Methods for detecting chromogranin a by mass spectrometry

Abstract

Provided are methods for detecting chromogranin A by mass spectrometry. In another aspect, provided herein are methods for quantitating chromogranin A by mass spectrometry. In another aspect, provided herein are methods for prognosis of or measuring the size of neuroendocrine tumors by mass spectrometry.

Description

(19) *EP004653862A2* (11) EP 4 653 862 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: 26.11.2025 Bulletin 2025 / 48 (21) Application number: 25203365.9 (22) Date of filing: 15.03.2019 (51) International Patent Classification (IPC): G01N 30 / 04 (2006.01) (52) Cooperative Patent Classification (CPC): G01N 33 / 6848; G01N 30 / 88; G01N 30 / 7233; G01N 2030 / 009; G01N 2030 / 045; G01N 2030 / 8831 (84) Designated Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR (30) Priority: 16.03.2018 US 201862644210 P (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: 19767849.3 / 3 765 858 (71) Applicant: Quest Diagnostics Investments LLC Secaucus, NJ 07094 (US) (72) Inventors: • WEBER, Darren Laguna Niguel, CA 92677 (US) • CAULFIELD, Michael P. Oceanside, CA 92057 (US) • MCPHAUL, Michael J. Capistrano Beach, CA 92624 (US) • GOLDMAN, Scott Laguna Niguel, CA 92677 (US) • CLARKE, Nigel San Clemente, CA 92673 (US) (74) Representative: Harrison IP Limited Mereside, Alderley Park Congleton Road Nether Alderley Macclesfield, Cheshire SK10 4TG (GB) Remarks: This application was filed on 19.09.2025 as a divisional application to the application mentioned under INID code 62. (54) METHODS FOR DETECTING CHROMOGRANIN A BY MASS SPECTROMETRY (57) Provided are methods for detecting chromogra- nin A by mass spectrometry. In another aspect, provided herein are methods for quantitating chromogranin A by mass spectrometry. In another aspect, provided herein are methods for prognosis of or measuring the size of neuroendocrine tumors by mass spectrometry. EP 4 65 3 86 2 A 2 Processed by Luminess, 75001 PARIS (FR) Description CROSS-REFERENCE TO RELATED PATENTAPPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 62 / 644,210, filed March 16, 2018, which is incorporated by reference herein in its entirety. BACKGROUND OF THE INVENTION

[0002] Chromogranin-A (CgA) is a 50kDa acidic glycoprotein expressed in the secretory granules of neuroendocrine tissue. Currently, blood levels of CgA are primarily measured using various immunoassays. However, as with any antibody-based assay, limitations arising from non-specific binding and a reduced dynamic range requiring sample dilution, pose hurdles in implementing such tests in diagnostic laboratories.

[0003] An accurate and sensitive assay for quantitating chromogranin A is needed. SUMMARY OF THE INVENTION

[0004] Provided herein are methods for detecting or determining the amount of chromogranin A (CgA) in a sample by mass spectrometry, including tandem mass spectrometry.

[0005] In certain embodiments, the methods provided herein are for detecting or determining the amount of chromo- granin A (CgA) comprising (a) purifying CgA in the sample; (b) ionizing CgA to produce ions detectable by mass spectrometry; and (c) detecting or determining the amount of theCgA ion(s) bymass spectrometry; wherein theamount of the CgA ion(s) is related to the amount of CgA in the sample.

[0006] In certain embodiments, the methods provided herein are for detecting or determining the amount of chromo- granin A (CgA) comprising (a) subjecting the sample to solid phase extraction; (b) enzymatically digesting the CgA; (c) subjecting the CgA to liquid chromatography; (d) ionizing CgA to produce ions detectable by mass spectrometry; and (e) detecting or determining the amount of the CgA ion(s) by mass spectrometry; wherein the amount of the CgA ion(s) is related to the amount of CgA in the sample.

[0007] In certain embodiments, methods provided herein comprise selected reaction monitoring (SRM) mass spectro- metry.

