Methods and systems for detecting protein biomarkers of rheumatoid arthritis in a biological sample using LC-ms / ms

Abstract

Disclosed are methods and systems using mass spectrometry for the detection and / or quantification of 14-3-3 proteins, or surrogate peptides thereof, in a biological sample.

Description

PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCTMETHODS AND SYSTEMS FOR DETECTING PROTEIN BIOMARKERS OF RHEUMATOID ARTHRITIS IN A BIOLOGICAL SAMPLE USING LC-MS / MSREFERENCE TO A SEQUENCE LISTING

[0001] The present application contains a Sequence Listing which has been submitted herewith and is hereby incorporated by reference in its entirety. The .xml copy of the Sequence Listing, created on June 3, 2025, is named 057618-1509623, and is 17,907 bytes in size.FIELD OF INVENTION

[0002] The present disclosure generally relates to improved methods and systems for detecting and / or quantifying one or more 14-3-3 proteins (e.g, 14-3-3q protein), or surrogate peptide thereof, in a sample using mass spectrometry (e.g, LC-MS / MS).BACKGROUND

[0003] Rheumatoid arthritis is one of the most common systemic autoimmune diseases. There are multiple pathophysiological factors that contribute to the onset of Rheumatoid arthritis and its symptoms, and there is high heterogeneity' among patients throughout the disease. If untreated, Rheumatoid arthritis results in severe joint destruction, leading to impaired physical activity and disability. The disease outcome and patient prognosis can be significantly improved by early identification of Rheumatoid arthritis and prediction of the disease severity at diagnosis for the implementation of an effective treatment strategy.

[0004] Early diagnosis of Rheumatoid arthritis is difficult due to limited clinical symptoms and an absence of physical signs. Laboratory data of patients with arthralgia, such as levels of rheumatoid factor (RF) and anticyclic citrullinated peptide (ACCP), are not remarkable for Rheumatoid arthritis. Therefore, there is a great need for the development and validation of new biomarkers that have predictive capacity' for Rheumatoid arthritis.

[0005] Additionally, there is a need for quantitative assays for protein biomarkers of Rheumatoid arthritis in various complex protein samples, e.g, in human serum. Conventionally these assays have been implemented as immunoassays, making use of specific antibodies against target proteins as specificity7and detection reagents. New methods that allow mass spectrometry' (MS) to provide quantitative peptide and protein detection are needed. However, there remains an issue of the dynamic range and sensitivity of MS assaysPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT when applied to very complex mixtures, such as those created by digestion of whole plasma protein to peptides.SUMMARY

[0006] In some embodiments, the present disclosure is related to methods and systems for detecting or quantifying a 14-3-3q protein in a sample using LC-MS / MS. The method may be embodied in a variety of ways.

[0007] In some embodiments, the method for determining the amount of 14-3-31} protein in a sample includes: (a) generating a precursor ion of 14-3-3 q protein; (b) optionally, generating one or more fragment ions of the precursor ion; and (c) detecting the amount of one or more ions in step (a) or (b) or both to determine the amount of 14-3-3q protein in the sample. In some embodiments, the method further comprises purifying the sample prior to step (a). In some embodiments, the purification step comprises liquid chromatography. In some embodiments, step (a) comprises generating one or more precursor ions of the 14-3-3q. In some embodiments, the method comprises enriching the sample in the 14-3-3q protein prior to step (a). In some embodiments, the sample can be enriched in the 14-3-3q protein using solid-phase extraction, precipitation, affinity enrichment, immunoaffinity enrichment, or combinations thereof to produce an enriched sample. The antibodies used for immunoaffinity enrichment can be specific to 14-3-3q protein or fragments thereof.

[0008] In some embodiments, the method for determining the amount of 14-3-3q protein in a sample includes: (a) generating a precursor ion of a surrogate peptide of 14-3-3q protein; (b) generating one or more fragment ions of the precursor ion; and (c) detecting the amount of one or more ions in step (a) or (b) or both to determine the amount of 14-3-3q protein in the sample. In some embodiments, the method further comprises purify ing the sample prior to step (a). In some embodiments, the purification step comprises liquid chromatography. In some embodiments, step (a) comprises generating one or more precursor ions of the 14-3-3q surrogate peptide having a mass to charge ratio selected from the group consisting of 408.7+0.5, 516.2+0.5, 739.9+0.5, 454.3+0.5, 308.2+0.5, 452.3+0.5, 433.2.7+0.5, 874.4+0.5, 652.8+0.5, 533.8+0.5, 634.3+0.5, 412.2+0.5, 412.9+0.5, 991.5+0.5, 720.0+0.5, 595.3+0.5, and 708.0+0.5. In some embodiments, the method comprises enriching the sample in the 14- 3-3q protein prior to step (a). In some embodiments, the sample can be enriched in the 14-3- 3q protein using solid-phase extraction, precipitation, affinity enrichment, immunoaffinity enrichment, or combinations thereof to produce an enriched sample. The antibodies used forPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT immunoaffinity enrichment can be specific to 14-3-3q protein or fragments thereof. In some embodiments, the antibody used for immunoaffinity enrichment can be specific to 14-3-3q protein and at least one other family member of 14-3-3 protein (e.g., 14-3-3(3, 14-3-3y, 14-3- 3a, etc.) or fragments thereof. In some embodiments, the antibody used for immunoaffinity enrichment is a pan- 14-3-3 protein antibody. In some embodiments, affinity enrichment may be performed with a binding partner of 14-3-3q or 14-3-3 protein family. In some embodiments, the method includes digesting the enriched sample to produce one or more surrogate peptides of 14-3-3q protein or 14-3-3 protein family.

[0009] In some embodiments, a method may include providing a biological sample comprising a plurality of proteins including 14-3-3q protein; adding a binding agent that specifically binds to 14-3-3q protein to the biological sample; separating 14-3-3q protein bound to the binding agent from unbound proteins in the biological sample to produce an enriched sample; contacting the enriched sample with a proteolytic enzyme to produce a proteolytic digest comprising peptides, wherein at least one of the peptides comprises a surrogate peptide of 14-3-3q protein; performing liquid chromatography on the proteolytic digest to purify the sample; and measuring the amount of 14-3-3q protein in the biological sample by detection of the surrogate peptide of 14-3-3q protein using mass spectrometry. In some embodiments, the binding agent that specifically binds to 14-3-3q protein comprises an antibody or an aptamer or a binding partner. In some embodiments, the proteolytic enzyme may be trypsin or an isozyme thereof. In some embodiments, the biological sample may be serum or plasma.

[0010] In some embodiments, the method may include providing a biological sample comprising a plurality of proteins including at least one 14-3-3 protein family member; adding a binding agent that specifically binds to one or more 14-3-3 protein family members to the biological sample; separating 14-3-3 protein family members bound to the binding agent from unbound proteins in the biological sample to produce an enriched sample; contacting the enriched sample with a proteolytic enzyme to produce a proteolytic digest comprising peptides, wherein at least one of the peptides comprises a surrogate peptide of one or more 14-3-3 protein family members; performing liquid chromatography on the proteolytic digest to purify the sample; and measuring the total amount of 14-3-3 protein family members in the biological sample by detection of the surrogate peptide of the one or more 14-3-3 protein family members using mass spectrometry. In some embodiments, the binding agent that specifically binds to 14-3-3 protein family members comprises an antibodyPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT or an aptamer or a binding partner. In some embodiments, the proteolytic enzyme may be trypsin. In some embodiments, the biological sample may be serum or plasma. In some embodiments, the total amount of 14-3-3 protein family members measured includes 14-3-3q protein.

[0011] In some embodiments, the 14-3-3q protein or 14-3-3 protein family members bound to the binding agent prior to liquid chromatography are separated by first coupling the binding agent to a solid phase substrate. The solid phase substrate can be washed to remove unbound proteins from the solid phase substrate and eluting the 14-3-3q protein or 14-3-3 protein family members bound to the binding agent from the solid phase substrate. In some embodiments, measuring the amount of 14-3-3q protein in the sample using mass spectrometry may comprise the steps of: (i) generating at least one precursor ion of 14-3 -3 q surrogate peptide; (ii) generating one or more fragment ions of the precursor ion; and (iii) detecting the presence or amount of the precursor ion generated in step (i) and / or the at least one or more fragment ions generated in step (ii), or both, and relating the detected ions to the presence or amount of the 14-3-3p protein in the biological sample. In some embodiments, the at least one precursor ion is formed by electrospray ionization. The electrospray ionization may be performed in positive or negative ion mode. In yet another embodiment, the mass spectrometry detection of the 14-3-3q protein surrogate peptide or 14-3-3 protein family surrogate peptide may be performed in selected reaction monitoring mode (SRM). In some embodiments, more than one surrogate peptide may be detected by mass spectrometry.

[0012] In some embodiments, the method may further comprise adding an internal standard to the biological sample, wherein the internal standard is a stable isotope of 14-3-3q protein. In some embodiments, the method may comprise adding an internal standard to the enriched sample, wherein the internal standard includes a stable isotope of the surrogate peptide and can be proteolyzed to form a stable isotope surrogate peptide. In other embodiments, the method may comprise adding an internal standard to the enriched or proteolyzed sample, wherein the internal standard is a stable isotope of the surrogate peptide. The detection of the 14-3-3q protein may be over a range of from 0.1 ng / mL to 100 ng / mL. In yet another embodiment, the mass spectrometry detection of the stable isotope of 14-3-3q protein may be performed in selected reaction monitoring (SRM) mode. The at least one precursor ion for a surrogate peptide 14-3-3q protein may have a mass / charge ratio (m / z) of about 516.2±0.5 and the one or more fragment ions comprise a fragment ion that may have an m / z of about 868.4±0.5, 753.3±0.5, 638.3±0.5, 507.3±0.5, 436.2±0.5, 279. H0.5, 394.H0.5, 136.1±0.5, orPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT251.1±0.5. In some embodiments, the liquid chromatography may include high performance liquid chromatography (HPLC). The liquid chromatography may be performed in reverse phase separation or hydrophilic interaction liquid chromatography.

[0013] In yet another embodiment, the disclosure provides a method for determining the presence or amount of at least one biomarker of interest in a biological sample, the method comprising: providing a biological sample comprising a plurality of proteins including 14-3- 3q protein; contacting a proteolytic enzy me with the biological sample to produce a proteolytic digest including peptides, wherein at least one of the peptides comprises a surrogate peptide of 14-3-3 q protein; adding one or more binding agent to the proteolytic digest of the biological sample, wherein the binding agent specifically binds to the surrogate peptide of 14-3-3 r] protein; separating the peptides bound to the binding agent from unbound peptides in the proteolytic digest to produce an enriched sample; purifying the sample enriched with the surrogate peptide of 14-3-3q protein using liquid chromatography; and measuring the amount of 14-3-3q protein in the biological sample by detection of the surrogate peptide of 14-3-3q protein using mass spectrometry. In some embodiments, the binding agent that specifically binds to the surrogate peptide of 14-3-3q protein comprises an antibody or an aptamer. The proteolytic enzy me may include try psin, and the biological sample may be serum or plasma.

[0014] In some embodiments, the disclosure provides a method for determining the presence or amount of at least one biomarker of interest in a biological sample, the method comprising: providing a biological sample comprising a plurality7of proteins including at least one 14-3-3 protein family member; contacting a proteolytic enzyme with the biological sample to produce a proteolytic digest comprising surrogate peptides of the plurality of proteins, wherein at least one of the surrogate peptides comprises a surrogate peptide of one or more 14-3-3 protein family members; adding one or more binding agents to the proteolytic digest of the biological sample, wherein the binding agent specifically binds to the surrogate peptides of one or more 14-3-3 protein family members; separating the surrogate peptides bound to the binding agent from unbound peptides in the proteolytic digest to produce an enriched sample; purifying the sample enriched with surrogate peptide of the one or more 14- 3-3 protein family members using liquid chromatography; and detecting the surrogate peptides of the one or more 14-3-3 protein family members using mass spectrometry to measure the total amount of the one or more 14-3-3 protein family members in the biological sample. In some embodiments, the total amount of 14-3-3 protein family members measuredPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT using surrogate peptides of one or more 14-3-3 protein family members is predominantly from 14-3-3q protein. In some embodiments, the binding agent that specifically binds to the surrogate peptides of the one or more 14-3-3 protein family members comprises an antibody or an aptamer. The proteolytic enzyme may include trypsin, and the biological sample may be serum or plasma.

[0015] In some embodiments, the method may include measuring the amount of 14-3-3q protein or 14-3-3 protein family members in the biological sample using mass spectrometry. The method may include the steps of: (i) generating a precursor ion of a 14-3-3r| surrogate peptide; (ii) generating one or more fragment ions of the precursor ion; and (iii) detecting the presence or amount of the precursor ion generated in step (i) and / or the at least one or more fragment ions generated in step (ii), or both, and relating the detected ions to the presence or amount of the 14-3-3q protein in the biological sample. In some embodiments, the precursor ions are formed by electrospray ionization wherein the electrospray ionization is performed in positive or negative ion mode. The method may further include adding an internal standard to the biological sample, wherein the internal standard is a stable isotope of 14-3-3q protein or fragment thereof.

[0016] In some embodiments, the mass spectrometry detection of surrogate peptides derived from 14-3-3i] protein or 14-3-3 protein family members is performed in selected reaction monitoring (SRM) mode. The detection of the 14-3-31] protein or 14-3-3 protein family members may be over a range of from 0. 1 ng / mL to 100 ng / mL. In some embodiments, the precursor ion for the surrogate peptides derived from 14-3-3i] protein has a mass / charge ratio (m / z) of about 412.2±0.5 and the one or more fragment ions comprise a fragment ion with a m / z of about 623.3±0.5, 476.3±0.5, 347.2±0.5, 234. l±0.5, or combinations thereof.

[0017] In some embodiments, the method may include performing liquid chromatography wherein the liquid chromatography includes high performance liquid chromatography (HPLC). The liquid chromatography technique may be performed in reverse phase separation or hydrophilic interaction liquid chromatography.

[0018] In some embodiments, the present disclosure provides systems for performing the methods of the invention. For example, disclosed is a system for determining the presence and / or amount of a biomarker of interest in a biological sample, the system comprising: a station for providing a biological sample comprising 14-3-3q protein; a station for partially- purifying 14-3-3q protein from other components in the sample; a station for chromatographically separating 14-3-3q protein from other components in the sample; and aPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT station for analyzing the chromatographically separated 14-3-3q protein by mass spectrometry to determine the presence or amount of 14-3-3q protein in the biological sample.

[0019] In some embodiments, the present disclosure provides a computer-program product tangibly embodied in a non-transitory machine-readable storage medium, including instructions configured to cause one or more computers to perform actions to measure a presence or amount of 14-3-3q protein in a biological sample comprising at least one of the following steps: (a) obtaining a biological sample from a subject; (b) optionally adding a stable isotope-labeled 14-3-3q protein to the sample as an internal standard; (c) performing liquid chromatography; and (d) measuring 14-3-31] protein by tandem mass spectrometry.

[0020] In some cases, the biomarker of interest is a protein, and the biological sample may be subjected to proteolytic digestion to generate a peptide from the protein.

[0021] A variety of mass spectrometry techniques may be used. For example, the station for mass spectrometry may comprise a tandem mass spectrometer.

[0022] As noted above, the system may comprise a station for chromatographic purification of the biomarker of interest prior to mass spectrometry, as for example by high performance liquid chromatography (HPLC). Thus, in alternate embodiments the mass spectrometry may comprise liquid chromatography tandem mass spectrometry (LC-MS / MS), or 2 dimensional LC-MS / MS.

[0023] Also, in certain embodiments, as for example where the biomarker of interest is a peptide, the system may comprise a station for subjecting the sample to protease digestion.

[0024] The system may comprise a computer such that at least one of the stations is controlled by the computer.

[0025] In some aspects, the present disclosure provides a method for determining a presence or amount of a l4-3-3q protein in a sample including: (a) providing one or more surrogate peptides of the 14-3-3q protein; (b) ionizing the one or more surrogate peptides of the 14-3-3q protein to generate one or more ions of the surrogate peptides of 14-3-3q protein detectable by mass spectrometry; (c) determining the presence or amount of the one or more ions of the surrogate peptides of the 14-3-3 i protein by mass spectrometry; and (d) determining the presence or amount of the 14-3-3q protein in the sample according to the presence or amount of the one or more ions determined in (c).

