispersion methods, such as vortexing, and/or (iv) changing the magnetic field direction, and thereby causing a local dispersion and re-aggregation of magnetically clustered particles as they re-align their dipoles with the new magnetic field direction, and allowing the locally dispersed magnetic particles to form specific binding interactions involving the target analyte. Para. 0442.
Thus, Neely discloses detecting and/or measuring the concentration of one or more analytes in a sample (e.g., a protein, a peptide, an enzyme, a polypeptide, an amino acid) (paras. 0270, 0314), for any of various purposes (such as for medical reasons or for study) (para. 0286, 0294, 0312). Neely teaches that the invention can be multiplexed, using an automated system or cartridge having magnetic particles with antibodies or protein biomarker on their surface to identify analytes (para. 0294, 0312).
Neely also teaches accelerating the kinetics of magnetic particle-analyte clustering by (i) reducing the spatial entropy of the binding interaction step by maintaining local concentration of the magnetic particles, (ii) introducing localized mixing by magnet mediated transportation of the pellet from position to position, (iii) reducing shearing of the specific-bound clusters by reducing the need for more energetic dispersion methods, such as vortexing, and/or iv) changing the magnetic field direction, and thereby causing a local dispersion and re-aggregation of magnetically clustered particles as they re-align their dipoles with the new magnetic field direction, and allowing the locally dispersed magnetic particles to form specific binding interactions involving the target analyte. Para. 0442.
It would have been obvious to one skilled in the art to perform the bead contacting step(s) in Zhang in a magnetic field that is changing since this provides the advantage of local dispersion and re-aggregation of magnetically clustered particles, allowing the locally dispersed magnetic particles to form specific binding interactions involving the target analyte, as taught by Neely. One skilled in the art would have recognized the desirability of facilitating the specific binding interactions for a more thorough and sensitive assay.
As to claim 2, Zhang teaches that the characterization comprises analyzing the plurality of peptides comprising LC-MS/MS [see paras. 0011, 0023, 0025-0026.]
As to claim 17, Zhang teaches that the first magnetic beads do not comprise an immobilized endopeptidase enzyme. [See para. 0022, 0024, 0049, disclosing blank beads.]
As to claim 18, the changing magnetic field taught by Neely is considered an electromagnetic mixer.
As to claim 19, Neely teachings providing a changing magnetic field as discussed above. It would have been within the skills of the ordinary artisan to provide a plurality of electromagnets capable of generating an AC driven oscillating magnetic field since such well-known elements would have resulted in the predictable outcome of providing the changing [equivalent to an oscillating magnetic field] taught by Neely.
As to claim 20, the protein analyte disclosed by Zhang (or Neely) is considered a complex protein sample or an isolated protein.
Claims 3-6, 21, 23, 25 and 28 is/are rejected under 35 U.S.C. 103 as being unpatentable over CN 103675115 (hereinafter “Zhang”) in view of US 20170233798 (hereinafter “Neely”), as applied to claim 1 above, and further in view of US 20190086306 (hereinafter “Lopez”).
Lopez teaches the following, which suggests the missing limitations of Zhang in view of Neely as follows.
Lopez teaches a method for preparing a protein-containing sample for analysis by mass spectrometry includes introducing the sample into a reaction vessel. The reaction vessel contains a reagent mixture including pre-measured quantities of an immobilized proteolytic enzyme, a reducing agent and an alkylating agent. See abstract and paras. 0006-0008.
Lopez teaches a method for preparing a protein-containing sample for mass spectrometry analysis, comprising: concurrently reacting the sample with an alkylating agent, a reducing agent and an immobilized proteolytic enzyme; and activating the sample by performing at least one of heating the sample, sonicating the sample, and applying pressure to the sample. Para. 0009.
The term “alkylating efficiency” is defined as the quantitative relation between the number of detected cysteines that have been modified by the alkylating agent during the reaction and the total number of cysteines detected in the sample, as determined by analyzing the sample using mass spectrometry followed by a database search. A specific and non-limiting example includes alkylation of cysteine with chloroacetamide (CAA) or Iodacetic acid (IAC) that increases the mass of the peptide by 57.021 or 58.005 Daltons, respectively. This increase in mass is measured using a mass spectrometer and quantified during the database search. Para. 0028.