[0008] In some embodiments, the methods provided herein are fully automated.

[0009] In some embodiments, the methods provided herein are antibody-free methods.

[0010] In some embodiments, purifying provided herein comprises extraction of serum using solid phase extraction (SPE). In some embodiments, SPE is an anion exchange solid-phase extraction. In some embodiments, SPE is amixed- mode anion exchange solid-phase extraction. In some embodiments, extracted samples are concentrated.

[0011] In some embodiments, purifying provided herein comprises liquid chromatography. In some embodiments, the liquid chromatography comprises high performance liquid chromatography (HPLC). In some embodiments, the liquid chromatography comprises high turbulence liquid chromatography (HTLC).

[0012] In some embodiments, extracted samples are enzymatically digested. In some embodiments, extracted samples are enzymatically digested by trypsin.

[0013] In some embodiments, the ionization comprises electrospray ionization (ESI). In some embodiments, the ionization comprises ionizing in positivemode. In someembodiments, the ionization comprises ionizing in negativemode.

[0014] In some embodiments, the ionization comprises atmospheric pressure chemical ionization (APCI). In some embodiments, the ionizationcomprises ionizing inpositivemode. Insomeembodiments, the ionizationcomprises ionizing in negative mode.

[0015] In some embodiments, methods provided herein comprise measuring the amount of precursor ion having a mass-to-charge ratio of 593.2 ± 0.5.

[0016] In some embodiments, methods provided herein comprise measuring the amount of precursor ion having a mass-to-charge ratio of 729.6 ± 0.5.

[0017] In someembodiments,methods providedherein comprisemeasuring theamount of a fragment of chromogranin A. In some embodiments, the CgA fragment measured comprises a sequence ELQDLALQGA (SEQ ID NO:1). In some embodiments, the CgA fragment measured comprises a sequence RRPEDQELESLSAIEAELEK (SEQ ID NO:4).

[0018] In someembodiments,methodsprovidedherein comprisemeasuring theamountof fragment ionhavingamass- to-charge ratio of 516.3 ± 0.5 or 815.5 ± 0.5 or both.

[0019] In someembodiments,methodsprovidedherein comprisemeasuring theamountof fragment ionhavingamass- to-charge ratio of 831.5 ± 0.5 or 989.5 ± 0.5 or both.

[0020] In some embodiments, methods provided herein further comprise adding an internal standard. In some embodiments, the internal standard is isotopically labeled. In some embodiments, the internal standard comprises 2 EP 4 653 862 A2 5 10 15 20 25 30 35 40 45 50 55 C13N15 labeled amino acids. In some embodiments, the internal standard is labeled on a leucine (L) or lysine (K). In some embodiments, the internal standard comprises a sequence ILSILRHQNLLKELQDLAL*QGAK*ERAHQQK (SEQ ID NO:2), wherein * is a C13N15 labeled amino acid. In some embodiments, the internal standard comprises a sequence RRPEDQELESL*SAIEAELEK* (SEQ ID NO:5), wherein * is a C13N15 labeled amino acid.

[0021] In some embodiments,methods provided herein comprisemeasuring the amount of internal standard precursor ion having amass-to-charge ratio of 600.8± 0.5 and / or product ion having amass-to-charge ratio of 602.4± 0.5, 830.6± 0.5, or 958.7 ± 0.5.

[0022] In some embodiments,methods provided herein comprisemeasuring the amount of internal standard precursor ion having amass-to-charge ratio of 600.8± 0.5 and / or product ion having amass-to-charge ratio of 734.6± 0.5, 839.5± 0.5, or 997.6 ± 0.5.

[0023] In certain embodiments, the limit of quantitation of the methods is less than or equal to 100 ng / mL. In some embodiments, the limit of quantitation of themethods is less than or equal to 90 ng / mL. In some embodiments, the limit of quantitationof themethods is less thanor equal to 80ng / mL. In someembodiments, the limit of quantitationof themethods is less than or equal to 70 ng / mL. In some embodiments, the limit of quantitation of themethods is less than or equal to 60 ng / mL. In some embodiments, the limit of quantitation of the methods is less than or equal to 50 ng / mL.