[0026] In some embodiments, the sample further comprises one or more surrogate peptides derived from other 14-3-3 protein family members. In some embodiments, providing the onePATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT or more peptides of (a) comprises contacting a biological sample with a proteolytic enzyme to produce a proteolytic digest comprising the one or more surrogate peptides of the 14-3-3q protein. In some embodiments, the method includes, prior to (a), and / or prior to producing the proteolytic digest, enriching the biological sample for one or more 14-3-3 protein family members. In some embodiments, the one or more 14-3-3 protein family members comprises the 14-3-3q protein. In some embodiments, the enriching of the biological sample comprises use of one or more binding agents that bind to one or more of the 14-3-3 protein family members or one or more binding agents that bind specifically to the 14-3-3q protein. In some embodiments, the enriching of the biological sample comprises an immunoprecipitation method. In some embodiments, the one or more binding agents comprise one or more monoclonal antibodies. In some embodiments, the proteolytic enzyme comprises trypsin or an isozyme thereof. In some embodiments, providing the one or more peptides of (a) comprises enriching the proteolytic digest for the one or more surrogate peptides of 14-3-3q. In some embodiments, the enriching of the proteolytic digest comprises contacting the proteolytic digest with one or more binding agents that bind to the one or more surrogate peptides of the 14-3-3q protein. In some embodiments, binding agents comprise one or more antibodies. In some embodiments, the one or more antibodies comprise one or more monoclonal antibodies. In some embodiments, the enriching of the proteolytic digest comprises an immunoprecipitation method. In some embodiments, the one or more binding agents comprise a binding agent that specifically binds to a peptide sequence selected from the peptide sequences of SEQ ID NOs. 2, 3, 7-15 and 17. In some embodiments, the one or more binding agents comprise a binding agent that specifically binds to a peptide sequence selected from the peptide sequences of SEQ ID NOs. 2. 7 and 12. In some embodiments, the proteolytic digest is enriched for a surrogate peptide comprising the sequence of one or more of SEQ ID NOs. 2, 7 and 12. In some embodiments, the one or more surrogate peptides provided of (a) comprise one or more of SEQ ID NOs. 2, 7 and 12. In some embodiments, the one or more surrogate peptides provided in (a) comprise one or more internal standard peptides. In some embodiments, one or more of the internal standard peptides comprise a stable isotope label. In some embodiments, enriching the biological sample or enriching the proteolytic digest comprises solid-phase extraction, precipitation, affinity enrichment, immunoaffimly enrichment, or combinations thereof. In some embodiments, ionizing the one or more surrogate peptides of 14-3-3q protein comprises: (i) generating at least one precursor ion of the one or more surrogate peptides of 14-3-3q protein; (ii) generating one or morePATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT product ions of the precursor ion; and detecting the presence or amount of the at least one precursor ion generated in step (i) and / or the one or more product ions generated in step (ii), or both, to determine the presence or amount of the 14-3-3i] protein in the biological sample. In some embodiments, the at least one precursor ion has a mass / charge ratio of 408.7±0.5. In some embodiments, the one or more product ions of the precursor ion has a mass / charge ratio of one or more of 703.3±0.5, 632.3±0.5, 503.3±0.5, 375.2±0.5, 304.2±0.5, 175.1±0.5, 185.1±0.5, 314.2±0.5, and 442.2±0.5. In some embodiments, the one or more ions of the surrogate peptides of 14-3-3r| protein comprise a mass / charge ratio selected from the group consisting of 703.3±0.5, 632.3±0.5, 503.3±0.5, and 185.1±0.5. In some embodiments, the one or more ions of the surrogate peptides of the 14-3-3i] protein comprise a mass / charge ratio selected from the group consisting of 868.4±0.5, 753.3±0.5, 507.3±0.5, and 279.1±0.5. In some embodiments, the one or more ions of the surrogate peptides of the 14-3-3p protein comprises a mass / charge ratio selected from the group consisting of 804.4±0.5. 691.4±0.5, 213.2±0.5, and 185.2±0.5. In some embodiments, the one or more ions of the surrogate peptides of the 14-3-3p protein comprises a mass / charge ratio selected from the group consisting of 1066.5±0.5, 967.5±0.5, 868.4±0.5, and 301.2±0.5. In some embodiments, the one or more ions of the surrogate peptides of the 14-3-3q protein comprises a mass / charge ratio selected from the group consisting of 623.3±0.5. 476.3±0.5, 347.2±0.5, and 234. l±0.5. In some embodiments, the mass spectrometry comprises tandem mass spectrometry. In some embodiments, the mass spectrometry comprises LC-MS, LC-MS / MS, or 2D-LC-MS / MS. In some embodiments, the biological sample comprises serum or plasma. In some embodiments, the biological sample is derived from a subject. In some embodiments, the subject is a mammal or wherein the subject is a human. In some embodiments, the subject has or is suspected of having rheumatoid arthritis. In some embodiments, the presence or amount of 14-3-3i] determined in (d) indicates the subject has rheumatoid arthritis. In some embodiments, the amount of 14-3-3i] determined in (d) is detected in a biological sample with a lower limit of detection of about 1 ng / ml. 0. 1 ng / ml or about 0. 1 ng / ml. In some embodiments, wherein the amount of the one or more ions of the surrogate peptides of (c) are detected with a lower limit of detection of about 1 ng / ml, 0.1 ng / ml or about 0.1 ng / ml.

[0027] In some aspects, a method for determining a presence or amount of 14-3-3i] protein in a biological sample comprises: providing a biological sample comprising a plurality of proteins including one or more 14-3-3 protein family members; contacting the biological sample with a proteolytic enzyme to produce a proteolytic digest comprising peptides,PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT wherein at least one of the peptides comprises a surrogate peptide derived from a 14-3 -3q protein; contacting the proteolytic digest with a binding agent, wherein the binding agent specifically binds the surrogate peptide derived from the 14-3-3q protein; separating unbound peptides from the surrogate peptide to produce a sample enriched for the surrogate peptide; purifying the surrogate peptide from the enriched sample using liquid chromatography; generating at least one precursor ion of the surrogate peptide; generating one or more product ions of the precursor ion; and detecting the presence or amount of the at least one precursor ion, the one or more product ions of the precursor ion, or both, to determine the presence or amount of the 14-3-3q protein in the biological sample.

[0028] In some embodiments, the proteolytic enzyme comprises trypsin or an isozyme thereof. In some embodiments, the at least one precursor ion is formed by electrospray ionization. In some embodiments, the electrospray ionization is performed in positive ion mode. In some embodiments, the detecting the presence or amount of the at least one precursor ion, the one or more product ions of the precursor ion, or both, is performed by a process comprising mass spectrometry’. In some embodiments, the mass spectrometry detection of the surrogate peptide is performed in selected reaction monitoring mode (SRM). In some embodiments, the at least one precursor ion of the surrogate peptide has a mass / charge ratio (m / z) of about 516.2±0.5. In some embodiments, the one or more product ions comprise an ion with a m / z of about 868.4±0.5, 753.3±0.5, 638.3±0.5, 507.3±0.5, 436.2±0.5, 279.1±0.5, 394.1±0.5, 136.1±0.5, or 251.1±0.5. In some embodiments, the mass spectrometry’ comprises LC-MS or LC-MS / MS. In some embodiments, the liquid chromatography comprises high performance liquid chromatography (HPLC). In some embodiments, the liquid chromatography is performed in reverse phase liquid chromatography or by hydrophilic interaction liquid chromatography.

[0029] In some aspects, a method for determining a presence or amount of 14-3-3q protein in a biological sample comprises: subjecting a surrogate peptide of 14-3-3q protein from a biological sample to an ionization source under conditions suitable to generate one or more precursor ions with a mass to charge ratio (m / z) of 452.3±0.5; fragmenting at least one of the precursor ions to generate one or more fragment ions detectable by mass spectrometry, wherein the one or more fragment ions comprise one or more ions selected from the group consisting of ions with m / z of 804.4±0.5, 691.4±0.5, 213.2±0.5, and 185.2±0.5; and determining the amount of one or more of the precursor ions and / or fragment ions by mass spectrometry to determine the amount of 14-3-3q protein in the sample.PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT

[0030] In some embodiments, the surrogate peptide of 14-3-3q protein is at least 80 % identical to any one of SEQ ID NOs. : 1 -17.

[0031] In some aspects, a method for determining a presence or amount of 14-3-3q protein in a biological sample comprises: providing a biological sample comprising one or more 14- 3-3 protein family members or fragments thereof; enriching the one or more 14-3-3 protein family members in the biological sample to produce an enriched sample; contacting a proteolytic enzyme with the enriched sample to produce a proteolytic digest comprising peptides, wherein the peptides comprise a surrogate peptide derived from a 14-3-3q protein; generating at least one precursor ion of the surrogate peptide; generating one or more product ions of the precursor ion; and detecting the presence or amount of the at least one precursor ion and / or the one or more fragment ions, or both, and determining a presence or amount of the 14-3-31] protein in the biological sample according to the detecting.

[0032] In some embodiments, the detecting comprises use of mass spectrometry. In some embodiments, the proteolytic enzyme comprises trypsin or an isozyme thereof. In some embodiments, the biological sample comprises serum or plasma. In some embodiments, the one or more precursor ions of the surrogate peptide has a mass to charge (m / z) ratio selected from the group consisting of 408.7±0.5, 516.2±0.5, 739.9±0.5, 454.3±0.5, 308.2±0.5, 452.3±0.5, 433.2.7±0.5, 874.4±0.5, 652.8±0.5, 533.8±0.5, 634.3±0.5. 412.2±0.5. 412.9±0.5, 991.5±0.5, 720.0±0.5, 595.3±0.5, and 708.0±0.5. In some embodiments, the method further comprises purifying the enriched sample using liquid chromatography, wherein the liquid chromatography is performed in reverse phase separation or hydrophilic interaction liquid chromatography.

[0033] In some aspects, the present disclosure provides a system for conducting the method of any one of the embodiments described herein, wherein the system comprises: a station for providing a biological sample comprising a 14-3-3q protein; a station for partially purifying the 14-3-3q protein from other components in the sample; a station for chromatographically separating 14-3-3q protein from other components in the sample; and a station for analyzing the chromatographically separated 14-3-3q protein by mass spectrometry to determine the presence or amount of the 14-3-3q protein in the biological sample.

[0034] In some embodiments, the sy stem further comprises a station for contacting the biological sample with a proteolytic enzyme, and optionally a station for partially purifying a surrogate peptide of the 14-3-3q protein from a proteolytic digest. In some embodiments, a computer-program product tangibly embodied in a non-transitory machine-readable storagePATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT medium, including instructions configured to cause one or more computers and / or devices to perform the methods described herein.

[0035] Further aspects, objects, and advantages will become apparent upon consideration of the detailed description and figures that follow.BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The drawings illustrate embodiments of the technology and are not limiting. Reference to figures and / or features of a figure in this document are not limiting. For clarity and ease of illustration, the drawings are not made to scale and. in some instances, various aspects may be shown exaggerated or enlarged to facilitate an understanding of particular embodiments.

[0037] FIG. 1 shows a flowchart for a method for detecting or quantifying 14-3-3q protein in a biological sample using LC-MS / MS.

[0038] FIG. 2 shows a flowchart for another method for detecting or quantifying 14-3-3q protein in a biological sample using LC-MS / MS.

[0039] FIG. 3 shows a system in accordance with an embodiment of the disclosure.

[0040] FIG. 4 shows an exemplary sequence of full-length human 14-3-3p protein (SEQ ID NO. 19). The bold sequences correspond to surrogate peptides formed from trypsin digestion of 14-3-3p (i.e., YDDMASAMK (SEQ ID NO. 2), VISSIEQK (SEQ ID NO. 6), TMADGNEK (SEQ ID NO. 7), and EAFEISK (SEQ ID NO 12). SEQ ID NOs. 2, 7 and 12 correspond to surrogate peptides produced from 14-3-3p which are not found in other 14-3-3 protein family members. SEQ ID NO. 6 is found in multiple 14-3-3 protein family members (alpha / beta, gamma, eta, and theta / tau). The underlined sequence SYKDSTLIMQLLRDNLTLWT (SEQ ID NO. 18) is conserved across all 14-3-3 family members and contains an epitope for the anti-14-3-3 (pan) antibody used for 14-3-3 protein enrichment workflows (e.g., workflow' 2 of Example 2).

[0041] FIGs. 5A-5F show7exemplary chromatograms for a blank / zero calibrator (FIGs. 5 A and 5B), a 20 ng / mL calibrator (FIGs. 5C and 5D), and native serum sample (FIGs. 5E and 5F) for the 14-3-3_VIS surrogate peptide (SEQ ID NO. 6) shown in top panels FIGs. 5 A, 5C and 5E) and its stable isotope internal standard (shown in bottom panels FIGs. 5B, 5D andPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT5F) produced from the workflow of Example 1. All native serum samples measured > 70 ng / mL for 14-3-3_VIS using the workflow of Example 1.

[0042] FIGs. 6A-6F show exemplary chromatograms for a blank / zero calibrator (FIGs. 6A and 6B), a 1 ng / mL calibrator (FIGs. 6C and 6D), and native serum sample (FIGs. 6E and 6F) for the 14-3-3_TMA surrogate peptide (SEQ ID NO. 7) shown in top panels FIGs. 6A, 6C and 6E) and its stable isotope internal standard (shown in bottom panels FIGs. 6B, 6D and 6F) produced from the workflow of Example 1. All native serum samples measured > 9 ng / mL for 14-3-3_TMA using the workflow of Example 1.

[0043] FIGs. 7A-7F show exemplary chromatograms for a blank / zero calibrator (FIGs. 7A and 7B), a 1 ng / mL calibrator (FIGs. 7C and 7D), and native serum sample (FIGs. 7E and 7F) for the 14-3-3 YDD surrogate peptide (SEQ ID NO. 2) shown in top panels FIGs. 7A. 7C and 7E) and its stable isotope internal standard (shown in bottom panels FIGs. 7B, 7D and 7F) produced from the workflow of Example 1. All native serum samples measured > 7 ng / mL for 14-3-3_YDD using the workflow of Example 1.

[0044] FIGs. 8A-8F show exemplary chromatograms for a blank / zero calibrator (FIGs. 8A and 8B), a 10 ng / mL calibrator (FIGs. 8C and 8D), and native serum sample (FIGs. 8E and 8F) for the 14-3-3_EAF surrogate peptide (SEQ ID NO. 12) shown in top panels FIGs. 8 A, 8C and 8E) and its stable isotope internal standard (shown in bottom panels FIGs. 8B, 8D and 78F) produced from the workflow of Example 1. All native serum samples measured > 11 ng / mL for 14-3-3_EAF using the workflow of Example 1.

[0045] FIGs. 9A-9C show Demming regression plots for the measured values of surrogate peptides of 14-3-3 proteins in serum: 14-3-3_VIS, SEQ ID NO. 6 (FIG. 9 A); 14-3-3 YDD, SEQ ID NO. 2 (FIG. 9B); and 14-3-3 EAF (SEQ ID NO. 12)[Y-axis] vs. 14-3-3 TMA, SEQ ID NO. 7 [X-axis] produced from the workflow of Example 1.

[0046] FIG. 10 shows a mean and standard deviation box plot of the measured values of surrogate peptides of 14-3-3 proteins in serum (14-3-3 VIS, 14-3-3 TMA. 14-3-3 YDD, and 14-3-3_EAF) produced from the workflow of Example 1 and Example 2.

[0047] FIGs. 11A-11D show example chromatograms for the surrogate peptides (top panel) and corresponding stable isotope labeled peptides (bottom panel) measured according to the workflow of Example 2 (FIGs. HA and 11C) and a hybrid workflow of Example 3 (FIGs. 1 IB and 1 ID). FIGs. 11 A and 1 IB show example chromatograms for the 14-3-PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT3 TMA peptide and FIGs. 11C and 1 ID shows example chromatograms for the 14-3-3 YDD peptide.DETAILED DESCRIPTION

[0048] Described herein, in some embodiments, is an improved method and / or system for detecting target analytes in a sample, often indicative of rheumatoid arthritis, using liquid chromatography and tandem mass spectrometry (LC-MS / MS). In some embodiments, the target analytes in a sample are 14-3-3 proteins (e.g. 14-3-3p). The quantification or detection of specific 14-3-3 proteins in a sample can be used to aid in the diagnosis, prognosis, and / or monitoring of disease (e.g., rheumatoid arthritis) in a subject. In some embodiments, a target analyte for detection via LC-MS / MS is a peptide used as a surrogate for detection (e.g., quantification) of a specific 14-3-3 protein. For example, surrogate peptides may be produced from a target protein by digestion with a protease (e.g, trypsin). In certain embodiments, one or more surrogate peptides may be used to detect and / or quantify a specific 14-3-3 protein in a sample. Beneficially, the methods and systems described herein improve sensitivity, selectivity, and accuracy for quantifying and / or detecting one or more 14-3-3 proteins in a sample. Additionally, the methods described herein can reduce auto-antibody interferences caused by antibodies in a sample that interfere with other methods used to detect 14-3-3 proteins in a sample, often leading to false-positive or false-negative results.

[0049] A sample can be obtained from a suitable subject. A sample can be isolated or obtained directly from a subject or part thereof. In some embodiments, a sample is obtained indirectly from an individual or medical professional. In some embodiments, a sample is derived from a subject. A sample can be any specimen that is isolated or obtained from a subject or part thereof. In some embodiments, a sample comprises a bodily fluid obtained from a subject. In some embodiments, a sample comprises an extract or lysate of a tissue or cells. Non-limiting examples of a sample include blood or a blood product (e.g, serum, plasma, platelets, buffy coats, or the like), umbilical cord blood, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., lung, gastric, peritoneal, ductal, ear, arthroscopic), urine, sputum, saliva, nasal mucous, prostate fluid, lavage, semen, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, a liquid biopsy, as well as lysates or extracts obtained from cells (blood cells, lymphocytes, placental cells, stem cells, bone marrow derived cells, embryo or fetal cells), feces, tissues, the like or combinations thereof. In some embodiments, a sample is blood or a blood product, e.g., serum. In some embodiments, a sample may bePATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT processed prior to, or while performing a method described herein. For example, a sample may be partially purified, enriched, filtered, concentrated, and / or diluted.

[0050] The term “subject” refers to an animal, typically a mammalian animal. In some embodiments a subject is a mammal. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In some embodiments, a subject is a primate. In some embodiments, a subject is a human.

[0051] In some embodiments, the methods and systems described herein are configured to detect a presence or absence of, and / or determine an amount of. a 14-3-3 protein in a sample. In some embodiments, the methods and systems described herein are configured to detect a presence, absence, and / or amount of one or more of 14-3-3q, 14-3-3(3, 14-3-3e, 14-3-3y, 14- 3-3T, 14-3-3O, and 14-3-3^ in a sample. In some embodiments, the methods and systems described herein are configured to detect a presence or absence of, and / or determine an amount of, surrogate peptides derived from one or more of 14-3-3p, 14-3-3(3, 14-3-3e, 14-3- 3y, 14-3-31, 14-3-3o, and 14-3-3 .

[0052] In some embodiments, a method described herein has a limit of detection (LOD) of a 14-3-3 protein in a sample in a range of 50 ng / ml to about 0.01 ng / ml, or 20 ng / ml to about 0.001 ng / ml. In some embodiments, a method described herein has a limit of detection for a 14-3-3 protein in a sample of 20 ng / ml or less, 10 ng / ml or less, 5 ng / ml or less, 11 ng / ml or less, 0.1 ng / ml or less, or about 0.01 ng / ml or less. In some embodiments, a method described herein has a limit of detection for a 14-3-3 protein in a sample of about 5 ng / ml, about 1 ng / ml, about 0.1 ng / ml or about 0.01 ng / ml.

[0053] In some embodiments, a method described herein has a limit of detection for a surrogate peptide of a 14-3-3 protein (e.g., 14-3-3p) in a range of 50 ng / ml to about 0.001 ng / ml, or 20 ng / ml to about 0.001 ng / ml. In some embodiments, a method described herein has a limit of detection for a surrogate peptide of a 14-3-3 protein of 20 ng / ml or less, 10 ng / ml or less, 5 ng / ml or less, 11 ng / ml or less, 0.1 ng / ml or less, or about 0.01 ng / ml or less. In some embodiments, a method described herein has a limit of detection for surrogate peptides of a 14-3-3 protein of about 5 ng / ml, about 1 ng / ml, about 0.1 ng / ml or about 0.01 ng / ml.

[0054] The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying description and drawings, in which some, but not allPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT embodiments of the presently disclosed subject matter are shown. The presently disclosed subject matter can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.