Referring now to FIG. 1A, shown is an overview of the workflows of three prior art methodologies for preparing a protein-containing sample for subsequent analysis by mass spectrometry, as well as the workflow of a new one-step protocol according to an embodiment. The so-called “classic method” includes the steps of cell lysis, protein abundance determination,
denaturation/reduction/alkylation, and digestion, all of which are performed sequentially. In the cell lysis step a lysis buffer solution, containing salts such as Tris-HCl or EDTA and optionally detergents such as Triton X-100 or SDS, is added to the sample to break open the cells contained therein. The “protein abundance” step provides a quantitative measure of protein in the sample, so that the concentration of trypsin used for the subsequent digestion step may be adjusted to yield a trypsin-to-protein ratio of about 1:50 (wt:wt). The proteins are then denatured with strong chaotropic agents such as urea or thiourea. This step is either followed by or combined with disulfide reduction using a reducing agent such as tris(2-carboxyethyl) phosphine (TCEP) or dithiothreitol (DTT). The free sulfhydryl groups on the cysteine residues are then alkylated with reagents such as iodoacetamide or iodoacetic acid to irreversibly prevent the free sulfhydryls from reforming disulfide bonds. The denatured, reduced and alkylated proteins are then digested by endoproteinases, (e.g., trypsin, chymotrypsin, Glu-C and Lys-C), which hydrolytically break peptide bonds to fragment proteins into peptides. The last two steps of the classic method are particularly time-consuming, accounting for most of the six-and-a-half to twenty-and-a-half hours required to complete a sample preparation. Para. 0031.
Referring now to FIG. 1B, the “Classic Method” workflow is shown in more detail. Each step that was discussed with reference to FIG. 1A comprises a series of sub-steps, each requiring various reagent additions and/or other user intervention. The cell lysis step includes the addition of a lysis buffer and the addition of benzoate, after which sonication occurs. The protein concentration in the resulting solution is then determined in a separate step. Next, a protein denaturation step is performed by adding a reducing agent and an alkylating agent. The duration of the protein denaturation step is about 2 hours. Finally, a digestion step is performed by adding Lys C, usually at a 1:100 enzyme:substrate ratio, and incubating for about 2 hours, after which a buffer is added and trypsin is added, in this case the enzyme:substrate ratio tend to vary from 1:10 up to 1:100, and the sample is incubated again for an additional 4 to 16 hours. After an acid addition step to stop the enzymatic activity, the sample is ready to be cleaned up and analyzed by mass spectrometry. Para. 0033.
Referring now to FIG. 1C, the two-step protocol according to an embodiment is shown in more detail. Samples that need an extra energy input during the protein extraction process are e.g., processed using the two-step protocol described below. In step 1 of the two-step protocol the sample is added to a sonication tube containing a lysis buffer. The lysis buffer contains a buffered solution, e.g. ammonium bicarbonate 50 mM pH 8.2 and suitable chaotropic agents or detergents that are capable of disrupting the hydrogen bonding network between water molecules and reducing the stability of the native state of proteins and membranes by weakening the hydrophobic effect. Usually, 6M urea, 1% SDS, 1% SDC are the typical additives. Once the lysis buffer has been added to the sample, the resulting solution is sonicated for e.g., about 5 minutes. High intensity ultrasound energy helps disrupt DNA as well as cell membranes, allowing proteins to be extracted efficiently from cellular matrices of the sample. In step 2 of the two-step method, the lysis buffer containing the sample is added to a reaction tube containing a reagent mixture including pre-measured quantities of immobilized proteolytic enzymes, a reducing agent, and an alkylating agent. By way of a specific and non-limiting example, 120 uL of immobilized trypsin slurry is added to the vessel, the beads are cleaned three times with ammonium bicarbonate, and then the supernatant is removed from the tube. A solution containing 5 mM of TCEP, 15 mM CAA in 50 mM ammonium bicarbonate pH 8.2 is then added to the beads and the mixture is ready for the sample to be added. Once the sample is added, the tube contents are heated at e.g., 50° C. for an additional e.g., 60 minutes. The tube or vessel can be centrifuged or the solution filtered out to remove the beads, after which the sample is ready to be analyzed by mass spectrometry. Para. 0034.
Referring now to FIG. 1D, the one-step protocol according to an embodiment is shown in more detail. In step 1 of the one-step protocol the user adds the sample into a reaction vessel containing a reagent mixture including a lysis buffer, pre-measured quantities of an immobilized proteolytic enzyme, a reducing agent and an alkylating agent. The contents of the reaction vessel are then activated, such as for instance by sonication for a period of about 5 minutes or by heating between 37° C. and 50° C. for between five and ninety minutes. As used herein, the term “activating” and its cognates refers to the act of adding energy to the contents of the reacting vessel so as to accelerate the rate of the desired reaction of the sample (or components thereof) with the various reagents. This workflow is envisioned for use with, for instance, mammalian cells and other types of samples such as purified proteins where exhaustive protein extraction is not needed, and the ultrasound step can be skipped during the protein extraction step. Para. 0035.