[0024] In some embodiments, the limit of detection of the methods is less than or equal to 50 ng / mL. In some embodiments, the limit of detection of the methods is less than or equal to 40 ng / mL. In some embodiments, the limit of detection of the methods is less than or equal to 35.5 ng / mL.

[0025] In some embodiments, methods provided herein comprise linearity of quantitation across a range between 50 ng / mL to 50,000 ng / mL.

[0026] In some embodiments, methods provided herein comprise inter‑ and intra-assay reproducibility of CV ≤15%.

[0027] In some embodiments, CgA is not derivatized prior to mass spectrometry.

[0028] In certain embodiments, the sample is a body fluid. In some embodiments, the sample is cerebrospinal fluid (CSF). In some embodiments, the sample is plasma or serum. In some embodiments, the sample is whole blood. In some embodiments, the sample is saliva or urine.

[0029] In some embodiments, the methods may include adding an agent to the sample in an amount sufficient to deproteinate the sample.

[0030] In someembodiments, elevated levels of chromograninA as compared to a reference range indicates increased risk of neuroendocrine tumors (NET). In someembodiments, quantitated levels of chromograninA indicates the sizeof the neuroendocrine tumor. In some embodiments, quantitated levels of chromogranin A indicates the tumor burden of the neuroendocrine tumor. In someembodiments, quantitated levelsof chromograninA indicates the response to treatment of the neuroendocrine tumor. In some embodiments, quantitated levels of chromogranin A indicates the prognosis of the neuroendocrine tumor.

[0031] Asused herein, unless otherwise stated, the singular forms "a," "an," and "the" include plural reference. Thus, for example, a reference to "a protein" includes a plurality of protein molecules.

[0032] Asusedherein, the term "purification" or "purifying" doesnot refer to removingallmaterials from thesampleother than the analyte(s) of interest. Instead, purification refers to a procedure that enriches the amount of one ormore analytes of interest relative to other components in the sample that may interfere with detection of the analyte of interest. Samples are purified herein by variousmeans to allow removal of one ormore interfering substances, e.g., one ormore substances that would interfere with the detection of selected CgA parent and daughter ions by mass spectrometry.

[0033] Asusedherein, the term "test sample" refers to anysample thatmay containCgA.Asusedherein, the term "body fluid" means any fluid that can be isolated from the body of an individual. For example, "body fluid" may include blood, plasma, serum, bile, saliva, urine, tears, perspiration, and the like.

[0034] As used herein, the term "derivatizing" means reacting two molecules to form a new molecule. Derivatizing agents may include isothiocyanate groups, dinitro-fluorophenyl groups, nitrophenoxycarbonyl groups, and / or phthalal- dehyde groups, and the like.

[0035] As used herein, the term "chromatography" refers to a process in which a chemical mixture carried by a liquid or gas is separated into components asa result of differential distributionof the chemical entities as theyflowaroundor over a stationary liquid or solid phase.

[0036] Asusedherein, the term "liquid chromatography" or "LC"meansaprocessof selective retardation of oneormore components of a fluid solution as the fluid uniformly percolates through a column of a finely divided substance, or through capillary passageways.The retardation results from thedistributionof thecomponentsof themixturebetweenoneormore stationary phases and the bulk fluid, (i.e.,mobile phase), as this fluidmoves relative to the stationary phase(s). Examples of "liquid chromatography" include reverse phase liquid chromatography (RPLC), high performance liquid chromato- graphy (HPLC), and high turbulence liquid chromatography (HTLC).