[0055] Many modifications and other embodiments of the presently disclosed subject matter set forth herein may come to mind to one skilled in the art having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The disclosure herein often utilizes the abbreviations shown below.AbbreviationsDefinitions

[0056] While the following terms are believed to be well understood by one of ordinary skill in the art. the following definitions are set forth to facilitate explanation of the presentlyPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT disclosed subject mater. Other definitions are found throughout the specification. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this presently described subject mater belongs.

[0057] Notwithstanding that the numerical ranges and parameters seting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of “1 to 10” should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, e.g, 1 to 6. 1, and ending with a maximum value of 10 or less, e.g., 5.5 to 10. Additionally, any reference referred to as being "‘incorporated herein” is to be understood as being incorporated in its entirety.

[0058] The terms “a,” ‘‘an,” and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a cell” includes a pl urality of such cells, unless the context clearly is to the contrary (e.g.. a plurality of cells), and so forth.

[0059] As used herein, the term "biomarker” or a “biomarker of interest” is any biomolecule that may provide biological information about the physiological state of an organism. In certain embodiments, the presence or absence of a biomarker may be informative. In other embodiments, the level of a biomarker may be informative. In an embodiment, the biomarker of interest may comprise a peptide, a hormone, a nucleic acid, a lipid or a protein.

[0060] As used herein, the term “biological sample” refers to a sample obtained from a biological source.

[0061] As used herein, the term “body fluid” refers to a liquid sample obtained from a biological source, including, but not limited to. an animal, a cell culture, an organ culture, and the like.

[0062] As used herein, the term “preferentially binds” refers to a reagent that predominantly binds to a protein. Preferential binding of a reagent is meant to include at least 90% of the times a reagent will bind to a protein and discriminate between other proteins.The term "specifically binds" refers to a binding agent that binds to a target analyte (e.g.. one or more 14-3-3 protein family members) in preference to binding other molecules or otherPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT peptides as determined by. for example, a suitable in vitro assay (e.g., an Elisa. Immunoblot. Flow cytometry, and the like). A specific binding interaction discriminates over non-specific binding interactions by about 2-fold or more, often about 10-fold or more, and sometimes about 100-fold or more, 1000-fold or more, 10,000- fold or more, 100,000-fold or more, or 1,000,000-fold or more.

[0063] As used herein, a “surrogate peptide” refers to a peptide produced through proteolysis and which can be derived from a single protein or collection of proteins based on its unique amino acid sequence. As such, detection of a surrogate peptide following proteolysis often provides evidence to the presence of the protein or collection of proteins from which it was derived. If calibration is performed, measurement of a surrogate peptide following proteolysis can be used to determine the amount of protein or the collective amount of protein from which it was derived in the sample prior to proteolysis.

[0064] As used herein, the terms “purify” or “separate” or derivations thereof do not necessarily refer to the removal of all materials other than the analyte(s) of interest from a sample matrix. Instead, in some embodiments, the terms “purify” or “separate” refer to a procedure that enriches the amount of one or more analytes of interest relative to one or more other components present in the sample matrix. In some embodiments, a “purification” or “separation” procedure can be used to remove one or more components of a sample that could interfere with the detection of the biomarker of interest, for example, one or more components that could interfere with detection of an analyte by mass spectrometry.

[0065] As used herein, the term “within group variability” or “CVg” refers to coefficient of variability of a measurement between subjects in a group.

[0066] As used herein, a “calibrator” is a sample created with known biomarker concentration in a matrix which is ideally but not necessarily free from a biomarker of interest. Calibrators are often used to generate a dose response curve used to determined concentrations of biomarkers in unknown samples.

[0067] As used herein, a “quality control” is a sample with a target concentration range which is used to verify the quality of results from an experiment.

[0068] As used herein, "chromatography" refers to a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of the chemical entities as they flow around or over a stationary liquid or solid phase.

[0069] As used herein, "liquid chromatography" (LC) means a process of selective retardation of one or more components of a fluid solution as the fluid uniformly percolatesPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid, (i.e., mobile phase), as this fluid moves relative to the stationary phase(s). "Liquid chromatography" includes reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC), high turbulence liquid chromatography (HTLC), and hydrophilic interaction liquid chromatography (HILIC).

[0070] As used herein, the term "HPLC" or "high performance liquid chromatography" refers to liquid chromatography in which the degree of separation is increased by forcing the mobile phase under pressure through a stationary phase, typically a densely packed column.The chromatographic column typically includes a medium (i.e.. a packing material) to facilitate separation of chemical moieties (i.e., fractionation). The medium may include minute particles. The particles may include a bonded surface that interacts with the various chemical moieties to facilitate separation of the chemical moieties such as the biomarker analytes quantified in the experiments herein. One suitable bonded surface is a hydrophobic bonded surface such as an alkyl bonded surface. Alkyl bonded surfaces may include C-4, C- 8, or C-18 bonded alkyl groups, preferably C-18 bonded groups. The chromatographic column may include an inlet port for receiving a sample and an outlet port for discharging an effluent that includes the fractionated sample. In the method, the sample (or pre-purified sample) may be applied to the column at the inlet port, eluted with a solvent or solvent mixture, and discharged at the outlet port. Different solvent modes may be selected for eluting different analytes of interest. For example, liquid chromatography may be performed using a gradient mode, an isocratic mode, or a polytyptic (i.e., mixed) mode. In one embodiment. HPLC may performed on a multiplexed analytical HPLC system with a Cl 8 solid phase using isocratic separation with water: methanol as the mobile phase.

[0071] As used herein, the term “analytical column” refers to a chromatography column having sufficient chromatographic plates to effect a separation of the components of a test sample matrix. Preferably, the components eluted from the analytical column are separated in such a way to allow the presence or amount of an analyte(s) of interest to be determined. In some embodiments, the analytical column comprises particles having an average diameter of about 5 pm. In some embodiments, the analytical column is a functionalized silica or polymer-silica hybrid, or a polymeric particle or monolithic silica stationary phase, such as a phenyl-hexyl functionalized analytical column.PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT

[0072] Analytical columns can be distinguished from “extraction columns / ’ which typically are used to separate or extract retained materials from non-retained materials to obtain a “purified” sample for further purification or analysis. In some embodiments, the extraction column is a functionalized silica or polymer-silica hybrid or polymeric particle or monolithic silica stationary phase, such as a Poroshell SBC-18 column.

[0073] The term “heart-cutting” refers to the selection of a region of interest in a chromatogram and subjecting the analytes eluting within that region of interest to a second separation, e.g., a separation in a second dimension.

[0074] The term "electron ionization" as used herein refers to methods in which an analyte of interest in a gaseous or vapor phase interacts with a flow of electrons. Impact of the electrons with the analyte produces analyte ions, which may then be subjected to a mass spectrometry technique.

[0075] The term "chemical ionization" as used herein refers to methods in which a reagent gas (e.g., ammonia) is subjected to electron impact, and analyte ions are formed by the interaction of reagent gas ions and analyte molecules.

[0076] The term "field desorption" as used herein refers to methods in which anon-volatile test sample is placed on an ionization surface, and an intense electric field is used to generate analyte ions.

[0077] The term “matrix-assisted laser desorption ionization,” or “MALDI” as used herein refers to methods in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample by various ionization pathways, including photo-ionization, protonation, deprotonation, and cluster decay. For MALDI, the sample is mixed with an energy-absorbing matrix, which facilitates desorption of analyte molecules.

[0078] The term “surface enhanced laser desorption ionization,” or “SELDI” as used herein refers to another method in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample by various ionization pathways, including photoionization, protonation, deprotonation, and cluster decay. For SELDI, the sample is typically bound to a surface that specifically retains one or more analytes of interest. As in MALDI, this process may also employ an energy-absorbing material to facilitate ionization.

[0079] The term “electrospray ionization,” or “ESI,” as used herein refers to methods in which a solution is passed along a short length of capillary tube, to the end of which is applied a high positive or negative electric potential. Upon reaching the end of the tube, the solution may be vaporized (nebulized) into ajet or spray of very small droplets of solution in solventPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT vapor. This mist of droplet can flow through an evaporation chamber which is heated slightly to prevent condensation and to evaporate solvent. As the droplets get smaller the electrical surface charge density increases until such time that the natural repulsion between like charges causes ions as well as neutral molecules to be released.

[0080] The term “Atmospheric Pressure Chemical Ionization,’7or “APCI,” as used herein refers to mass spectroscopy methods that are similar to ESI, however, APCI produces ions by ion-molecule reactions that occur within a plasma at atmospheric pressure. The plasma is maintained by an electric discharge between the spray capillary and a counter electrode.Then, ions are typically extracted into a mass analyzer by use of a set of differentially pumped skimmer stages. A counterflow of dry and preheated N2 gas may be used to improve removal of solvent. The gas-phase ionization in APCI can be more effective than ESI for analyzing less-polar species.

[0081] The term “Atmospheric Pressure Photoionization’’ (“APPI”) as used herein refers to the form of mass spectroscopy where the mechanism for the photoionization of molecule M is photon absorption and electron ejection to form the molecular M+. Because the photon energy typically is just above the ionization potential, the molecular ion is less susceptible to dissociation. In many cases it may be possible to analyze samples without the need for chromatography, thus saving significant time and expense. In the presence of water vapor or protic solvents, the molecular ion can extract H to form MH+. This tends to occur if M has a high proton affinity. This does not affect quantitation accuracy because the sum of M+ and MH+ is constant. Drug compounds in protic solvents are usually observed as MH+, whereas nonpolar compounds such as naphthalene or testosterone usually form M+ (see e ., Robb et al., 2000. Anal. Chem. 72(15): 3653-3659).

[0082] The term “inductively coupled plasma” as used herein refers to methods in which a sample is interacted wi th a partially ionized gas at a sufficiently high temperature to atomize and ionize most elements.

[0083] The term “ionization” and “ionizing” as used herein refers to the process of generating an analyte ion having a net electrical charge equal to one or more electron units. Negative ions are those ions having a net negative charge of one or more electron units, while positive ions are those ions having a net positive charge of one or more electron units.

[0084] The term “desorption” as used herein refers to the removal of an analyte from a surface and / or the entry of an analyte into a gaseous phase.PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT

[0085] As used herein, the term “hemolyzed” refers to the rupturing of the red blood cell membrane, which results in the release of hemoglobin and other cellular contents into the plasma or serum and the term “lipemic” refers to an excess of fats or lipids in blood.

[0086] As used herein, “liquid plasma” is plasma that is obtained from drawing blood from a patient and that is separated from the red blood cells but that remains in a liquid state. Liquid plasma is generally obtained from subjects by phlebotomy or venipuncture.

[0087] As used herein, “dried plasma” is plasma that has been allowed to dry. Dried plasma may be produced following separation from red blood cells by migration of the plasma through pores of a solid-phase substrate which restrict migration of cells as is described in more detail herein.

[0088] As used herein, a “protein variant” is a protein that has an amino acid sequence that is different from the most common or wild-type sequence.

[0089] As used herein, a “protein family” refers to a collection of different oligopeptides, derived from expression of different genes, but which have homologous or analogous amino acid sequences. In some cases, a “protein family” could also be used to define a collection of protein isoforms derived from a single gene, but which differ in their amino acid sequence through, for example, alternative splicing and / or post-translational processing.Methods of Detecting or Quantifying 14-3-3r] Protein Using LC-MS / MS

[0090] The present disclosure is directed to a method for detecting or determining the presence or amount of at least one biomarker of interest in a biological sample using liquid chromatography and tandem mass spectrometry (LC-MS / MS). The methods described herein may be employed for detecting and / or measuring one or more 14-3-3 proteins in a biological sample. 14-3-3 proteins are a family of proteins found in all eukaryotic cells, from plants to humans. 14-3-3 proteins regulate a vast array of cellular functions, and dysregulation of 14-3- 3 proteins or their interactions is implicated in numerous diseases. In humans and most mammals, there are seven distinct, but homologous isoforms: beta (|3), epsilon (s), eta (r|), gamma (y), tau (T), sigma (o), and zeta (0, each encoded by a separate gene.

[0091] In some embodiments, a method of detecting or determining an amount of one or more 14-3-3 proteins in a sample includes detecting the amount of intact 14-3-3 proteins, surrogate peptides derived from 14-3-3 proteins, or combinations thereof. In some embodiments, a method of detecting or determining an amount of one or more 14-3-3 proteins includes detecting one or more intact 14-3-3 proteins (e.g., proteins that are notPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT subject to proteolysis) using LC-MS / MS. In some embodiments, a method of detecting or determining an amount of one or more 14-3-3 proteins does not include directly detecting or measuring an amount of one or more intact 14-3-3 proteins (<?.g., proteins that were not subject to proteolysis) using mass spectrometry (e.g., LC-MS / MS). In some embodiments, a method of detecting or determining an amount of one or more 14-3-3 proteins includes detecting one or more surrogate peptides of 14-3-3 proteins using LC-MS / MS.

[0092] In some embodiments, a method of detecting or determining an amount of one or more 14-3-3 proteins in a sample includes purifying or enriching a sample (e.g., a biological sample). A 14-3-3 proteins may be derived, isolated, extracted, purified or partially purified from one or more subjects, one or more samples or one or more sources. In some embodiments, purification of the sample may include one or more of liquid chromatography, solid-phase extraction, precipitation (e.g, immunoprecipitation), affinity enrichment (e.g., affinity chromatography), immunoaffinity enrichment, or combinations thereof to produce a purified or enriched sample.

[0093] In some embodiments, a method includes purifying a sample using liquid chromatography. Liquid chromatography is a process of selective retardation of one or more components in a fluid solution as the fluid moves through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid, (z.e., mobile phase), as this fluid moves relative to the stationary phase(s). Any suitable method can be used for liquid chromatography including reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC), high turbulence liquid chromatography (HTLC), hydrophilic interaction liquid chromatography (HILIC), and normal phase liquid chromatography (NPLC). The terms HILIC and NPLC are used interchangeably and relate to a chromatographic separation whereby the stationary phase is polar in nature (z.e., silica, cyano, amino, and similar types of hydrophilic functionalized packing).

[0094] A method may include analyzing a chromatographically separated analyte (e.g, 14- 3-3 proteins) by mass spectrometry to determine the presence or amount of the analyte in the sample. Mass spectrometry may include filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "in / / .." In some mass spectrometry’ techniques, one or more analytes of interest are ionized, and the ions are subsequently introduced into a mass spectrometer where, due to a combination of electric fields, the ions follow a path in spacePATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT that is dependent upon mass (“m”) and charge (“z”). For example, in tandem MS / MS spectrometry, the precursor ion is selected following ionization, and that precursor ion is subjected to fragmentation to generate product (z.e., fragment) ions, whereby one or more product ions are selected for detection. Each precursor ion is known as a transition, and monitoring of one or more transitions is known as selected reaction monitory (SRM) or multiple reaction monitoring (MMR). Thus, in certain embodiments, the MS / MS analysis comprises at least one of SRM or MMR.

[0095] A variety of mass spectrometry techniques may be used to measure and quantify an analyte in the methods and systems described herein. In certain embodiments, the mass spectrometer uses a quadrupole or quadrupole ion trap system. In a quadrupole mass spectrometer, ions in an oscillating radio frequency (RF) field experience a force proportional to the direct current (DC) potential applied between electrodes and the amplitude of the RF signal. The voltage and amplitude can be selected so that only ions having a particular m / z travel the length of the quadrupole, while all other ions are deflected. Thus, quadrupole instruments can act as both a “mass filter” and as a “mass detector” for the ions injected into the instrument.

[0096] In certain embodiments, an MS method comprises tandem mass spectrometry or MS / MS. In some embodiments, an MS methods or systems herein comprises use of a triple quadrupole MS / MS. Triple quadrupole MS / MS instruments typically consist of two quadrupole mass filters separated by a fragmentation means. In sone embodiments, a triple quadrupole MS / MS instrument may comprise a quadrupole mass filter operated in the RF only mode as an ion containment or transmission device. In another embodiment, a quadrupole may further comprise a collision gas at a pressure of between 1 and 10 millitorr. Many other types of “hybrid” tandem mass spectrometers are contemplated for use in the methods and systems of the present invention including various combinations of magnetic sector analyzers and quadrupole filters.

[0097] A tandem MS / MS may be operated in a variety of modes. For the methods and systems of the described herein, ions can be produced using a variety of methods, nonlimiting examples of which include electron ionization, chemical ionization, fast atom bombardment, field desorption, and matrix-assisted laser desorption ionization (“MALDI”), surface enhanced laser desorption ionization (“SELDI”), photon ionization, electrospray ionization (“ESI”), atmospheric pressure ionization (“ACPI”), nanospray, and inductivelyPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT coupled plasma. In one embodiment, a tandem MS / MS spectrometer is operated in a positive or negative ion electrospray ionization mode.

[0098] In some embodiments, a mass spectrometry may isolate precursor ions for further fragmentation. For example, collision-induced dissociation (“CID”) may be used to generate fragment ions for further detection. In CID, precursor ions gain energy through collisions with an inert gas and subsequently fragment by a process referred to as “unimolecular decomposition.”

[0099] In some embodiments, MS comprises 2D-LC-MS / MS. In some embodiments, 2D- LC-MS / MS comprises a multiplex system comprising staggered multiplexed LC and MS sample inlet systems. Also, the methods and systems of the present invention may comprise multiple column switching protocols, and / or heart-cutting (LC-LC or 2D-LC) techniques, and / or LC separations prior to MS detection. In some embodiments, the methods and systems of the present invention may include a multiplexed two-dimensional liquid chromatographic system coupled with a tandem mass spectrometer (MS / MS) system, for example a triple quadrupole MS / MS system. Such embodiments provide for staggered, parallel sample input into the MS system.

[0100] An analyte of interest (e.g., a 14-3-3 protein or surrogate peptide thereof) may be quantified based upon an amount of the characteristic transitions measured by a mass spectrometer. In some embodiments, the tandem mass spectrometer comprises a triple quadrupole mass spectrometer. In some embodiments, the tandem mass spectrometer is operated in an electrospray ion (ESI) mode. In some embodiments, the electrospray is operated in a positive ion mode. In some embodiments, a nanospray emitter is used. In some embodiments, the quantification of an analyte and internal standards is performed in a selected reaction monitoring mode (SRM). In some embodiments, other methods of ionization such as the use of inductively coupled plasma, or MALDI, or SELDI, APCI, or APPI may be used for ionization. In some embodiments, the quantification of an analyte and / or internal standards is performed in a selected reaction monitoring mode (SRM).