A specific and non-limiting example of the one-step protocol according to an embodiment uses a closeable reaction vessel, such as for instance an Eppendorf tube, which contains a reagent mixture prepared using 1 μL tris(2-carboxyethyl)phosphine (TCEP) 0.2 M solution, 0.2 mg of CCA, 0.8 mg of CaCl2 and 50 μL Poroszyme® immobilized trypsin beads. In this example, the reagent mixture includes a proteolytic enzyme in the form of immobilized trypsin (such as Poroszyme® beads, available from Thermo Fisher Scientific), however, other suitable immobilized enzymes may be used, as long as the immobilization process helps to stabilize the secondary and tertiary structure of the protein without decreasing its activity. Importantly, having the enzyme immobilized to a solid support allows the enzyme to be present in a very high enzyme:substrate ratio (i.e. 100:1), forcing the enzyme kinetics to speed up due to its dependence on enzyme concentration. Advantageously, the immobilized enzyme does not contaminate the sample and it can easily be removed from the sample, since the solid support is very easy to separate from the solution. A non-limiting list of alternative reagents and/or concentrations includes at least the following: buffer pH: 7.8-8.2; reducing agent TCEP 1-5 mM; alkylating agent CAA 10 mM-25 mM; alkylating agent IAC 1-5 mM; alkylating agent IAA 1-15 mM; and CaCl.sub.2 0.1-1 mM. As suggested above, the reagent mixture optionally includes a proteolytic enzyme other than trypsin, such as for instance one of LysC, LysN, AspN, GluC, ArgC or chymotrypsin. Optionally, the reagent mixture is dried down and is stored below room temperature prior to use, such as for instance stored at 4° C., −20° C. or −80° C. Para. 0036.
In certain embodiments of the invention, the reagent mixture contained within the prepared reaction vessel includes an isobaric labeling reagent that reacts with the peptides formed by protein digestion to produce isotopically labeled peptides. For example, a kit prepared in accordance with an embodiment may include a set of reaction vessels containing the above-described reagent mixture (comprising reducing and alkylating agents and immobilized proteolytic enzyme), each one of which further contains a different isotopologue of an isobaric labeling reagent, such that differentially labeled samples may be prepared and subsequently combined so as to determine relative quantities of peptides of interest via analysis by liquid chromatography-mass spectrometry, using techniques well known in the art. Isobaric labeling reagents are available from Thermo Fisher Scientific under the trade name Tandem Mass Tags. Para. 0037.
The stability of the reaction mixture that is employed in the new protocol, which includes immobilized trypsin, a reducing agent (TCEP), and an alkylating agent (CAA), was investigated and the results are presented in FIGS. 8-10. More particularly, the reagent mixture was prepared and then stored at various temperatures between −80° C. and 24° C. for a period of three weeks, prior to being used to incubate samples containing one million HELA cells. The HELA cells were added to previously stored reaction mixture, and digested using the new protocol according to an embodiment of the invention (i.e., with heating for 1 h @50° C.). After the digestion, lug of digested peptides were transferred to an autosampler vial, and then analyzed by LC-MS/MS using a 2 h gradient in a Thermo Scientific EASY-nLC1200 system coupled to a Thermo Scientific Q Exactive HF mass spectrometer. Para. 0053.
Thus, the above shows that Lopez teaches and/or suggests the following limitations as part of a method for preparing a protein-containing sample for analysis by mass spectrometry:
a chaotropic agent to provide a denature protein solution (as in Applicant’s claim 3);
a reducing agent to the denatured protein solution to provide a reduced, denatured protein analyte (as in Applicant’s claim 4);
the mixing comprises exposing the denatured protein analyte to an alkylating agent to provide
an alkylated protein analyte (as in Applicant’s claim 5);
quenching excess alkylating agent by adding a reducing agent to the alkylated protein analyte to provide the pretreated protein analyte (as in Applicant’s claim 6);
a chaotropic agent that is an organic chaotropic agent (as in Applicant’s claim 21, 23);
an alkylating agent that is iodoacetic acid (as in Applicant’s claim 25); and
pretreating and enzymatically digesting are performed sequentially (as in Applicant’s claim 28).
It would have been obvious to one skilled in the art to utilize the reagents and steps disclosed by Lopez as part of the protein analysis by mass spectrometry in the Zhang invention since Lopez teaches that these are known and classic methods for preparing a protein-containing sample for analysis by mass spectrometry.