[0037] Asusedherein, the term "high performance liquid chromatography" or "HPLC" 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. 3 EP 4 653 862 A2 5 10 15 20 25 30 35 40 45 50 55

[0038] As used herein, the term "high turbulence liquid chromatography" or "HTLC" refers to a form of chromatography that utilizes turbulent flow of the material being assayed through the column packing as the basis for performing the separation.HTLChasbeenapplied in thepreparation of samples containing twounnameddrugs prior to analysis bymass spectrometry. See, e.g., Zimmer et al., J. Chromatogr. A 854: 23‑35 (1999); see also, U.S. Patents No. 5,968,367, 5,919,368,5,795,469,and5,772,874,which furtherexplainHTLC.Personsofordinaryskill in theart understand "turbulent flow". When fluid flows slowly and smoothly, the flow is called "laminar flow". For example, fluid moving through an HPLC column at low flow rates is laminar. In laminar flow the motion of the particles of fluid is orderly with particles moving generally in straight lines. At faster velocities, the inertia of the water overcomes fluid frictional forces and turbulent flow results. Fluid not in contact with the irregular boundary "outruns" that which is slowed by friction or deflected by an uneven surface.When a fluid is flowing turbulently, it flows in eddies andwhirls (or vortices), withmore "drag" thanwhen the flow is laminar. Many references are available for assisting in determining when fluid flow is laminar or turbulent (e.g., Turbulent Flow Analysis: Measurement and Prediction, P.S. Bernard & J.M. Wallace, John Wiley & Sons, Inc., (2000); An Introduction to Turbulent Flow, Jean Mathieu & Julian Scott, Cambridge University Press (2001)).

[0039] As used herein, the term "gas chromatography" or "GC" refers to chromatography inwhich the samplemixture is vaporizedand injected into a streamof carrier gas (as nitrogenor helium)moving throughacolumncontaininga stationary phasecomposedof a liquidor aparticulate solid and is separated into its component compoundsaccording to theaffinity of the compounds for the stationary phase.

[0040] As used herein, the term "large particle column" or "extraction column" refers to a chromatography column containing anaverageparticle diameter greater thanabout 35µm.Asused in this context, the term "about"means±10%. In a preferred embodiment the column contains particles of about 60 µm in diameter.

[0041] As used herein, the term "analytical column" refers to a chromatography column having sufficient chromato- graphic plates toeffect a separationofmaterials in a sample that elute from thecolumnsufficient toallowadeterminationof the presence or amount of an analyte. Such columns are often distinguished from "extraction columns", which have the general purpose of separating or extracting retained material from non-retained materials in order to obtain a purified sample for further analysis. As used in this context, the term "about" means ± 10%. In a preferred embodiment the analytical column contains particles of about 4 µm in diameter.

[0042] As used herein, the term "on-line" or "inline", for example as used in "on-line automated fashion" or "on-line extraction" refers to a procedure performed without the need for operator intervention. In contrast, the term "off-line" as used herein refers to a procedure requiring manual intervention of an operator. Thus, if samples are subjected to precipitation, and the supernatants are thenmanually loaded into an autosampler, the precipitation and loading steps are off-line from the subsequent steps. In various embodiments of themethods, oneormore stepsmaybeperformed in an on- line automated fashion.

[0043] As used herein, the term "mass spectrometry" or "MS" refers to an analytical technique to identify compounds by their mass. MS refers tomethods of filtering, detecting, andmeasuring ions based on their mass-to-charge ratio, or "m / z". MS technology generally includes (1) ionizing the compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating a mass-to-charge ratio. The compounds may be ionized and detected by any suitablemeans. A "mass spectrometer" generally includes an ionizer and an ion detector. In general, one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrographic instrument where, due to a combination of magnetic and electric fields, the ions follow a path in space that is dependent upon mass ("m") and charge ("z"). See, e.g., U.S. Patent Nos. 6,204,500, entitled "Mass Spectrometry From Surfaces;" 6,107,623, entitled "Methods and Apparatus for Tandem Mass Spectrometry;" 6,268,144, entitled "DNA Diagnostics Based On Mass Spectrometry;" 6,124,137, entitled "Surface-Enhanced Photolabile Attachment And Release For Desorption And Detection Of Analytes;" Wright et al., Prostate Cancer and Prostatic Diseases 2:264‑76 (1999); and Merchant and Weinberger, Electrophoresis 21:1164‑67 (2000).