[0101] The specificity and sensitivity provided by the methods and systems described herein may allow for the analysis of analytes from a variety of biological materials. For example, LC -MS / MS and 2D-LC-MS / MS methods can be applied to the quantification of an analyte of interest in a complex sample biological matrices, including, but not limited to. blood, serum, plasma, urine, saliva, and the like. Thus, the methods and systems of the present invention are suitable for clinical applications and / or clinical trials.PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT

[0102] As additional potential advantages, in certain embodiments, the systems and methods of the present invention provide approaches for addressing isobaric interferences, varied sample content, including hemolyzed and lipemic samples, while attaining low ng / mL limits of detection (LOD) of target analytes. Accordingly, embodiments of the methods and systems of the present invention may provide for the quantitative, sensitive, and specific detection of clinical analytes used in the clinical diagnosis of disorders.

[0103] In some embodiments, a method includes purifying or enriching one or more surrogate peptides of a 14-3-3q protein, e.g., using immunoprecipitation and / or liquid chromatography, ionizing the one or more surrogate peptides and detecting the one or more surrogate peptides of by mass spectrometry. In some embodiments, the presence or amount of a 14-3-3q protein in a sample is determined according to the presence or amount of the one or more surrogate peptides detected by mass spectrometry. In some embodiments, one or more ions of a surrogate peptide comprise a precursor ion with a mass to charge ratio of 408.7±0.5 and / or one or more fragment ions selected from the group of ions having a mass to charge ratio of 703.3±0.5, 632.2±0.5, 503.3±0.5, 375.2±0.5, 304.2±0.5, 175.1±0.5, I85.1±0.5, 314.2±0.5, and 442.2±0.5.

[0104] In some embodiments, a method of detecting or determining an amount of a 14-3-3q protein in a biological sample includes purifying a sample comprising one or more surrogate peptides of 14-3 -3 iq protein using liquid chromatography, ionizing the one or more surrogate peptides of 14-3-3q protein to generate one or more ions of the surrogate peptides of 14-3-3q protein detectable by mass spectrometry', and determining the amount of the one or more ions of the surrogate peptides of 14-3-3q protein by tandem mass spectrometry' to determine the amount of 14-3-3q protein in the sample. In some embodiments, the one or more ions comprise a precursor ion with a mass to charge ratio of 408.7±0.5 and one or more fragment ions selected from the group of ions with mass to charge ratios consisting of 703.3±0.5, 632.2±0.5, 503.3±0.5, 375.2±0.5, 304.2±0.5, 175.1±0.5, 185.1±0.5, 314.2±0.5, and 442.2±0.5.

[0105] In some embodiments, the method for determining the amount of 14-3-3q protein in a sample includes: (a) generating a precursor ion of a surrogate peptide of 14-3-3q protein;(b) generating one or more fragment ions of the precursor ion; and (c) detecting the amount of one or more ions in step (a) or (b) or both to determine the amount of 14-3-3q protein in the sample. In some embodiments, step (a) comprises generating one or more precursor ions of the 14-3-3q protein having a mass to charge ratio selected from the group consisting ofPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT408.7±0.5, 516.2±0.5, 739.9±0.5, 454.3±0.5, 308.2±0.5, 452.3±0.5, 433.2.7±0.5. 874.4±0.5. 652.8±0.5, 533.8±0.5, 634.3±0.5, 412.2±0.5, 412.9±0.5, 991.5±0.5, 720.0±0.5, 595.3±0.5, and 708.0±0.5. In some embodiments, the method further comprises purifying the sample prior to step (a). In some embodiments, the purification step comprises liquid chromatography.

[0106] In some embodiments, a method comprises enriching a sample for one or more 14- 3-3 protein family members prior to generating surrogate peptides of a 14-3-3 r] protein. For example, a sample (e.g, a biological sample) is often enriched for one or more 14-3-3 protein family members prior to generating surrogate peptides by digestion (e.g., protease digestion). In some embodiments, a method comprises enriching a sample for one or more 14-3-3 protein family members prior to subjecting a sample to LC-MS / MS. In some embodiments, a sample can be enriched for one or more 14-3-3 protein family members using solid-phase extraction, precipitation, affinity enrichment, immunoaffmity enrichment, or combinations thereof to produce an enriched sample.

[0107] In some embodiments, a sample is contacted with a binding agent (e.g. , an antibody) that binds specifically to one or more 14-3-3 protein family members. The bound 14-3-3 protein family members can then be isolated from a sample using a suitable method. In some embodiments, bound complexes of one or more 14-3-3 protein family members are isolated by an affinity purification method.

[0108] In some embodiments, a binding agent comprises a pan-antibody that binds to two, three, four, five or six or more 14-3-3 protein family members in a sample. For example, a pan antibody may specifically bind to a 14-3-3q protein, a 14-3-3a protein, and a 14-3-3y protein, or fragments thereof. In some embodiments, a pan antibody specifically binds to a 14-3-3r] protein and at least one other 14-3-3 protein family member or a fragment thereof. In some embodiments, a pan antibody comprises a polyclonal antibody, or a binding fragment thereof. In some embodiments, a pan antibody comprises a monoclonal antibody, or binding fragment thereof.

[0109] In some embodiments, a sample is contacted with a binding agent (e.g. , an antibody) that binds specifically to a 14-3-3q protein. The bound 14-3-3q protein can then be isolated from a sample using a suitable method. In some embodiments, a bound protein is isolated by an affinity purification method. For example, an antibody -bound 14-3-3q protein can be immunoprecipitated using protein G beads, followed by washing, and optionally resuspension in a buffer suitable for protease digestion. In some embodiments, an antibody that bindsPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT specifically to a 14-3-3rj protein comprises a monoclonal antibody, or binding fragment thereof.

[0110] In some embodiments unbound proteins can be removed from a sample (e.g., using a washing step) to obtain a sample enriched for a desired protein or peptide. A sample enriched for one or more 14-3-3 family member proteins can be digested with a proteolytic enzyme to produce one or more surrogate peptide of a 14-3-3q protein.[OHl] In some embodiments, an enriched sample comprises one or more 14-3-3 proteins bound to one or more binding agents (z.e., referred to as bound proteins, e.g., antibody-bound proteins) that are attached to a suitable substrate (e.g., a bead or a surface).

[0112] In some embodiments, a process of enriching a sample comprising increasing a concentration or purity of one or more 14-3-3 protein family members or surrogate peptides in a sample. In some embodiments, a method of enriching may increase the concentration and / or purity of a 14-3-3 protein family member or surrogate peptide in a sample 2-fold to a 1000-fold. A method of enriching may increase the concentration and / or purity of a 14-3-3 protein family member or surrogate peptide in a sample greater than 2-fold, greater than 10- fold, greater than 100-fold, or greater than 1000-fold compared to the concentration or purity of the 14-3-3 protein family member or surrogate peptide prior to enrichment.

[0113] In some embodiments, a binding agent comprises or consists of a suitable antibody, an antibody fragment and / or an antigen binding portion thereof (e.g., a binding fragment). An antibody can refer to a natural antibody, polyclonal antibody, monoclonal antibody, recombinant antibody, a chimeric antibody, an antibody binding fragment (e.g.. an antigen binding portion of an antibody), a CDR-grafted antibody, a humanized antibody, a human antibody, or portions thereof. In some embodiments a binding agent comprises or consists of one or more suitable antigen binding portions of an antibody, non-limiting examples of which include Fab, Fab', F(ab')2, Fv fragment, single-chain Fv (scFv), diabody (Dab), synbody, the like and / or a combination or portion thereof. In some embodiments a binding agent comprises TandAbs, aptamers, nanobodies. BiTEs. SMIPs, DARPins, DNLs, affibodies, Duocalins, adnectins, tynomers, Kunitz Domains AlbudAbs, DARTs, DVD-IG, Covx-bodies, peptibodies, scFv-Igs, SVD-Igs, dAb-Igs, Knob-in-Holes, triomAbs, the like or combinations thereof. In some embodiments, a binding agent comprises a single-chain polypeptide comprising one or more antigen binding portions of an antibody.

[0114] In some embodiments, a binding agent specifically binds to at least one 14-3-3 protein or to at least one surrogate peptide of a 14-3-3 protein (e.g., a 14-3-3 r, protein).PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT

[0115] In some embodiments, a binding agent is coated or bound to a suitable substrate. In some embodiments, a substrate is a solid-phase substrate. A solid-phase substrate can comprise a bead, particle or a surface. In some embodiments, a solid-phase substrate comprises a magnetic particle. In some embodiments, a solid-phase substrate comprises a coating. For example, a bead can be coated with streptavidin. In some embodiments, a binding agent is attached to a magnetic bead. In some embodiments, a binding agent can be biotinylated and conjugated to magnetic beads coated with streptavidin.

[0116] In some embodiments, bound complexes of one or more 14-3-3 protein family members are bound to a suitable substrate. For example, antibody-bound 14-3-3 protein family members can be immunoprecipitated using protein G beads, followed by washing, and optionally resuspension in a buffer suitable for protease digestion.

[0117] In some embodiments, a binding agent attached to a substrate can be washed to purify and / or enrich a bound protein (e.g., a 14-3-3 protein or surrogate protein) using a suitable washing method. In some embodiments, a protein bound to a binding agent that is attached to a substrate can be eluted using a suitable method to provide a purified or enriched protein.

[0118] In some embodiments, surrogate peptides are produced by digesting proteins of a sample with a proteolytic reagent, thereby producing a proteolytic digest. In some embodiments, surrogate peptides are produced by contacting a sample with a proteolytic reagent, thereby producing a proteolytic digest. Non-limiting examples of proteolytic reagents include a protease and proteolytic chemicals such as cyanogen bromide and acids. In some embodiments, any suitable proteolytic reagent or method can be used for a method herein with a requirement that (1) at least one of the surrogate peptides produced by the proteolytic digestion process is defined by a specific peptide sequence, and (2) the proteolytic digestion process is reproducible in that the at least one surrogate peptide can be reliably be produced upon repeating the proteolytic digestion process with another identical or similar sample. In certain embodiments, surrogate peptides are produced prior to an enrichment method. In certain embodiments, surrogate peptides are produced after an enrichment method.

[0119] In some embodiments, surrogate peptides of a 14-3-3 protein (e.g., 14-3-3q) are produced by enzymatic digestion (e.g.. protease digestion) of a sample using a suitable proteolytic enzyme. Non-limiting examples of a proteolytic enzyme that can be used for a proteolytic digestion process herein include trypsin, LysN, LysC, Glu-C, Asp-N, ArgC,PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT pepsin, proteinase K. elastase, thermolysin, papain or subtilisin. or any combination thereof. In some embodiments, a proteolytic enzyme comprises trypsin, or an isozyme thereof

[0120] In some embodiments, surrogate peptides of a 14-3-3 protein are produced by chemical digestion. In some embodiments, a method of generating surrogate peptides includes contacting a sample or a sample enriched for one or more 14-3-3 proteins, with one or more suitable chemical reagents, non-limiting examples of which include an acid, cyanogen bromide, the like or combinations thereof. In some embodiments, a 14-3-3 protein (e.g., 14-3-3i]) can be contacted with a chemical reagent to produce surrogate peptides. In some embodiments, a chemical reagent can be any suitable chemical that produces reproducible and predicable fragments from a 14-3-3 protein. For example, a protein sequence can be cleaved with a specific chemical reagent such at least one predetermined peptide is generated. Surrogate peptides produced in such a manner can be isolated and / or separated from a substrate using a suitable method.

[0121] In some embodiments, bound proteins of an enriched sample are contacted with a proteolytic reagent to produce surrogate peptides. For example, a 14-3-3 protein (e.g., 14-3- 3q) can be bound to an antibody and immunoprecipitated using protein G beads, followed by contacting the immunoprecipitated complexes with a proteolytic reagent. This method often does not require elution of a 14-3-3 protein from a binding agent or from a substrate (e.g., protein G beads). Accordingly, in certain embodiments, an enriched sample comprising a bound 14-3-3 protein is contacted with a proteolytic enzyme thereby producing surrogate peptide. Surrogate peptides produced in such a manner can be isolated and / or separated from a substate using a suitable method.

[0122] In some embodiments, a sample is not enriched for one or more 14-3-3 proteins and the sample is digested with a protease or chemical reagent to generate surrogate peptides prior to mass spectrometry'. For example, a sample, including all proteins, can be digested with a proteolytic enzyme or chemical reagent to produce a digest that includes a plurality of peptides. The peptides in the digest may include one or more surrogate peptides of a 14-3-3q and / or other 14-3-3 protein family members.

[0123] In some embodiments, a proteolytic digest can be enriched for one or more surrogate peptides. A proteolytic digest can be enriched for one or more surrogate peptides derived from 14-3 -3 iq or another 14-3-3 protein family members by contacting a proteolytic digest with a binding agent that specifically binds to a surrogate peptide of interest.PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT

[0124] In some embodiments, a sample can be enriched for one or more surrogate peptides using solid-phase extraction, precipitation, affinity enrichment, immunoaffinity enrichment, or combinations thereof to produce an enriched sample.

[0125] In some embodiments, a sample is contacted with a binding agent (e.g., an antibody) that binds specifically to one or more surrogate peptides. In some embodiments, a sample is contacted with a binding agent (e.g., an antibody) that binds specifically to a surrogate peptide generated by proteolytic digestion of a l4-3-3q protein. Bound surrogate peptides can then be isolated from a proteolytic digest using a suitable method. In some embodiments, bound complexes of one or more surrogate peptides are isolated by an affinity purification method. For example, an antibody-bound surrogate peptide can be immunoprecipitated using protein G beads, followed by washing, and optionally elution using a suitable method. In some embodiments, an antibody that binds specifically to a surrogate peptide comprises a monoclonal antibody, or binding fragment thereof.

[0126] In embodiments where trypsin, or an isozyme thereof, is used as the proteolytic enzyme, surrogate peptides may include one or more tryptic peptides of 14-3-3 protein family members. In some embodiments, when using trypsin to conduct a digestion, the surrogate peptide(s) may be selected from try ptic peptides derived uniquely from 14-3-3q to differentiate 14-3-3 i from other 14-3-3 protein family members in a sample, as well as from other serum proteins. For example, in Table 1 below, SEQ ID NOs: 2, 3, 7-15, and 17 are examples of surrogate peptides derived uniquely from 14-3-3q.Table 1. Surrogate peptides derived from 14-3-3q protein and 14-3-3 protein family members.PATENT APPLICATIONAttorney Docket No 057618-1509623Client Reference No. LC 2024-01-WO-PCTX - sequence present in protein with requisite N-terminal tryptic site O - sequence present in protein but lacks requisite N-terminal tryptic site.

[0127] In some embodiments, a presence or amount of a 14-3-3q protein in a biological sample can be determined by using LC-MS / MS following enrichment and / or enzymatic digestion. For example, one or more 14-3-3q surrogate peptides that are unique to 14-3-3q can be measured using MS (e.g., LC-MS / MS), and the amount of surrogate peptides of the 14-3-3i] protein may be used to quantify the amount of 14-3-3q protein in a biological sample. In some embodiment, a presence or amount of 14-3-3n in a sample is determined by determining a presence or amount of one or more of SEQ ID NOs: 2, 3, 7-15, and 17 in a sample (e.g., an enriched proteolytic digest) by mass spectrometry'.

[0128] In some embodiments, a method may include measuring surrogate peptides shared among one or more 14-3-3 protein family member or fragments thereof. In this embodiment, a surrogate peptide may be shared among one or more 14-3-3 protein family members. Measurements derived from surrogate peptides shared among one or more 14-3-3 protein family members may produce a measurement that is a composite measurement of the corresponding 14-3-3 protein family members and not from 14-3-31] protein alone. For example, SEQ ID NOs: 1, 4-6, and 16 in Table 1 provide surrogate peptides shared among one or more 14-3-3 protein family members. However, measurements derived from a shared surrogate peptide may effectively measure solely 14-3-3q in serum when 14-3-3q is substantially more abundant than other 14-3-3 protein family members. To facilitate selective measurements of the 14-3-3q surrogate peptides by LC-MS / MS, enrichment steps may be performed prior to LC-MS / MS. Thus, the methods described herein and in further detail below' may be adapted to use any one of the surrogate peptides in Table 1.

[0129] In one embodiment, a method may comprise ionizing one or more surrogate peptides of a 14-3-3q protein to generate one or more ions of the surrogate peptides of the 14-3-3q protein detectable by mass spectrometry7; and determining the amount of the one or more ions of the surrogate peptides of the 14-3-3q protein by tandem mass spectrometry7. For example,PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT generating one or more ions of the surrogate peptides of 14-3-31] protein detectable by mass spectrometry may include (a) generating a precursor ion of a surrogate peptide of 14- 3 -31] protein; (b) generating one or more fragment ions of the precursor ion; and (c) detecting the amount of one or more ions in step (a) or (b) or both to determine the amount of 14-3-31] protein in the sample.

[0130] In some embodiments, the precursor ion of a surrogate peptide of a 14-3-3 protein family member has a m / z ratio of 408.7±0.5 and the product ions produced from the precursor ion have a m / z ratio of 703.3±0.5, 632.3±0.5, 503.3±0.5, 375.2±0.5, 304.2±0.5, 175.1±0.5, 185.1±0.5, 314.2±0.5, 442.2±0.5, or combinations thereof. In some embodiments, the product ions produced from the precursor ion have a m / z ratio selected from the group consisting of 703.3±0.5, 632.3±0.5, 503.3±0.5, and 185. l±0.5.

[0131] In some embodiments, the precursor ion of a surrogate peptide of a 14-3-3 protein family member has a m / z ratio of 516.±0.5 and the product ions produced from the precursor ion have a m / z ratio of 868.4±0.5, 753.3±0.5, 638.3±0.5, 507.3±0.5, 436.2±0.5, 279.1±0.5. 394.1±0.5, 136.1±0.5, 251.1±0.5, or combinations thereof. In some embodiments, the product ions produced from the precursor ion have a m / z ratio selected from the group consisting of 868.4±0.5, 753.3±0.5, 507.3±0.5, and 279.1±0.5.

[0132] In some embodiments, the precursor ion of a surrogate peptide of a 14-3-3 protein family member has a m / z ratio of 452.3. ±0.5 and the product ions produced from the precursor ion have a m / z ratio of 804.4±0.5, 691.4±0.5, 604.3±0.5, 517.3±0.5, 404.2±0.5, 275.2±0.5, 213.2±0.5, 258.1±0.5, 185.2±0.5, or combinations thereof. In some embodiments, the product ions produced from the precursor ion have a m / z ratio selected from the group consisting of 804.4±0.5, 691.4±0.5, 213.2±0.5, and 185.2±0.5.