Claims 7-16, 26, 27 is/are rejected under 35 U.S.C. 103 as being unpatentable over CN 103675115 (hereinafter “Zhang”) in view of US 20170233798 (hereinafter “Neely”), and US 20190086306 (hereinafter “Lopez”), as applied to claims 1 and 3 above, and further in view of US 20070128729 (hereinafter “Puente”).
As to claims 7 and 8, Zhang, Neely, and Lopez, discussed above, are silent as to an endopeptidase enzyme being immobilized.
However, Lopez does teach a method for preparing a protein-containing sample for analysis by mass spectrometry which includes introducing an immobilized proteolytic enzyme, a reducing agent and an alkylating agent (see for example, paras. 0006-0008).
Moreover, Puente show that endopeptidase such as trypsin is used for enzyme pretreatment [digesting] for protein analysis by mass spectrometry (see claim 1 in Puente), and thus it would have been obvious to one skilled in the art to utilize endopeptidase, or specifically trypsin, in the Zhang-Neely-Lopez invention in order to analyte protein by mass spectrometry.
As to claim 9, Puente does not requires that the endopeptidase enzyme be a free endopeptidase enzyme or an immobilized endopeptidase enzyme, and therefore it would have been obvious that it could be either. Moreover, Lopez discloses an immobilized proteolytic enzyme (see for example para. 006-008.)
As to claim 10, multiple beads are taught by Zhang (see discussion of Zhang above).
As to claim 11, Neely teaches that the beads [and therefore the claimed first magnetic beads and the second magnetic beads independently comprise superparamagnetic particle (see Neely, para. 0017).
As to claim 12, while Zhang is silent as to the first magnetic beads and the second magnetic beads independently have a high magnetic response having a Bmax in a range of from about 20 emu/g to about 250 emu/g, 40 emu/g to 200 emu/g, 50 emu/g to 150 emu/g. or 80 emu/g to 100 emu/g, such a magnetic response falls within a workable or optimum range, and thus its discovery would have required ordinary skills in the art, given that Zhang, Neely, Lopez, and Puente teaches the general conditions of the claims, as discussed above.
As to claim 13, while Zhang is silent as to the first magnetic beads and/or the second magnetic beads independently have a high surface area of> 5 m2/g, such range falls within a workable or optimum range, and thus its discovery would have required ordinary skills in the art, given that Zhang, Neely, Lopez, and Puente teaches the general conditions of the claims, as discussed above.
As to claim 14, the enzyme taught by Lopez and Puente is equivalent to the claimed coating.
As to claim 15, enzyme taught by Lopez and Puente is equivalent to the claimed functional group-coated surface.
As to claim 16, the protein or antibody or any part of its chemical composition taught by Zhang, Neely, Lopez, or Puente is equivalent to the claimed bioaffinity adsorbent.
As to claim 26, Zhang teaches that the pretreating is completed in a period of time within a range of from 5 to 90 minutes.
As to claim 27, Zhang teaches that the enzymatically digesting is completed in a period of time from 5 to 60 minutes.
Claim 22 is rejected under 35 U.S.C. 103 as being unpatentable over CN 103675115 (hereinafter “Zhang”) in view of US 20170233798 (hereinafter “Neely”), as applied to claim 1 above, and further in view of US 20190086306 (hereinafter “Lopez”), as applied to claim 3 above, and further in view of WO 2015055825 (hereinafter “Poglitsch”).
While Zhang, Neely, and Lopez, discussed above, are silent as to the chaotropic agent being ethanol, Poglitsch discloses that this is a known chaotropic agent (see para. 0104), and thus its use requires ordinary skills in the art as a known chaotropic agent, as shown by Poglitsch, with reasonable expectation of success as a substitute chaotropic agent in the modified Zhang invention.
Claim 24 is rejected under 35 U.S.C. 103 as being unpatentable over CN 103675115 (hereinafter “Zhang”) in view of US 20170233798 (hereinafter “Neely”), as applied to claim 1 above, and further in view of US 20190086306 (hereinafter “Lopez”), as applied to claim 3 above, and further in view of US 20150126402 (hereinafter “Shabb”).
While Zhang, Neely, and Lopez (discussed above) is silent as to iodoacetic acid being the alkylating agent, Shabb discloses that this is a known alkylating agent (para. 0289), and thus its use requires ordinary skills in the art as a known alkylating agent, as shown by Shabb, with reasonable expectation of success as a substitute alkylating agent in the modified Zhang invention.
Conclusion
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/Ann Montgomery/ Primary Examiner, Art Unit 1678