[0044] As used herein, the term "operating in negative ion mode" refers to those mass spectrometry methods where negative ions are generated and detected. The term "operating in positive ionmode" as used herein, refers to thosemass spectrometry methods where positive ions are generated and detected.

[0045] As used herein, the term "ionization" or "ionizing" refers to the process of generating an analyte ion having a net electrical charge equal to one ormore electron units. Negative ions are those having a net negative charge of one ormore electron units, while positive ions are those having a net positive charge of one or more electron units.

[0046] Asusedherein, the term "electron ionization" or "EI" refers tomethods inwhichananalyte of interest in agaseous or vapor phase interactswith a flowof electrons. Impact of the electronswith theanalyte producesanalyte ions,whichmay then be subjected to a mass spectrometry technique.

[0047] As used herein, the term "chemical ionization" or "CI" refers tomethods 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.

[0048] As used herein, the term "fast atom bombardment" or "FAB" refers to methods in which a beam of high energy atoms (often Xe or Ar) impacts a non-volatile sample, desorbing and ionizing molecules contained in the sample. Test samples are dissolved in a viscous liquid matrix such as glycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown‑6 crown 4 EP 4 653 862 A2 5 10 15 20 25 30 35 40 45 50 55 ether, 2-nitrophenyloctyl ether, sulfolane, diethanolamine, and triethanolamine. The choice of an appropriate matrix for a compound or sample is an empirical process.

[0049] As used herein, the term "matrix-assisted laser desorption ionization" or "MALDI" 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 photoionization, protonation, deprotonation, and cluster decay. For MALDI, the sample is mixed with an energy-absorbing matrix, which facilitates desorption of analyte molecules.

[0050] As used herein, the term "surface enhanced laser desorption ionization" or "SELDI" 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 preferentially retains one ormore analytes of interest. As inMALDI, this processmay also employ an energy-absorbing material to facilitate ionization.

[0051] Asusedherein, the term "electrospray ionization"or "ESI," refers tomethods inwhichasolution ispassedalonga short length of capillary tube, to the end of which is applied a high positive or negative electric potential. Solution reaching the end of the tube is vaporized (nebulized) into a jet or spray of very small droplets of solution in solvent vapor. Thismist of droplets flows throughanevaporationchamber,which isheatedslightly toprevent condensationand toevaporate 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.

[0052] As used herein, the term "atmospheric pressure chemical ionization" or "APCI," 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 themassanalyzer by useof a set of differentially pumpedskimmer stages. A counterflow of dry and preheated N2 gasmay be used to improve removal of solvent. The gas-phase ionization in APCI can be more effective than ESI for analyzing less-polar species.

[0053] The term "Atmospheric Pressure Photoionization" or "APPI" as used herein refers to the form of mass spectro- scopy where the mechanism for the photoionization of molecule M is photon absorption and electron ejection to form the molecular ion 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 andexpense. In the presence ofwater vapor or protic solvents, themolecular ion can extractH to formMH+. This tends to occur ifM 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 formM+. Robb, D.B., Covey, T.R. and Bruins, A.P. (2000): See, e.g., Robb et al., Atmospheric pressure photoionization: An ionization method for liquid chromatography-mass spectro- metry. Anal. Chem. 72(15): 3653‑3659.

[0054] Asusedherein, the term "inductively coupled plasma" or "ICP" refers tomethods inwhich a sample interactswith a partially ionized gas at a sufficiently high temperature such that most elements are atomized and ionized.

[0055] As used herein, the term "field desorption" refers to methods in which a non-volatile test sample is placed on an ionization surface, and an intense electric field is used to generate analyte ions.