[0133] In some embodiments, the precursor ion of a surrogate peptide of a 14-3-3 protein family member has a m / z ratio of 433.2±0.5 and the product ions produced from the precursor ion have a m / z ratio of 764.3±0.5, 633.3±0.5, 562.2±0.5, 447.2±0.5, 276.2±0.5, 233.1±0.5, 373.2±0.5, 205.1±0.5, or combinations thereof. In some embodiments, the product ions produced from the precursor ion have a m / z ratio selected from the group consisting of 633.3±0.5, 562.2±0.5, 233.1±0.5, and 205.1±0.5.

[0134] In some embodiments, the precursor ion of a surrogate peptide of a 14-3-3 protein family member has a m / z ratio of 634.3±0.5 and the product ions produced from the precursor ion have a m / z ratio of 1066.5±0.5, 967.5±0.5, 868.4±0.5, 739.4±0.5, 668.3±0.5. 202.1±0.5, 301.2±0.5, or combinations thereof. In some embodiments, the product ionsPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT produced from the precursor ion have a m / z ratio selected from the group consisting of 1066.5±0.5, 967.5±0.5, 868.4±0.5, and 301 ,2±0.5.

[0135] In some embodiments, the precursor ion of a surrogate peptide of a 14-3-3 protein family member has a m / z ratio of 412.2±0.5 and the product ions produced from the precursor ion have a m / z ratio of 623.3±0.5. 476.3±0.5, 347.2±0.5, 234. l±0.5, 147. l±0.5, 312.2±0.5, 459.2±0.5, or combinations thereof. In some embodiments, the product ions produced from the precursor ion have a m / z ratio selected from the group consisting of 623.3±0.5, 476.3±0.5, 347.2±0.5, and 234.1±0.5.

[0136] In alternate embodiments the mass spectrometry is tandem mass spectrometry’, liquid chromatography tandem mass spectrometry (LC-MS / MS), or 2 dimensional LC-MS / MS.

[0137] In certain embodiments, the sample is subjected to a purification step prior to mass spectrometry. For example, the purification step may comprise liquid-liquid extraction of the sample, protein precipitation or dilution of the sample prior to mass spectrometry. In one embodiment, the sample is diluted into a solvent or solvent mixture that may be used for LC and / or MS (e.g. LC-MS / MS or 2D-LC-MS / MS).

[0138] Additionally and / or alternatively, a further purification step may comprise liquid chromatography, such as high-performance liquid chromatography (HPLC). In some cases, the chromatography comprises extraction and analytical liquid chromatography. Additionally, and / or alternatively, high turbulence liquid chromatography (HTLC) (also known as high throughput liquid chromatography) may be used. In alternate embodiments, the liquid chromatography separation technique may include reverse phase separation or hydrophilic interaction liquid chromatography separation (HILIC).

[0139] In one embodiment, the method may comprise purifying one or more surrogate peptides of a 14-3-3q protein using liquid chromatography; ionizing the one or more surrogate peptides of the 14-3-3q protein to generate one or more ions of the surrogate peptides of 14-3-3q protein detectable by mass spectrometry; and determining the amount of the one or more ions of the surrogate peptides of the 14-3 -3 rq protein by tandem mass spectrometry.

[0140] For example, generating one or more ions of the surrogate peptides of 14-3-3q protein detectable by mass spectrometry may include (a) generating a precursor ion of a surrogate peptide of 14-3-3 q protein; (b) generating one or more fragment ions of the precursor ion; and (c) detecting the amount of one or more ions in step (a) or (b) or both to determine the amount of 14-3 -3iq protein in the sample.PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT

[0141] In one embodiment of method 100 for determining the presence or amount of at least one biomarker of interest in a biological sample may be represented by FIG. 1 . For example, at block 110 the method may include first providing a biological sample comprising a plurality of proteins including at least one biomarker of interest. The biomarker of interest may be at least 14-3-3ty protein. The biomarker of interest may be 14-3-3q protein and at least one other 14-3-3 protein family member. In some embodiments, the biological sample may be serum or plasma.

[0142] At block 120, the method may further include adding a binding agent that binds specifically to 14-3-3q protein in the biological sample. In some embodiments, the binding agent is an antibody or an aptamer. In some embodiments, the biological sample, comprising at least one of the biomarkers of interest, is treated with an internal standard. The internal standard may be a stable isotope of 14-3-3q protein or a surrogate peptide of 14-3-31} protein.

[0143] At block 130, the biological sample may be subsequently treated with a proteolytic enzyme to produce a proteolytic digest comprising a plurality of peptides. The plurality of peptides may include at least one surrogate peptide derived from 14-3-3p protein. In some embodiments, the peptides produced may include surrogate peptides derived from other 14-3- 3 protein family members. In some embodiments, the proteolytic enzy me may be trypsin. One skilled in the art may understand that a substitute proteolytic enzyme may be used in the methods described herein. In such an event, the cleavage of the 14-3-3q peptides in the biological sample may be different and thus alternate antibodies or aptamers may be employed.

[0144] At block 140, the surrogate peptides derived from 14-3-3q protein are separated from other peptides in the proteolytic digest to produce an enriched sample. In some embodiments, the proteolytic digest can be enriched in the surrogate peptides derived from 14-3-3p protein using solid-phase extraction, precipitation, affinity enrichment, immunoaffinity enrichment, or combinations thereof to produce an enriched sample. The antibodies used for immunoaffinity enrichment can specifically bind to 14-3-3 protein family members or fragments thereof. In some embodiments, the separation may include separating, based upon the antibody, via techniques such as precipitation or solid phase extraction. In some embodiments, separating the surrogate peptides derived from 14-3-3q protein bound to the binding agent prior to liquid chromatography may include (1) binding the binding agent to a solid phase substrate; (2) washing unbound peptides from the solid phase substrate; and (3)PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT eluting the surrogate peptides derived from 14-3-3q protein bound to the binding agent from the solid phase substrate.

[0145] At block 150, liquid chromatography may be optionally performed on the separated sample to purify the sample. The liquid chromatography may include high performance liquid chromatography (HPLC). In some cases, the chromatography comprises extraction and analytical liquid chromatography. Additionally, and / or alternatively, high turbulence liquid chromatography (HTLC) (also known as high throughput liquid chromatography) may be used. In alternate embodiments, the liquid chromatography separation technique may include reverse phase separation or hydrophilic interaction liquid chromatography separation (H1LIC).

[0146] At block 160, the amount or quantify of 14-3-3q protein in the sample is measured using mass spectrometry'. In other embodiments, the analytical technique used to measure the biomarker may be mass spectrometry, but the analytical technique used to measure the internal standard may not be mass spectrometry. For example, the internal standard may be measured by immunometric methods, colorimetric methods, electrochemical methods, or fluorometric methods. Or the analytical technique used to measure the biomarker may not be mass spectrometry (but may be one of the other methods disclosed herein). In alternate embodiments the mass spectrometry is tandem mass spectrometry’, liquid chromatography tandem mass spectrometry (LC-MS / MS), or 2 dimensional LC-MS / MS.

[0147] In some embodiments, the step of measuring the amount of 14-3-3q protein in the sample using mass spectrometry' may include the steps of (1) generating at least one precursor ion of the surrogate peptide of 14-3-3q protein; (2) generating one or more fragment ions of the precursor ion; and (3) detecting the presence or amount of the precursor ion generated in step (1) and / or the at least one or more fragment ions generated in step (2), or both, and relating the detected ions to the presence or amount of the 14-3-3q protein in the biological sample. In some embodiments, the precursor ions and / or fragment ions may be selected from any one of the ions listed in Table 2.

[0148] In some embodiments, the at least one precursor ion is formed by methods such as atmospheric pressure chemical ionization (APCI) mode or Electrospray Ionization (ESI). In electrospray ionization, the mode may be selected from either negative ion mode or positive ion mode. In some embodiments, positive ion mode ESI is utilized to produce the at least one precursor ion. For example, tandem mass spectrometry would generally include electrospray ionization of the liquid chromatography eluent to generate gas-phase precursor ions of the 14-PATENT APPLICATIONAttorney Docket No 057618-1509623Client Reference No. LC 2024-01-WO-PCT3-3q surrogate peptides. The precursor ions would be isolated in the first stage of mass analysis and subsequently fragmented (or dissociated) to derive product ions (fragment ions), that would be subjected to a second stage of mass analysis. Isolation of the precursor ion in the first stage of mass analysis can be performed using a quadrupole mass analyzer or ion trap mass analyzer. Fragmentation of the isolated precursor ion can be performed in a number of ways, such as by collision-induced dissociation (CID) or Electron-capture dissociation (ECD). In some embodiments, the second stage of mass analysis can be performed using any number of mass analyzers such as a quadrupole, ion trap, time-of-flight, or orbitrap. Precursor and product ions which may be suitable for tandem mass spectrometry analysis of the 14-3-3q surrogate peptides are listed in Table 2.Table 2. 14-3-3q surrogate peptide precursor and product ions.PATENT APPLICATIONAttorney Docket No 057618-1509623Client Reference No. LC 2024-01-WO-PCT

[0149] In an alternate embodiment, the methods described herein may include a method according to FIG. 2. For example, the method 200 described in FIG. 2 may be a different workflow than the method described in FIG. 1. At block 210, the method includes providing a biological sample that includes a plurality of proteins including one or more 14-3-3 protein family members. In some embodiments, the one or more 14-3-3 protein family members can be 14-3-3i] protein and at least one other family member of 14-3-3 protein (e.g, 14-3-3|3, 14- 3-3y, 14-3-3a, etc.) or fragments thereof.

[0150] At block 220, the biological sample may be subsequently treated with a proteolytic enzyme to produce a proteolytic digest comprising a plurality of peptides. The plurality of peptides may include at least one surrogate peptide derived from the 14-3-3 protein family. For example, the plurality of peptides may include at least one surrogate peptide derived from 14-3-3q protein. In some embodiments, the peptides may include surrogate peptides derived from other 14-3-3 protein family members. In some embodiments, the proteolytic enzyme may be trypsin. In some embodiments, the biological sample is treated with an internal standard prior to proteolysis, wherein the internal standard contains proteolytic sites as well as stable isotopes of any one of the SEQ ID NOs: 1-17, such that once proteolyzed produce stable isotopes of the corresponding SEQ ID NOs: 1-17.

[0151] At block 230, the method may further include adding a binding agent to the proteolytic digest. For example, the binding agent binds specifically to surrogate peptides of 14-3-3q protein and / or 14-3-3 protein family members. The binding agent may bind specifically to 14-3-3q protein and at least one other 14-3-3 protein family member. In some embodiments, the binding agent is an anti-peptide antibody or an aptamer. In some embodiments, the anti-peptide antibody may bind with any surrogate peptide with at least 80 % identical to any one of SEQ ID NOs.: 1-17 provided in Table 1 (e.g, at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %. or 99 % Identity).

[0152] In some embodiments, the method may further include adding an internal standard to the biological sample prior to digestion of the sample. The internal standard may be aPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT stable isotope of 14-3-3i] protein and / or other 14-3-3 protein family members. The proteolytic digest comprising the plurality of peptides may include surrogate peptides of the stable isotope of 14-3-3q.

[0153] In some embodiments, the method may comprise adding an internal standard to the enriched sample, wherein the internal standard includes a stable isotope of the surrogate peptide. The stable isotope of the surrogate peptide can be proteolyzed to form a stable isotope surrogate peptide. In other embodiments, the method may comprise adding an internal standard to the enriched or proteolyzed sample, wherein the internal standard is a stable isotope of the surrogate peptide.

[0154] In some embodiments, the method may include using two or more different stable isotope labeled internal standards. For example, for each target analyte to be detected and quantified, two different stable isotope internal standards are used, that allow for the target analyte, first stable isotope labeled internal standard and second stable isotope internal standard to be distinguished from one other using mass spectrometry. For example, one stable isotope labeled internal standard could include a single label (e.g.. have a single stable isotope-labeled amino acid), while the second stable isotope labeled internal standard could include a different single label (e.g., a different stable isotope-labeled amino acid), include two or more stable isotopes (e.g., two different stable isotope-labeled amino acids). In some embodiments, the stable isotope labeled internal standards are the same as the target analyte, but for the presence of the stable isotope label(s). For example, if the target molecule is one or more of the 14-3-3 protein family members, the first stable isotope labeled molecule can be a 14-3-3 protein family member containing a stable isotope and the second stable isotope labeled molecule can be a 14-3-3 protein family member containing a stable isotope distinguishable from the stable isotope on the first stable isotope labeled 14-3-3 protein family member.

[0155] If a peptide is the target (e.g., used as a surrogate for detecting the presence of a protein), the same principles apply. For example, if the target analyte is 14-3-3q protein, and the peptide used to determine the presence of 14-3-3q protein is YDDMASAMK (SEQ ID NO: 2), the first stable isotope labeled molecule can be a YDDMASAMK (SEQ ID NO: 2) containing a stable isotope and the second stable isotope labeled molecule can be YDDMASAMK (SEQ ID NO: 2) containing a stable isotope distinguishable from the stable isotope on the first stable isotope labeled 14-3-3q protein. For example, the YDDMASAMK (SEQ ID NO: 2) containing the first stable isotope could have a stable isotope on a singlePATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT amino acid, while the YDDMASAMK (SEQ ID NO: 2) containing the second stable isotope could have a stable isotope on two amino acids or have a single amino acid with two stable isotopes. Variations of this labeling are possible, if the first and second stable isotope labeled molecules are distinguishable from one another and from the native molecules via mass spectrometry.

[0156] At block 240, the method may include the step of enriching the proteolytic digest with surrogate peptides of 14-3-3q protein, and optionally one or more other surrogate peptides of 14-3-3 protein family members, in the proteolyzed digest to produce a sample enriched with surrogate peptides of l4-3-3q protein. In some embodiments, the enrichment methods may include immunoaffinity enrichment, affinity enrichment, and abundant protein depletion.

[0157] At block 250, the sample enriched with surrogate peptides of 14-3-3q protein, and optionally one or more other surrogate peptides of 14-3-3 protein family members, can be purified. For example, liquid chromatography may be performed on the enriched sample to purify the sample. Liquid chromatography may include high performance liquid chromatography (HPLC). In some cases, liquid chromatography comprises extraction and analytical liquid chromatography. Additionally, and / or alternatively, high turbulence liquid chromatography (HTLC) (also known as high throughput liquid chromatography) may be used. In alternate embodiments, the liquid chromatography separation technique may include reverse phase separation or hydrophilic interaction liquid chromatography separation (HILIC).

[0158] At block 260, the amount of 14-3-3 protein in the sample is measured using mass spectrometry. For example, the amount of surrogate peptides of 14-3-3q protein can be measured using mass spectrometry to quantify the amount of 14-3-3q protein in the sample. In some embodiments, the step of measuring the amount of 14-3-3q protein and / or other 14- 3-3 protein family members in the sample using mass spectrometry7may include the steps of (1) generating at least one precursor ion of a surrogate peptide of 14-3-3q protein and optionally 14-3-3 protein family members; (2) generating one or more fragment ions of the precursor ion; and (3) detecting the presence or amount of the precursor ion generated in step (1) and / or the one or more fragment ions generated in step (2), or both, and relating the detected ions to the presence or amount of the 14-3-3q protein in the biological sample. In some embodiments, the precursor ions and / or fragment ions may be selected from any one of the ions listed in Table 2.PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT

[0159] In other embodiments, the analytical technique used to measure the biomarker may be mass spectrometry, but the analytical technique used to measure the internal standard may not be mass spectrometry. For example, the internal standard may be measured by immunometric methods, colorimetric methods, electrochemical methods, or fluorometric methods. Or the analytical technique used to measure the biomarker may not be mass spectrometry (but may be one of the other methods disclosed herein). In alternate embodiments the mass spectrometry is tandem mass spectrometry, liquid chromatography tandem mass spectrometry (LC-MS / MS), or 2 dimensional LC-MS / MS.

[0160] In some embodiments, the native 14-3-3q protein in a biological sample is measured. For example, the method for detecting 14-3-3q protein in a biological sample may include generating a precursor ion of the 14-3-3q protein and detecting the amount of precursor ion of the 14-3-3q protein to determine the amount of 14-3-3q protein in the sample by mass spectrometry'. In this example, a surrogate peptide of 14-3-3q protein is not used. The method may include purifying the sample prior to mass spectrometry, for example, using liquid chromatography. In some embodiments, one or more precursor ions of the 14-3-3q are generated. In some embodiments, the method comprises enriching the sample in the 14-3-3q protein prior to generating precursor ions.Systems for Detecting or Quantifying 14-3-3r] Protein Using LC-MS / MS

[0161] Other disclosed embodiments comprise systems. For example, the disclosure herein provides a system for determining the presence and / or amount of 14-3-3q protein in a biological sample, the system comprising: a device for providing a test sample comprising a body fluid; and a station for analyzing the body fluid by mass spectrometry to determine the presence or amount of 14-3-3q protein in the biological sample. For example, in certain embodiments, the system may comprise: a station for providing a biological sample believed to contain 14-3-3q protein wherein the biological sample comprises serum or plasma; a station for at least partially purify ing the 14-3-3q protein from the biological sample; a station for chromatographically separating the 14-3-3q protein from the biological sample; and a station for measuring the 14-3-3q protein by mass spectrometry to determine the presence or amount of the at least one biomarker of interest in the biological sample.

[0162] In an embodiment, the mass spectrometry' is operated in an atmospheric pressure chemical ionization (APCI) mode. Also in certain embodiments, at least one of the stations is automated and / or controlled by a computer. For example, as described herein, in certainPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT embodiments, at least some of the steps are automated such that little to no manual intervention is required.

[0163] In one embodiment, the station for chromatographic separation comprises at least one apparatus to perform liquid chromatography (LC). In one embodiment, the station for liquid chromatography comprises a column for extraction chromatography. Additionally, or alternatively, the station for liquid chromatography comprises a column for analytical chromatography. In certain embodiments, the column for extraction chromatography and analytical chromatography comprises a single station or single column. For example, in one embodiment, liquid chromatography is used to purify the biomarker of interest from other components in the sample that co-purify with the biomarker of interest after extraction or dilution of the sample. In some embodiments, the separation technique used in the liquid chromatography may include reverse phase separation or HILIC.

[0164] The system may also include a station for analyzing the chromatographically separated one or more biomarkers of interest by mass spectrometry to determine the presence or amount of the one or more biomarkers in the test sample. In certain embodiments, tandem mass spectrometry is used (MS / MS). For example, in certain embodiments, the station for tandem mass spectrometry comprises an Applied Biosystems API4000 or API5000 or thermo quantum or Agilent 7000 triple quadrupole mass spectrometer or an Applied Biosystems API5500 or API6500 triple quadrupole or thermo Q-Exactive mass spectrometer.

[0165] The system may also comprise a station for partially purifying at least one biomarker of interest from the biological sample and / or diluting the sample. In an embodiment, the station for purifying comprises a station for supported liquid extraction (SLE) and / or liquidliquid extraction (e.g, for smaller molecules and / or lipids). For example, the station for liquid-liquid extraction may comprise equipment and binding agents for addition of solvents to the sample and removal of waste fractions. In some embodiments, the station for purifying may comprise protein precipitation. In another embodiment, the station for purifying may comprise immunoaffinity enrichment (e.g., for proteins or peptides).

[0166] In certain embodiments, the methods and systems of the present invention may comprise multiple liquid chromatography steps. Thus, in certain embodiments, a two- dimensional liquid chromatography (LC) procedure is used. For example, in one embodiment, the method and systems of the present invention may comprise transferring the sample, or surrogate peptides derived from the sample, from a LC extraction column to an analytical column. In one embodiment, the transferring from the extraction column to anPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT analytical column is done by a heart-cutting technique. In another embodiment, transfer from the extraction column to an analytical column by a chromatofocusing technique.Alternatively, transfer from the extraction column to an analytical column may be done by a column switching technique. These transfer steps may be done manually or may be part of an on-line system.

[0167] Various columns comprising stationary phases and mobile phases that may be used for extraction or analytical liquid chromatography are described herein. The column used for extraction liquid chromatography may be varied depending on the biomarker of interest. In some embodiments, the extraction column is a functionalized silica or polymer-silica hybrid or polymeric particle or monolithic silica stationary phase, such as a Poroshell SBC- 18 column. The column used for analytical liquid chromatography may be varied depending on the analyte and / or the column that was used for the extraction liquid chromatography step. For example, in certain embodiments, the analytical column comprises particles having an average diameter of about 5 pm. In some embodiments, the analytical column is a functionalized silica or polymer-silica hybrid, or a polymeric particle or monolithic silica stationary phase, such as a phenyl-hexyl functionalized analytical column. In some embodiments, reverse phase separation may include a column including particles functionalized with hydrocarbon chains (Cl 2 to Cl 8) or aromatic groups such as phenyl or bi-phenyl. The weak mobile phase may include predominately water and the strong mobile phase may include one or more organic solvents such as acetonitrile and methanol. As noted herein, in certain embodiments, the mass spectrometer may comprise a tandem mass spectrometer (MS / MS). For example, in one embodiment of the methods and systems of the present invention, the tandem MS / MS spectrometry comprises a triple quadrupole tandem mass spectrometer. In other embodiments, the tandem mass spectrometer may be a hybrid mass spectrometer, such as a quadrupole-orbit rap or a quadrupole-time-of-flight mass spectrometer.

[0168] The tandem MS / MS may be operated in a variety of modes. In one embodiment, the tandem MS / MS spectrometer is operated in an atmospheric pressure chemical ionization (APCI) mode or Electrospray Ionization (ESI). The electrospray ionization maybe performed in either negative ion mode or positive ion mode. In some embodiments, the quantification of the analytes and internal standards is performed in the selected reaction monitoring mode (SRM).PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT

[0169] The systems and methods of the present invention may. in certain embodiments, provide for a multiplexed or high throughput assay. For example, certain embodiments may comprise a multiplexed liquid chromatography tandem mass spectrometry (LC-MS / MS) or two-dimensional or tandem liquid chromatography -tandem mass spectrometry (LC)-LC- MS / MS) methods for the proteomic analysis.

[0170] In some embodiments, a tandem MS / MS system is used. The precursor ion is selected following ionization, and that precursor ion is subjected to fragmentation to generate product ions (z.e., fragment), whereby one or more product ions are subjected to a second stage of mass analysis for detection. For example, the second stage of mass analysis could be performed using any number of mass analyzers such as quadrupole, ion trap, time-of-flight, or orbitrap. Precursor and product ions which may be suitable for tandem mass spectrometry analysis of 14-3-3q surrogate peptides are listed in Table 2. For example, in tandem mass spectrometry, a first step may include electrospray ionization of the liquid chromatography eluent to generate gas-phase precursor ions of the 14-3-3q surrogate peptides. Those precursor ions may be isolated in the first stage of mass analysis and subsequently fragmented (or dissociated) to derive product ions, that would be subjected to a second stage of mass analysis. Isolation of the precursor ion in the first stage of mass analysis may be performed using a quadrupole mass analyzer or ion trap mass analyzer. Fragmentation of the isolated precursor ion may be performed in a number of ways including collision-induced dissociation (CID) or electron capture dissociation (ECD). The second stage of mass analysis may be performed using any number of mass analyzers including quadrupole, ion-trap, time- of-flight, or orbitrap.

[0171] The analyte of interest may then be detected and / or quantified based upon the amount of the characteristic transitions measured by tandem MS. In some embodiments, the tandem mass spectrometer comprises a triple quadrupole mass spectrometer. In some embodiments, the tandem mass spectrometer is operated in a positive ion Atmospheric Pressure Chemical Ionization (APCI) mode. In some embodiments, the quantification of the analytes is performed in the selected reaction monitoring mode (SRM). In some embodiments, an internal standard may be added to the sample and the internal standard and the analytes quantification is performed in SRM. In some embodiments, other methods of ionization such as the use of inductively coupled plasma, or MALDI, or SELDI, ESI, or APPI may be used for ionization.

[0172] In some embodiments, the back-calculated amount of each analyte in each sample may be determined by comparison of unknown sample response or response ratio when employingPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT internal standardization to calibration curves generated by spiking a known amount of purified analyte material into a standard test sample, e.g, charcoal stripped human serum. In one embodiment, calibrators are prepared at know n concentrations and analyzed as per the biomarker methodology' to generate a response or response ratio when employing internal standardization versus concentration calibration curve.

[0173] The temperature for heating the sample during ionization may, in alternate embodiments range from 100 °C to about 1000 °C and includes all ranges therein. In an embodiment, the dehydration step is performed within the interface of the mass spectrometer employed in APCI or electrospray mode at 500 degrees C ± 100 degrees. In an embodiment, the sample is heated for several microseconds at the interface for dehydration to occur. In alternate embodiments, the heating step is done for less than 1 second, or less than 100 milliseconds (msec), or less than 10 msec, or less than 1 msec, or less than 0.1 msec, or less than 0.01 msec, or less than 0.001 msec.

[0174] FIG. 3 shows an embodiment of a system (302) of the present invention. For the systems described herein, there may be additional stations and / or some of the stations may be combined. For example, as shown in FIG. 3, the system may comprise a station for processing a sample comprising serum or plasma (304) that may comprise a biomarker of interest into sampling containers (e.g.. 96 well microtiter assay wells). The station for processing the sample may include components for adding at least one of a quantity control (QC) and / or calibration standard to the bodily fluid. In some embodiments, the station for processing the sample may include performing a proteolytic digest and adding anti-peptide antibodies to the bodily fluid. In one embodiment, the sample is aliquoted into a container or containers to facilitate extraction at an extraction station (306) of the biomarker of interest. The station for aliquoting may comprise receptacles to discard the portion of the biological sample that is not used in the analysis.

[0175] The extraction station (306) may optionally comprise a station for adding an internal standard to the sample. In an embodiment, the extraction station (306) may comprise a station for adding anti-peptide antibodies to the bodily fluid to purify or enrich the sample. In an embodiment, the internal standard comprises the biomarker of interest labeled with a nonnatural isotope. Thus, the station for extraction and / or adding an internal standard may comprise safety features to facilitate adding an isotopically labeled internal standard solutions to the sample.PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT

[0176] Where LC-MS / MS is not used to measure the internal standard, the system may comprise a station for aliquoting portions of the extracted sample (308) for measurement of a normalization marker and measurement of the biomarker of interest. The system may also comprise a station (310) for measuring the normalization marker with a technique that is independent of mass spectrometry. The technique used will depend on the nature of the normalization (normalizing) standard, but may be one of any of the techniques known to one of skill in the art.

[0177] The system may also comprise a station for further processing of the biomarker. For example, the system may also, in some embodiments, comprise a station (312) for purification steps such as supported liquid extraction, liquid-liquid extraction, protein precipitation and / or dilution of the sample.

[0178] The system may also comprise a station for liquid chromatography (e.g., HPLC) of the sample (314). As described herein, in an embodiment, the station for liquid chromatography may comprise an extraction liquid chromatography column. The station for liquid chromatography may comprise a column comprising the stationary phase, as well as containers or receptacles comprising solvents that are used as the mobile phase. In an embodiment, the mobile phase comprises a gradient of methanol and water, acetonitrile and water, or other miscible solvents with aqueous volatile buffer solutions. Thus, in one embodiment, the station may comprise the appropriate lines and valves to adjust the amounts of individual solvents being applied to the column or columns. Also, the station may comprise a means to remove and discard those fractions from the LC that do not comprise the biomarker of interest. In an embodiment, the fractions that do not contain the biomarker of interest are continuously removed from the column and sent to a waste receptacle for decontamination and to be discarded. The system may also comprise an analytical LC column (314). The analytical column may facilitate further purification and concentration of the biomarker of interest as may be required for further characterization and quantification.

[0179] Also, the system may comprise a station for characterization and quantification of the biomarker of interest. In one embodiment, the system may comprise a station for mass spectrometry (MS) (316) of the biomarker. In an embodiment, the station for mass spectrometry comprises a station for tandem mass spectrometry (MS / MS). Also, the station for characterization and quantification may comprise a station for data analysis (318) for the biomarker and the normalization marker and / or a computer (320) and software for analysis ofPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT the results. In an embodiment, the analysis comprises both identification and quantification of the biomarker of interest.

[0180] In some embodiments, one or more of the purification or separation steps can be performed “on-line.’' As used herein, the term “on-line” refers to purification or separation steps that are performed in such a way that the test sample is disposed, e.g. injected, into a system in which the various components of the system are operationally connected and, in some embodiments, in fluid communication with one another. The on-line system may comprise an autosampler for removing aliquots of the sample from one container and transferring such aliquots into another container. For example, an autosampler may be used to transfer the sample after extraction onto an LC extraction column. Additionally, or alternatively, the on-line system may comprise one or more injection ports for injecting the fractions isolated from the LC extraction columns onto the LC analytical column. Additionally, or alternatively, the on-line system may comprise one or more injection ports for injecting the LC purified sample into the MS system. Thus, the on-line system may comprise one or more columns, including but not limited to, an extraction column, including an HTLC extraction column, and in some embodiments, an analytical column. Additionally, or alternatively, the system may comprise a detection system, e.g., a mass spectrometer system. The on-line system may also comprise one or more pumps; one or more valves; and necessary plumbing. In such “on-line” systems, the test sample and / or analytes of interest can be passed from one component of the system to another without exiting the system, e.g., without having to be collected and then disposed into another component of the system.

[0181] In some embodiments, the on-line purification or separation method can be automated. In such embodiments, the steps can be performed without the need for operator intervention once the process is set-up and initiated. Thus, in various embodiments, the system, or portions of the system may be controlled by a computer or computers (120). Thus, in certain embodiments, the present invention may comprise software for controlling the various components of the system, including pumps, valves, autosamplers, and the like. Such software can be used to optimize the extraction process through the precise timing of sample and solute additions and flow rate. For example, when employed on a multiplexing LC system, such as the ARIA Transcend™ TLX-4, injections may be run in parallel with injections staggered every 1.5 minutes to improve the duty cycle of the mass spectrometric analysis.PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT

[0182] In some embodiments, the computer architecture may be a server computer, workstation, desktop computer, laptop, tablet, network appliance, personal digital assistant (“PDA”), e-reader, digital cellular phone, or other computing device, and may be utilized to execute any aspects of the software components presented herein.

[0183] The computer may include a baseboard, or “motherboard.” which is a printed circuit board to which a multitude of components or devices may be connected by way of a system bus or other electrical communication paths. In one embodiment, one or more central processing units (“CPUs”) operate in conjunction with a chipset. The CPUs may be standard programmable processors that perform arithmetic and logical operations necessary for the operation of the computer.

[0184] The CPUs perform operations by transitioning from one discrete, physical state to the next through the manipulation of switching elements that differentiate between and change these states. Switching elements may generally include electronic circuits that maintain one of two binary states, such as flip-flops, and electronic circuits that provide an output state based on the logical combination of the states of one or more other switching elements, such as logic gates. These basic switching elements may be combined to create more complex logic circuits, including registers, adders-subtractors, arithmetic logic units, floating-point units, and the like.

[0185] The chipset provides an interface between the CPUs and the remainder of the components and devices on the baseboard. The chipset may provide an interface to a random access memory' (“RAM”), used as the main memory' in the computer. The chipset may further provide an interface to a computer-readable storage medium such as a read-only memory (“ROM”) or non-volatile RAM (“NVRAM”) for storing basic routines that help to startup the computer and to transfer information between the various components and devices. The ROM or NVRAM may also store other software components necessary' for the operation of the computer 1000 in accordance with the embodiments described herein.

[0186] The computer may operate in a networked environment using logical connections to remote computing devices and computer systems through a network, such as the local area network. The chipset may include functionality for providing network connectivity7through a NIC, such as a gigabit Ethernet adapter. The NIC can connect the computer to other computing devices over the network. It should be appreciated that multiple NICs may be present in the computer, connecting the computer to other types of networks and remote computer systems.PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT

[0187] The computer may be connected to a mass storage device that provides non-volatile storage for the computer. The mass storage device may store system programs, application programs, other program modules, and data, which have been described in greater detail herein. The mass storage device may be connected to the computer through a storage controller connected to the chipset. The mass storage device may consist of one or more physical storage units. The storage controller may interface with the physical storage units through a serial attached SCSI ("SAS") interface, a serial advanced technology attachment (“SATA”) interface, a fiber channel (“FC”) interface, or other ty pe of interface for physically connecting and transferring data between computers and physical storage units.

[0188] The computer may store data on the mass storage device by transforming the physical state of the physical storage units to reflect the information being stored. The specific transformation of physical state may depend on various factors, in different implementations of this description. Examples of such factors may include, but are not limited to, the technology used to implement the physical storage units, whether the mass storage device is characterized as primary or secondary storage, and the like.

[0189] For example, the computer may store information to the mass storage device by issuing instructions through the storage controller to alter the magnetic characteristics of a particular location within a magnetic disk drive unit, the reflective or refractive characteristics of a particular location in an optical storage unit, or the electrical characteristics of a particular capacitor, transistor, or other discrete component in a solid-state storage unit. Other transformations of physical media are possible without departing from the scope and spirit of the present description, with the foregoing examples provided only to facilitate this description. The computer may further read information from the mass storage device by detecting the physical states or characteristics of one or more particular locations within the physical storage units.

[0190] In addition to the mass storage device described above, the computer may have access to other computer-readable storage media to store and retrieve information, such as program modules, data structures, or other data. It should be appreciated by those skilled in the art that computer-readable storage media can be any available media that provides for the storage of non-transitory data and that may be accessed by the computer.

[0191] By way of example, and not limitation, computer-readable storage media may include volatile and non-volatile, removable and non-removable media implemented in any method or technology. Computer-readable storage media includes, but is not limited to, RAM, ROM,PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT erasable programmable ROM (“EPROM”), electrically-erasable programmable ROM (“EEPROM”), flash memory or other solid-state memory technology, compact disc ROM (“CD-ROM”), digital versatile disk (“DVD”), high definition DVD (“HD-DVD”), BLU- RAY, or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to store the desired information in a non-transitory fashion.

[0192] The mass storage device may store an operating system utilized to control the operation of the computer. According to one embodiment, the operating system comprises the LINUX operating system. According to another embodiment, the operating system comprises the WINDOWS® SERVER operating system from MICROSOFT Corporation. According to further embodiments, the operating system may comprise the UNIX or SOLARIS operating systems. It should be appreciated that other operating systems may also be utilized. The mass storage device may store other system or application programs and data utilized by the computer. The mass storage device might also store other programs and data not specifically identified herein.

[0193] In one embodiment, the mass storage device or other computer-readable storage media is encoded with computer-executable instructions which, when loaded into the computer, transforms the computer from a general-purpose computing system into a specialpurpose computer capable of implementing the embodiments described herein. These computer-executable instructions transform the computer by specifying how the CPUs transition between states, as described above. According to one embodiment, the computer has access to computer-readable storage media storing computer-executable instructions which, when executed by the computer, perform the various routines described above in the methods of detecting 14-3-3i]. The computer might also include computer-readable storage media for performing any of the other computer-implemented operations described herein.

[0194] The computer may also include one or more input / output controllers for receiving and processing input from a number of input devices, such as a keyboard, a mouse, a touchpad, a touch screen, an electronic stylus, or other type of input device. Similarly, the input / output controller may provide output to a display, such as a computer monitor, a flat-panel display, a digital projector, a printer, a plotter, or other Npe of output device. It will be appreciated that the computer may not include all of the components described above, may include other components that are not explicitly described, or may utilize an architecture completelyPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT different than that described herein. It should also be appreciated that many computers, might be utilized in combination to embody aspects of the various technologies disclosed herein.

[0195] Although some or all of the steps in the method and the stations comprising the system may be on-line, in certain embodiments, some or all of the steps may be performed “off-line / ’ In contrast to the term “on-line”, the term “off-line” refers to a purification, separation, or extraction procedure that is performed separately from previous and / or subsequent purification or separation steps and / or analysis steps. In such off-line procedures, the analytes of interest typically are separated, for example, on an extraction column or by liquid / liquid extraction, from the other components in the sample matrix and then collected for subsequent introduction into another chromatographic or detector system. Off-line procedures typically require manual intervention on the part of the operator.

[0196] Liquid chromatography may, in certain embodiments, comprise high turbulence liquid chromatography or high throughput liquid chromatography (HTLC). See, e.g, Zimmer et al., J. Chromatogr. A 854:23-35 (1999); see also, U.S. Pat. Nos. 5,968.367; 5,919,368;5,795,469; and 5,772,874. Traditional HPLC analysis relies on column packings in which laminar flow of the sample through the column is the basis for separation of the analyte of interest from the sample. In such columns, separation is a diffusional process. Turbulent flow, such as that provided by HTLC columns and methods, may enhance the rate of mass transfer, improving the separation characteristics provided. In some embodiments, high turbulence liquid chromatography (HTLC), alone or in combination with one or more purification methods, may be used to purify the biomarker of interest prior to mass spectrometry. In such embodiments, samples may be extracted using an HTLC extraction cartridge which captures the analyte, then eluted and chromatographed on a second HTLC column or onto an analytical HPLC column prior to ionization. Because the steps involved in these chromatography procedures can be linked in an automated fashion, the requirement for operator involvement during the purification of the analyte can be minimized. Also, in some embodiments, the use of a high turbulence liquid chromatography sample preparation method can eliminate the need for other sample preparation methods including liquid-liquid extraction. Thus, in some embodiments, the test sample, e.g., a biological fluid, can be disposed, e.g., injected, directly onto a high turbulence liquid chromatography system.