[0056] As used herein, the term "desorption" refers to the removal of an analyte from a surface and / or the entry of an analyte into a gaseous phase.

[0057] As used herein, the term "limit of quantification", "limit of quantitation" or "LOQ" refers to the point where measurements become quantitatively meaningful. The analyte response at this LOQ is identifiable, discrete and reproducible with a precision of 20% and an accuracy of 80% to 120%.

[0058] As used herein, the term "limit of detection" or "LOD" is the point at which the measured value is larger than the uncertainty associated with it. The LOD is defined arbitrarily as 2 standard deviations (SD) from the zero concentration.

[0059] As used herein, an "amount" of CgA in a body fluid sample refers generally to an absolute value reflecting the massofCgAdetectable in volumeof body fluid.However, anamount also contemplates a relativeamount in comparison to another CgA amount. For example, an amount of CgAin a body fluid can be an amount which is greater than or less than a control or normal level of CgA normally present.

[0060] The term "about" asusedherein in reference toquantitativemeasurementsnot including themeasurement of the mass of an ion, refers to the indicated value plus or minus 10%. Mass spectrometry instruments can vary slightly in determining themassof agivenanalyte. The term "about" in thecontext of themassof an ionor themass / charge ratio of an ion refers to + / ‑ 0.5 atomic mass unit.

[0061] Thesummaryof the inventiondescribedabove isnon-limitingandother featuresandadvantagesof the invention will be apparent from the following detailed description of the invention, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] 5 EP 4 653 862 A2 5 10 15 20 25 30 35 40 45 50 55 Figure 1 shows the analytical workflow for the CgA LC-MS / MS assay. Negatively charged CgA binds through electrostatic, lipophilic, and hydrophilic interactions with the mixed-mode anion exchange resin under conditions where it is retainedwhile other proteins arewashed away. After elution, the isolatedCgA is digestedwith trypsin and a representative peptide is quantified by LC-MS / MS analysis. Figure 2 shows three calibration curves run over three separate days. Linear range was demonstrated to be 50 to 50,000 ng / mL. CV was less than 10%. Figure 3 shows chromatograms corresponding to patient specimenswith normal (top) and high (bottom) CgAvalues. Figure 4 shows average ELISA immunoassay values as compared to the values obtained by LC-MS / MS. Figure 5 shows a comparison betweenCisBio immunoassay and LC-MS / MSCgA assay (Passing & Bablok curve fit, 308 samples). Figure 6 shows peak area graphs for normal and abnormal CgA levels determined by LC-MS / MS. Figure 7 shows an example chromatogram for chromogranin internal standard. DETAILED DESCRIPTION OF THE INVENTION

[0063] LevelsofCgAare increased in thepresenceof neuroendocrine-derived tumors (NET),makingCgAuseful serum marker for monitoring patients with NETs. Circulating levels of serum CgA are proportional to tumor burden, providing prognostic information in treatment response. Herewe describe a novel, fully automated, antibody-free LC-MS / MSassay to quantitate chromogranin-A out of serum. Exploiting the acidic properties of CgA, 100 uL of serum is extracted using an anion exchange solid-phase extraction plate followed by addition of internal standard. The extracted sample is then concentratedandenzymatically digestedusing trypsin. Auniquepeptide toCgA is thenchromatographically resolvedand analyzed by SRMon a Sciex 6500+QTrap. The ratio of the analyte peak area to the isotopically labeled internal standard peak area is used to achieve quantitation.CgAshows linearity across awide range (50‑50000ng / mL,R2≥0.99), aswell as inter‑ and intra-assay reproducibility (CV ≤15%). A cohort of 300 patient samples was analyzed to compare CgA serum values measured by the Cisbio CGA-ELISA-US immunoassay to the described LC-MS / MS assay. When comparing immunoassay and LC-MS / MS measurements for CgA in this cohort, an R2 of 0.71 was observed, showing a good correlation between the two assay platforms.