[0197] For example, in a typical high turbulence or turbulent liquid chromatography system, the sample may be injected directly onto a narrow (e.g., 0.5 mm to 2 mm internal diameter by 20 to 50 mm long) column packed with large (e.g., > 25 micron) particles. When a flowPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT rate (e.g., 3-500 mL per minute) is applied to the column, the relatively narrow width of the column causes an increase in the velocity of the mobile phase. The large particles present in the column can prevent the increased velocity from causing back pressure and promote the formation of vacillating eddies between the particles, thereby creating turbulence within the column.

[0198] In high turbulence liquid chromatography, the analyte molecules may bind quickly to the particles and typically do not spread out, or diffuse, along the length of the column. This lessened longitudinal diffusion typically provides better, and more rapid, separation of the analytes of interest from the sample matrix. Further, the turbulence within the column reduces the friction on molecules that typically occurs as they travel past the particles. For example, in traditional HPLC, the molecules traveling closest to the particle move along the column more slowly than those flowing through the center of the path between the particles. This difference in flow rate causes the analyte molecules to spread out along the length of the column. When turbulence is introduced into a column, the friction on the molecules from the particle is negligible, reducing longitudinal diffusion.

[0199] In some embodiments, the liquid chromatography method may comprise reverse phase separation. In reverse phase separation, particles may be functionalized with hydrocarbon chains (C12 or Cl 8) or aromatic groups (phenyl or bi-phenyl). The weak mobile phase may be comprised predominantly of water and the strong mobile phase may be comprised of one or more organic solvents such as acetonitrile and methanol. To improve retention of the 14-3-3q surrogate peptide(s) during reverse phase separations as well as other electrospray ionization, ion pairing binding agents, such as formic acid or acetic acid, may be added to the weak and or strong mobile phases.

[0200] In some embodiments, the liquid chromatography method may include hydrophilic interaction liquid chromatography (HILIC). HILIC separation may include particles functionalized with silanol groups, amide groups, or cyano groups. The weak mobile phase may be comprised predominately of aprotic organic solvents such as acetonitrile or ethyl acetate. The strong mobile phase may be comprised predominately of water and / or protic organic solvents such as methanol. To improve retention of 14-3-3q surrogate peptide(s) during HILIC separations, ion pairing binding agents such as ammonium formate or ammonium acetate may be added to the weak and / or strong mobile phase. In some embodiments, the ion pairing reagent may be added to both the weak and strong mobile phase.PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT

[0201] The methods and systems of the present invention may use mass spectrometry to detect and quantify the biomarker of interest. The terms “mass spectrometry” or “MS” as used herein generally refer to methods of filtering, detecting, and measuring ions based on their mass-to- charge ratio, or “m / z.” In MS techniques, one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrometer where, due to a combination of electric fields, the ions follow a path in space that is dependent upon mass (“m”) and charge (“z”).

[0202] In certain embodiments, the mass spectrometer uses a “quadrupole” system. In a “quadrupole” or “quadrupole ion trap” mass spectrometer, ions in an oscillating radio frequency (RF) field experience a force proportional to the direct current (DC) potential applied between electrodes, the amplitude of the RF signal, and m / z. The voltage and amplitude can be selected so that only ions having a particular m / z travel the length of the quadrupole, while all other ions are deflected. Thus, quadrupole instruments can act as both a “mass filter” and as a “mass detector” for the ions injected into the instrument.

[0203] In certain embodiments, tandem mass spectrometry is used. See, e.g., U.S. Pat. No. 6,107,623, entitled “Methods and Apparatus for Tandem Mass Spectrometry,” which is hereby incorporated by reference in its entirety. Further, the selectivity of the MS technique can be enhanced by using “tandem mass spectrometry,” or “MS / MS.” Tandem mass spectrometry (MS / MS) is the name given to a group of mass spectrometric methods wherein “parent or precursor” ions generated from a sample are fragmented to yield one or more “fragment or product” ions, which are subsequently mass analyzed by a second MS procedure. MS / MS methods are useful for the analysis of complex mixtures, especially biological samples, in part because the selectivity of MS / MS can minimize the need for extensive sample clean-up prior to analysis. In an example of an MS / MS method, precursor ions are generated from a sample and passed through a first mass filter to select those ions having a particular mass-to-charge ratio. These ions are then fragmented, typically by collisions with neutral gas molecules in a suitable ion containment device, to yield product (fragment) ions, the mass spectrum of which is recorded by an electron multiplier detector. The product ion spectra so produced are indicative of the structure of the precursor ion, and the two stages of mass filtering can eliminate ions from interfering species present in the conventional mass spectrum of a complex mixture.

[0204] In an embodiment, the methods and systems of the present invention use a triple quadrupole MS / MS (see e.g., Yost, Enke in Ch. 8 of Tandem Mass Spectrometry, Ed.PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCTMcLafferty. pub. John Wiley and Sons. 1983). Triple quadrupole MS / MS instruments typically consist of two quadrupole mass filters separated by a fragmentation means. In one embodiment, the instrument may comprise a quadrupole mass filter operated in the RF only mode as an ion containment or transmission device. In an embodiment, the quadrupole may further comprise a collision gas at a pressure of between 1 and 10 millitorr. Many other types of '‘hybrid’’ tandem mass spectrometers are also known and can be used in the methods and systems of the present invention including various combinations of orbitrap analyzers and quadrupole filters. These hybrid instruments often comprise high resolution orbitrap analyzers (see e.g., Hu Q, Noll RJ, Li H, Makarov A. Hardman M. Graham Cooks R. The Orbitrap: a new mass spectrometer. J Mass Spectrum. 2005;40(4):430-443) for the second stage of mass analysis. Use of high-resolution mass analyzer may be highly effective in reducing chemical noise to very' low levels.

[0205] For the methods and systems of the present invention, ions can be produced using a variety of methods including, but not limited to. electron ionization, chemical ionization, fast atom bombardment, field desorption, and matrix-assisted laser desorption ionization (‘'MALDI”), surface enhanced laser desorption ionization (“SELDI”), photon ionization, electrospray ionization, and inductively coupled plasma.

[0206] In those embodiments, such as MS / MS, where precursor ions are isolated for further fragmentation, collision-induced dissociation (“CID”) may be used to generate the fragment ions for further detection. In CID, precursor ions gain energy' through collisions with an inert gas and subsequently fragment by a process referred to as “unimolecular decomposition.” Sufficient energy must be deposited in the precursor ion so that certain bonds within the ion can be broken due to increased vibrational energy.

[0207] In other such embodiments, such as MS / MS, where precursor ions are isolated for further fragmentation, electron-capture dissociation (ECD) may be used to generate the fragment ions for further detection. In ECD, disulfide bonds may be specifically cleaved. Fragmentation may be fast and specific. In some examples, labile post-translational modifications and non-covalent bonds often remain intact after bond dissociation during ECD. In some embodiments, ECD may provide a high amount of sequence coverage as compared to CID. For example, at high electron energies, ECD may be able to distinguish between leucine and isoleucine.

[0208] In some embodiments, to attain the required analytical selectivity and sensitivity, the presently disclosed 2D-LC-MS / MS methods include multiplexed sample preparationPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT procedures. For example, in certain embodiments dialysis of the sample is performed using a 96 well plate having a dialysis membrane in each well or multiple sample tubes. Additionally, or alternatively, the multiplex system may comprise staggered multiplexed LC and MS sample inlet systems. Also, the methods and systems of the present invention may comprise multiple column switching protocols, and / or heart-cutting (LC-LC or 2D-LC) techniques, and / or LC separations prior to MS detection. In some embodiments, the methods and systems of the present invention may include a multiplexed two-dimensional liquid chromatographic system coupled with a tandem mass spectrometer (MS / MS) system, for example a triple quadrupole MS / MS system. Such embodiments provide for staggered, parallel sample input into the MS system.

[0209] Thus, multiple samples may each be applied to individual extraction columns. Once the samples have each run through the extraction column, they may each be transferred directly (e.g., by column switching) to a second set of analytical columns. As each sample elutes from the analytical column, it may be transferred to the mass spectrometer for identification and quantification.

[0210] A plurality of analytes can be analyzed simultaneously or sequentially by the presently disclosed LC-MS / MS and 2D-LC-MS / MS methods. Exemplary analytes amenable to analysis by the presently disclosed methods include, but are not limited to, peptides, steroid hormones, nucleic acids, vitamins and the like. One of ordinary skill in the art would recognize after a review of the presently disclosed subject matter that other similar analytes could be analyzed by the methods and systems disclosed herein. Thus, in alternate embodiments, the methods and systems may be used to quantify steroid hormones, protein and peptide hormones, peptide and protein biomarkers, drugs of abuse and therapeutic drugs. For example, optimization of key parameters for each analyte can be performed using a modular method development strategy' to provide highly tuned bioanalytical assays. Thus, certain steps may be varied depending upon the analyte being measured as disclosed herein.

[0211] Also, embodiments of the methods and systems of the present invention may provide equivalent sensitivity' attainable for many of the analytes being measured using much less sample. For example, through using this optimization procedure, an LLOQ of about 10 nmol / L for detection of a peptide biomarker for dried plasma corresponding to about 20 pL of liquid plasma. Such small sample sizes render sampling (often by finger-prick) much more accessible.PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT

[0212] All patents, publications and abstracts cited above are incorporated herein by reference in their entireties. Various embodiments of the invention have been described in fulfillment of the various objectives of the invention. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptions thereof will be readily apparent to those skilled in the art without departing from the spirit and scope of the present invention as defined in the following claims.

[0213] In the embodiments described herein, all open-ended transitional phrases such as “comprising,” “including,” and / or “comprises,” and the like, can be used interchangeably with closed or semi-closed transitional phrases such as “consisting of’ and "consisting essentially of’, respectively. Accordingly, the embodiments disclosed herein may be practiced in the absence of any element which is not specifically disclosed herein. Thus, for example, in each instance herein the term “comprising” can be replaced with “consisting essentially of or “consisting of.EXAMPLES

[0214] The examples below provide illustrative embodiments of the method described herein and do not limit the scope of the invention. The objective of the method described below' was to validate workflows of measuring 14-3-3 proteins using LC-MS / MS.Materials

[0215] Commercially available stable-isotope labeled peptides were used with sequences matching those of the indicated surrogate peptides in FIG. 4 (SEQ ID No. 2: YDDMASAMK, SEQ ID No. 6: VISSIEQK, SEQ ID No. 7: TMADGNEK, and SEQ ID No. 12: EAFEISK). Stable-isotope labeled peptide w'ere labeled with [13C6,15N2]-lysine or with [13C6.15N4]-arginine. The purity' of all synthesized peptides was verified to be >95% by high- performance liquid chromatography (215 nm) and concentrations were assigned by amino acid analysis. All peptides were initially reconstituted in water with 0.1% (v / v) formic acid and 0.001% (w / v) ZWITTERGENT® 3-16 detergent at a concentration of 100 pg / mL, then combined and diluted in the same solvent to produce a stable-isotope labeled peptide solution with each at a final, working concentration of 20 ng / mL.

[0216] Calibrators were prepared for the mass spectrometer. The calibrators were prepared using recombinant 14-3-3p spiked into phosphate buffered saline (PBS) containing 20PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT mg / mL human serum albumin (HSA) or 20 mg / mL bovine serum albumin (BSA). The recombinant 14-3-3p had >98% purity as verified by SDS-PAGE (Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis) and concentrations were determined using a Bradford assay.

[0217] For enrichment of the sample, monoclonal rabbit, anti-14-3-3 pan antibody was used. The monoclonal rabbit, anti-14-3-3 pan antibody was biotinylated and conjugated to magnetic beads coated with streptavidin for enrichment of 14-3-3p and other 14-3-3 protein family members. The immunogen used to produce this antibody was a peptide from the conserved region on the C-terminus of 14-3-3 family members (i.e., SEQ ID NO. 18).

[0218] Monoclonal mouse anti-peptide antibodies were developed for the four tryptic peptides of 14-3-3p (see FIG. 4, SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 12) and were denoted as anti-YDD (SEQ ID No. 2), anti -VIS (SEQ ID No. 6), anti-TMA (SEQ ID No. 7), and anti-EAF (SEQ ID No. 12) antibodies based on the first 3 amino acids of the corresponding tryptic peptide. Each anti-peptide antibody was conjugated to magnetic Protein G beads for enrichment of the corresponding peptide following trypsin digestion.LC-MS / MS Analysis Techniques

[0219] The samples were analyzed by LC-MS / MS using a Transcend TLX-4 Turboflow Aria multiplexing HPLC system (Thermo-Fisher Scientific, San Jose, CA), a fully automated on-line two-dimensional liquid chromatography system, coupled to a Sciex 7500+ Triple Quadrupole Mass Spectrometer (AB Sciex, LLC, Framingham. MA). Forty microliters of processed sample was loaded onto a 2.1x75 mm Poroshell SB-C18 (5 pm) column (Agilent Technologies) for 10 seconds at 100% mobile phase A (0.1% formic acid in water). Peptides were eluted with a linear gradient from 0% to 30% mobile phase B (0.1% formic acid in acetonitrile) over 100 seconds before washing and re-equilibrating the column.

[0220] The mass spectrometer was operated in positive electrospray ionization mode using a spray voltage of 2500 V, curtain gas of 40 psi, source gas 1 and 2 of 60 psi, and source temperature of 500 °C. Matching selected reaction monitoring (SRM) transitions were acquired for each natural and stable-isotope labeled peptide with 5 msec dwell times; unit resolution for both QI and Q3; EP, CXP, and Q0D settings of 10 V; and optimized collision energies at a CAD gas setting of 9. Data analysis and quantitation was performed in Skyline v24.1 using the total area of all transitions that did not have an identifiable chromatographic interferent. External calibration curves were produced with a linear, 1 / x-weighted regression.PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCTExample 1 (Workflow 1)

[0221] The following example provides a method of quantifying 14-3-3 proteins using surrogate peptide enrichment. In this example, the sample was digested with a digestion aid to produce surrogate peptides of the target analyte followed by enrichment of the target analyte.

[0222] Serum samples and calibrators (1.58 - 200 ng / mL 14-3-3p) were digested to produce surrogate peptides of 14-3-3 proteins with subsequent enrichment of the resulting 14- 3-3 surrogate peptides using anti-peptide antibodies. Initially, 200 uL of sample was diluted2-fold with 100 mM Tris-HCl (pH 8) containing 1% (w / v) sodium deoxycholate and 1 mM dithiothreitol, then denatured for 30 min at 56 °C before addition of 0.4 ng of each stable isotope labeled peptide. Next, TPCK-treated bovine trypsin (800 pg) (trypsin treated with N- tosyl-L-phenylalanine chloromethyl ketone (TPCK)) was added to denatured samples and allowed to digest for 30 min at 37 °C followed by addition of soybean try psin inhibitor (1 mg) to terminate the digestion. Finally, 2 ug of each anti-peptide antibody coupled to protein G magnetic beads were added to digested serum samples and mixed for 2 hours at room temperature. After removing the unbound fraction, magnetic beads containing the bound peptides were washed 3 times with 200 pL of PBS containing 0.03% (w / v) CHAPS (3-((3- cholamidopropyl) dimethylammonium)-! -propanesulfonate) detergent before elution of the bound peptides in 100 pL of buffer containing 100 mM Glycine, 2% (v / v) formic acid.0.001% (w / v) ZWITTERGENT® 3-16, and 1% (w / v) heptanesulfonic acid for LC-MS / MS analysis. Results are shown in Figs. 5A-5F, 6A-6F, 7A-7F and 8A-8F. The precursor ions and product ions of the detected surrogate peptides of 14-3-3 during mass spectrometry are provided in Table 2.

[0223] The quantities obtained from co-enriched 14-3-3 surrogate peptides were compared. Demming regression showed good agreement and correlation between surrogate peptides specific to 14-3-3q (z.e., 14-3-3_TMA, 14-3-3_YDD, and 14-3-3_EAF). 14-3-3_VIS results were systematically higher than those obtained for the other surrogate peptides, which may be explained by the contribution of other 14-3-3 family members to the measurement of the 14-3-3_VIS surrogate peptide (which is found in alpha / beta, gamma, eta, and theta / tau forms); however, the high degree of correlation between the 14-3-3_VIS and 14-3-3_TMA results suggests the same measurand is being detected / quantified and that the bias between measurements may be due to a calibration error due to the use of a recombinant protein for calibration, (see FIGs. 9A-9C)PATENT APPLICATIONAttorney Docket No 057618-1509623Client Reference No. LC 2024-01-WO-PCTExample 2 (Workflow 2)

[0224] The following example provides a method of quantifying 14-3-3 proteins using protein enrichment. In this example, the sample was enriched in one or more 14-3-3 proteins using immunoaffinity enrichment followed by digestion of 14-3-3 proteins to produce surrogate peptides of 14-3-3 proteins.

[0225] Serum samples and calibrators (1.58 - 200 ng / mL 14-3-3eta) were enriched for one or more 14-3-3 proteins using anti-14-3-3(pan) antibodies with subsequent on-bead digestion of the enriched 14-3-3 proteins to produce 14-3-3 surrogate peptides. 200 uL of sample was diluted 2-fold with phosphate buffered saline containing 1% (v / v) Tween-20 and 5 mg / mL bovine serum albumin. 4 ug of anti-14-3-3(pan) antibody were coupled to streptavidin coated magnetic beads. The buffered sample solution was added to 4 ug of anti-14-3-3(pan) antibody coupled to streptavidin magnetic beads and mixed for 1 hour at room temperature. After removing the unbound fraction, the magnetic beads containing the bound 14-3-3 protein were washed 3 times with 200 pL phosphate buffered saline containing 1% (v / v) Tween-20 and 5 mg / mL bovine serum albumin. Subsequently, the washed beads containing the bound 14-3- 3p were resuspended in 350 uL of 50 mM Tris-HCl (pH 8) containing 0.2% (w / v) sodium deoxy cholate and 1 mM dithiothreitol, then denatured for 30 min at 56 °C before addition of 0.4 ng of each stable isotope labeled peptide. Next, TPCK-treated bovine trypsin (62.5 pg) was added and allowed to digest for 30 min at 37 °C followed by the addition 500 uL of buffer containing 100 mM Glycine, 2% (v / v) formic acid, 0.001% (w / v) ZWITTERGENT® and 1% (w / v) heptanesulfonic acid. The digested samples containing residual beads and precipitated deoxy cholate were centrifuged for 10 minutes at 2,200 x g before collecting the resulting clarified supernatant for analysis by LC-MS / MS.

[0226] FIG. 10 shows a mean and standard deviation box plot of the measured values of surrogate peptides of 14-3-3p (14-3-3 VIS, 14-3-3_TMA, 14-3-3_YDD, and 14-3-3_EAF). 14-3-3 was measured in serum samples from ostensibly healthy donors using the workflow of Example 1 and Example 2. Regardless of surrogate peptide, 14-3-3 results were systematically lower using the workflow of Example 2 compared to the workflow of Example 1 . This could be explained by the workflow of Example 2 only measuring intact forms of 14-3-3 (z.e., forms of 14-3-3 that possess both the anti- 14-3 -3 (pan) epitope and respective surrogate peptides), while workflow of Example 1 measures both intact and truncated forms of 14-3-3. However, the high degree of correlation between 14-3-3_YDD and 14-3-3 EAF measurements observed with the workflow of Example 1 (see FIGs. 9B andPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT9C) indicates all forms of 14-3-3fi measured possess both surrogate peptides (amino acid residues 20 - 162), indicating such truncation would need to occur between residues 1 2 and the residue 214 (i.e., the start of the epitope for the anti- 14-3 -3 (pan) antibody used in workflow 2). Alternatively, the lower results with workflow 2 could be explained by the presence of autoantibodies against 14-3-3p or other family members, which inhibit the enrichment of 14-3-3 using anti-14-3-3(pan) antibodies in workflow 2 leading to low 14-3-3 measurements in the workflow of Example 2. Indeed, autoantibodies for 14-3-3p are well known and it has been previously show n that the workflow of Example 1 is not susceptible to autoantibody interference for other protein measurands.Example 3 (Hybrid Workflow)

[0227] The following example provides a method of quantifying 14-3-3 proteins using protein enrichment and surrogate peptide enrichment. In this example, the sample was in one or more 14-3-3 proteins using immunoaffinity enrichment followed by digestion of 14-3-3 proteins to produce surrogate peptides of 14-3-3 proteins.

[0228] Serum samples and calibrators (1.58 - 200 ng / mL 14-3-3eta) were enriched for 14- 3-3 protein using anti-14-3-3(pan) antibodies with subsequent on-bead digestion of the enriched 14-3-3 proteins to produce 14-3-3 surrogate peptides.200 uL of sample w as diluted 2-fold with phosphate buffered saline containing 1% (v / v) Tween-20 and 5 mg / mL bovine serum albumin before adding 4 ug of anti-14-3-3(pan) antibody coupled to streptavidin magnetic beads and mixing for 1 hour at room temperature. After removing the unbound fraction, the magnetic beads containing the bound 14-3-3 protein w as washed 3 times with 200 pL phosphate buffered saline containing 1% (v / v) Tw een-20 and 5 mg / mL bovine serum albumin. Subsequently, the washed beads contained the bound 14-3-3p were resuspended in 350 uL of 50 mM Tris-HCl (pH 8) containing 0.2% (w / v) sodium deoxy cholate and 1 mM dithiothreitoL then denatured for 30 min at 56 °C before addition of 0.4 ng of each stable isotope labeled peptide. Next, TPCK-treated bovine trypsin (62.5 pg) w as added and allowed to digest for 30 min at 37 °C followed by the addition soybean try psin inhibitor (1 mg) to terminate the digestion. Finally, 2 ug of each anti-peptide antibody coupled to protein G magnetic beads were added to the digested samples and mixed for 2 hours at room temperature. After discarding the unbound fraction, the magnetic beads containing the bound peptides w ere w ashed 3 times with 200 pL of PBS containing 0.03% (w / v) CHAPS before elution of the bound peptides in 100 pL of buffer containing 100 mM Glycine, 2% (v / v)PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT formic acid, 0.001% (w / v) Zwittergent 3-16 and 1% (w / v) heptanesulfonic acid for LC- MS / MS analysis.

[0229] FIGS. 11 A-B and 11C-D show example chromatograms for the 14-3-3 TMA surrogate peptide (FIG. 11 A-B) and 14-3-3 YDD surrogate peptide (FIG. 11C-D) measured according to the workflow of Example 1 (Figs. 11 A and 11C) and Example 3 (Figs. 1 IB and 1 ID). The natural and stable isotope labeled peptides are shown in each case with the indicated transitions, which were suitable for both workflows. Chromatograms are shown for (11 A-B) the 14-3-3_TMA surrogate peptide and (11C-D) the 14-3-3_YDD surrogate peptide obtained for a 12.5 ng / mL calibrator processed with the workflow of Example 2 and separately processed with the workflow of Example 3. Given the lower 14-3-3 concentrations measured with the workflow' of Example 2, other techniques were investigated to improve the sensitivity of a workflow' using 14-3-3 protein enrichment follow ed by digestion. To do so, Example 3 was devised whereby following 14-3-3 protein enrichment and digestion of the enriched 14-3-3 protein, the resulting 14-3-3 surrogate peptides were further enriched using anti-peptide antibodies. In doing this, >10-fold enhancement in sensitivity was observed. It is expected that other forms of surrogate peptide enrichment, such as solid phase extraction, may be suitable following 14-3-3 protein enrichment and digestion of enriched 14-3-3 protein.

Claims

PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCTWHAT IS CLAIMED:

1. A method for determining a presence or amount of a 14-3 -3i] protein in a sample, the method comprising:(a) providing one or more surrogate peptides of the 14-3-3p protein;(b) ionizing the one or more surrogate peptides of the 14-3-3q protein to generate one or more ions of the surrogate peptides of 14-3-3i] protein detectable by mass spectrometry;(c) determining the presence or amount of the one or more ions of the surrogate peptides of the 14-3-31] protein by mass spectrometry; and(d) determining the presence or amount of the 14-3 -3i protein in the sample according to the presence or amount of the one or more ions determined in (c).

2. The method of claim 1, wherein the sample further comprises one or more surrogate peptides derived from other 14-3-3 protein family members.

3. The method of claim 1 or 2, wherein providing the one or more peptides of (a) comprises contacting a sample with a proteolytic enzyme to produce a proteolytic digest comprising the one or more surrogate peptides of the 14-3-31] protein, and / or optionally contacting the sample with a chemical reagent to produce a proteolytic digest comprising the one or more surrogate peptides of the 14-3 -3i] protein.

4. The method of any one of claims 1 to 3, further comprising, prior to (a), and / or prior to producing the proteolytic digest, enriching the biological sample for one or more 14-3-3 protein family members.

5. The method of claim 4, wherein the one or more 14-3-3 protein family members comprise the 14-3-3i] protein.

6. The method of claim any one of claims 4 to 5. wherein the enriching of the biological sample comprises use of one or more binding agents that bind to one or more of the 14-3-3 protein family members or one or more binding agents that bind specifically to the 14-3-31] protein.PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT7. The method of claim any one of claims 4 to 5, wherein the enriching of the biological sample comprises an immunoprecipitation method.

8. The method of claims 6 or 7. wherein the one or more binding agents comprise one or more monoclonal antibodies.

9. The method of any one of claims 3 to 8, wherein the proteolytic enzyme comprises trypsin or an isozyme thereof.

10. The method of any one of claims 3 to 9, wherein providing the one or more peptides of (a) comprises enriching the proteolytic digest for the one or more surrogate peptides of 14- 3-3q.1 1. The method of claim 10, wherein the enriching of the proteolytic digest comprises contacting the proteolytic digest with one or more binding agents that bind to the one or more surrogate peptides of the 14-3-3q protein.

12. The method of claim 11, wherein binding agents comprise one or more antibodies.

13. The method of claim 12, wherein the one or more antibodies comprise one or more monoclonal antibodies.

14. The method of any one of claims 10 to 13, wherein the enriching of the proteolytic digest comprises an immunoprecipitation method.

15. The method of any one of claims 10 to 14. wherein the one or more binding agents comprise a binding agent that specifically binds to a peptide sequence selected from the peptide sequences of SEQ ID NOs. 2, 3, 7-15 and 17.

16. The method of any one of claims 10 to 15. wherein the one or more binding agents comprise a binding agent that specifically binds to a peptide sequence selected from the peptide sequences of SEQ ID NOs. 2, 7 and 12.PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT17. The method of any one of claims 10 to 16, wherein the proteolytic digest is enriched for a surrogate peptide comprising the sequence of one or more of SEQ ID NOs. 2, 7 and 12.

18. The method of any one of claims 10 to 17. wherein the one or more surrogate peptides provided of (a) comprise one or more of SEQ ID NOs. 2, 7 and 12.

19. The method of any one of claims 1 to 18, wherein the one or more surrogate peptides provided in (a) comprise one or more internal standard peptides.

20. The method of claim 19, wherein one or more of the internal standard peptides comprise a stable isotope label.

21. The method of any one of claims 3 to 20, wherein enriching the biological sample or enriching the proteolytic digest comprises solid-phase extraction, precipitation, affinity enrichment, immunoaffinity enrichment, or combinations thereof.

22. The method of any one of claims 1 to 21, wherein ionizing the one or more surrogate peptides of 14-3-3q protein comprises:(i) generating at least one precursor ion of the one or more surrogate peptides of 14-3- 3q protein;(ii) generating one or more product ions of the precursor ion; and(iii) detecting the presence or amount of the at least one precursor ion generated in step (i) and / or the one or more product ions generated in step (ii), or both, to determine the presence or amount of the 14-3-3q protein in the biological sample.

23. The method of claim 22, wherein the at least one precursor ion has a mass / charge ratio of 408.7±0.5.

24. The method of claim 22 or 23, wherein the one or more product ions of the precursor ion has a mass / charge ratio of one or more of 703.3±0.5, 632.3±0.5, 503.3±0.5, 375.2±0.

5. 304.2±0.

5. 175.1±0.5, 185.1±0.5, 314.2±0.5, and 442.2±0.5.PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT25. The method of any one of claims 1 to 24, wherein the one or more ions of the surrogate peptides of 14-3-3q protein comprise a mass / charge ratio selected from the group consisting of 703.3±0.5, 632.3±0.5, 503.3±0.5, and 185.1±0.5.

26. The method of any one of claims 1 to 25, wherein the one or more ions of the surrogate peptides of the 14-3-3i] protein comprise a mass / charge ratio selected from the group consisting of 868.4±0.5, 753.3+0.

5. 507.3+0.5, and 279. 1+0.5.

27. The method of any one of claims 1 to 26, wherein the one or more ions of the surrogate peptides of the 14-3-3i] protein comprises a mass / charge ratio selected from the group consisting of 804.4+0.5, 691.4±0.5, 213.2+0.5, and 185.2+0.5.

28. The method of any one of claims 1 to 27, wherein the one or more ions of the surrogate peptides of the 14-3-3r) protein comprises a mass / charge ratio selected from the group consisting of 1066.5±0.5, 967.5+0.5, 868.4+0.5, and 301.2+0.5.

29. The method of any one of claims 1 to 28, wherein the one or more ions of the surrogate peptides of the 14-3-3p protein comprises a mass / charge ratio selected from the group consisting of 623.3+0.5, 476.3±0.5, 347.2±0.5, and 234.1+0.5.

30. The method of any one of claims 1 to 29, wherein the mass spectrometry comprises tandem mass spectrometry.31 . The method of any one of claims 1 to 30, wherein the mass spectrometry comprises LC-MS, LC-MS / MS, or 2D-LC-MS / MS.

32. The method of any one of claims 1 to 30, wherein the biological sample comprises serum or plasma.

33. The method of any one of claims 1 to 32, wherein the biological sample is derived from a subject.PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT34. The method of claim 33. wherein the subject is a mammal or wherein the subject is a human.

35. The method of claims 33 or 34, wherein the subject has or is suspected of having rheumatoid arthritis.

36. The method of any one of claims 33 to 35, wherein the presence or amount of 14-3-3q determined in (d) indicates the subject has rheumatoid arthritis.

37. The method of any one of claims 1 to 36, wherein the amount of 14-3-3p determined in (d) is detected in a biological sample with a lower limit of detection of about 1 ng / ml, 0.1 ng / ml or about 0.1 ng / ml.

38. The method of any one of claims 1 to 37, wherein the amount of the one or more ions of the surrogate peptides of (c) are detected with a lower limit of detection of about 1 ng / ml, 0.1 ng / ml or about 0.1 ng / ml.

39. A method for determining a presence or amount of 14-3-3q protein in a biological sample, the method comprising: providing a biological sample comprising a plurality of proteins including one or more 14-3-3 protein family members; contacting the biological sample with a proteolytic enzyme to produce a proteolytic digest comprising peptides, wherein at least one of the peptides comprises a surrogate peptide derived from a 14-3-3q protein; contacting the proteolytic digest with a binding agent, wherein the binding agent specifically binds the surrogate peptide derived from the 14-3-3r| protein: separating unbound peptides from the surrogate peptide to produce a sample enriched for the surrogate peptide; purifying the surrogate peptide from the enriched sample using liquid chromatography ; generating at least one precursor ion of the surrogate peptide; generating one or more product ions of the precursor ion; andPATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT detecting the presence or amount of the at least one precursor ion. the one or more product ions of the precursor ion, or both, to determine the presence or amount of the 14-3-3q protein in the biological sample.

40. The method of claim 39, wherein the proteolytic enzyme comprises trypsin or an isozyme thereof.

41. The method of claim 40, wherein the at least one precursor ion is formed by electrospray ionization.

42. The method of claim 41, wherein the electrospray ionization is performed in positive ion mode.

43. The method of any one of claims 39 to 42. wherein the detecting the presence or amount of the at least one precursor ion. the one or more product ions of the precursor ion. or both, is performed by a process comprising mass spectrometry.

44. The method of claim 43, wherein the mass spectrometry detection of the surrogate peptide is performed in selected reaction monitoring mode (SRM).

45. The method of any one of claims 39 to 44, wherein the at least one precursor ion of the surrogate peptide has a mass / charge ratio (m / z) of about 516.2±0.5.

46. The method of any one of claims 39 to 45, wherein the one or more product ions comprise an ion with a m / z of about 868.4±0.5, 753.3±0.5, 638.3±0.5, 507.3±0.5, 436.2±0.5, 279.1±0.5, 394.1±0.5, 136.1±0.5, or 251.1±0.5.

47. The method of any one of claims 43 to 46. wherein the mass spectrometry comprises LC-MS, LC-MS / MS, or 2D-LC-MS / MS.

48. The method of claim 47, wherein the liquid chromatography comprises high performance liquid chromatography (HPLC).PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT49. The method of claim 47 or 48. wherein the liquid chromatography is performed in reverse phase liquid chromatography or by hydrophilic interaction liquid chromatography.

50. A method for determining a presence or amount of 14-3-3q protein in a biological sample, the method comprising: subjecting a surrogate peptide of 14-3-3q protein from a biological sample to an ionization source under conditions suitable to generate one or more precursor ions with a mass to charge ratio (m / z) of 452.3±0.5; fragmenting at least one of the precursor ions to generate one or more fragment ions detectable by mass spectrometry, wherein the one or more fragment ions compnse one or more ions selected from the group consisting of ions with m / z of 804.4±0.5, 691.4±0.5, 213.2±0.5, and 185.2±0.5; and determining the amount of one or more of the precursor ions and / or fragment ions by mass spectrometry to determine the amount of 14-3-31] protein in the sample.

51. The method of claim 50, wherein the surrogate peptide of 14-3-31] protein is at least 80 % identical to any one of SEQ ID NOs.: 1-17.

52. A method for determining a presence or amount of 14-3-3q protein in a biological sample, the method comprising: providing a biological sample comprising one or more 14-3-3 protein family members or fragments thereof: enriching the one or more 14-3-3 protein family members in the biological sample to produce an enriched sample; contacting a proteolytic enzy me with the enriched sample to produce a proteolytic digest comprising peptides, wherein the peptides comprise a surrogate peptide derived from a 14-3-3i] protein; generating at least one precursor ion of the surrogate peptide; generating one or more product ions of the precursor ion; and detecting the presence or amount of the at least one precursor ion and / or the one or more fragment ions, or both, and determining a presence or amount of the 14-3-3q protein in the biological sample according to the detecting.PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT53. The method of claim 52. wherein the detecting comprises use of mass spectrometry.

54. The method of claim 52 or 53, wherein the proteolytic enzyme comprises trypsin or an isozy me thereof.

55. The method of any one of claims 52 to 54, wherein the biological sample comprises serum or plasma.

56. The method of claim any one of claims 52 to 55, the one or more precursor ions of the surrogate peptide has a mass to charge (m / z) ratio selected from the group consisting of 408.7±0.5, 516.2±0.5, 739.9±0.5, 454.3±0.5, 308.2±0.5, 452.3±0.5, 433.2.7±0.5, 874.4±0.5, 652.8±0.5, 533.8±0.5, 634.3±0.5, 412.2±0.5, 412.9±0.5, 991.5±0.5, 720.0±0.5, 595.3±0.5, and 708.0±0.5.

57. The method of any one of claims 52 to 56, further comprising purifying the enriched sample using liquid chromatography, wherein the liquid chromatography is performed in reverse phase separation or hydrophilic interaction liquid chromatography.

58. A system for conducting the method of any one of claims 1 to 57, the system comprising: a station for providing a biological sample comprising a 14-3-3q protein; a station for partially purifying the 14-3-3q protein from other components in the sample; a station for chromatographically separating 14-3-3q protein from other components in the sample; and a station for analyzing the chromatographically separated 14-3-3q protein by mass spectrometry to determine the presence or amount of the 14-3-3q protein in the biological sample.

59. The system of claim 58, further comprising a station for contacting the biological sample with a proteolytic enzyme, and optionally a station for partially purifying a surrogate peptide of the 14-3-3q protein from a proteolytic digest.PATENT APPLICATION Attorney Docket No 057618-1509623 Client Reference No. LC 2024-01-WO-PCT60. A computer-program product tangibly embodied in a non-transitory machine-readable storage medium, including instructions configured to cause one or more computers and / or devices to perform the method of any one of claims 1 to 57.

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