[0064] In certain embodiments, the methods provided herein are for detecting or determining the amount of chromo- granin A (CgA) comprising (a) purifying CgA in the sample; (b) ionizing CgA to produce ions detectable by mass spectrometry; and (c) detecting or determining the amount of theCgA ion(s) bymass spectrometry; wherein theamount of the CgA ion(s) is related to the amount of CgA in the sample.

[0065] In certain embodiments, the methods provided herein are for detecting or determining the amount of chromo- granin A (CgA) comprising (a) subjecting the sample to solid phase extraction; (b) enzymatically digesting the CgA; (c) subjecting the CgA to liquid chromatography; (d) ionizing CgA to produce ions detectable by mass spectrometry; and (e) detecting or determining the amount of the CgA ion(s) by mass spectrometry; wherein the amount of the CgA ion(s) is related to the amount of CgA in the sample.

[0066] In certain embodiments, methods provided herein comprise selected reaction monitoring (SRM) mass spectro- metry.

[0067] In some embodiments, the methods provided herein are fully automated.

[0068] In some embodiments, the methods provided herein are antibody-free methods.

[0069] In some embodiments, purifying provided herein comprises extraction of serum using solid phase extraction (SPE). In some embodiments, SPE is an anion exchange solid-phase extraction. In some embodiments, SPE is amixed- mode anion exchange solid-phase extraction. In some embodiments, extracted samples are concentrated.

[0070] In some embodiments, purifying provided herein comprises liquid chromatography. In some embodiments, the liquid chromatography comprises high performance liquid chromatography (HPLC). In some embodiments, the liquid chromatography comprises high turbulence liquid chromatography (HTLC).

[0071] In some embodiments, extracted samples are enzymatically digested. In some embodiments, extracted samples are enzymatically digested by trypsin.

[0072] In some embodiments, the ionization comprises electrospray ionization (ESI). In some embodiments, the ionization comprises ionizing in positivemode. In someembodiments, the ionization comprises ionizing in negativemode.

[0073] In some embodiments, the ionization comprises atmospheric pressure chemical ionization (APCI). In some embodiments, the ionizationcomprises ionizing inpositivemode. Insomeembodiments, the ionizationcomprises ionizing 6 EP 4 653 862 A2 5 10 15 20 25 30 35 40 45 50 55 in negative mode.

[0074] In some embodiments, methods provided herein comprise measuring the amount of precursor ion having a mass-to-charge ratio of 593.2 ± 0.5.

[0075] In some embodiments, methods provided herein comprise measuring the amount of precursor ion having a mass-to-charge ratio of 729.6 ± 0.5.

[0076] In someembodiments,methods providedherein comprisemeasuring theamount of a fragment of chromogranin A. In some embodiments, the CgA fragment measured comprises a sequence ELQDLALQGA (SEQ ID NO:1). In some embodiments, the CgA fragment measured comprises a sequence RRPEDQELESLSAIEAELEK (SEQ ID NO:4).

[0077] In someembodiments,methodsprovidedherein comprisemeasuring theamountof fragment ionhavingamass- to-charge ratio of 516.3 ± 0.5 or 815.5 ± 0.5 or both.

[0078] In someembodiments,methodsprovidedherein comprisemeasuring theamountof fragment ionhavingamass- to-charge ratio of 831.5 ± 0.5 or 989.5 ± 0.5 or both.

[0079] In some embodiments, methods provided herein further comprise adding an internal standard. In some embodiments, the internal standard is isotopically labeled. In some embodiments, the internal standard comprises C13N15 labeled amino acids. In some embodiments, the internal standard is labeled on a leucine (L) or lysine (K). In some embodiments, the internal standard comprises a sequence ILSILRHQNLLKELQDLAL*QGAK*ERAHQQK (SEQ ID NO:2), wherein * is a C13N15 labeled amino acid. In some embodiments, the internal standard comprises a sequence RRPEDQELESL*SAIEAELEK* (SEQ ID NO:5), wherein * is a C13N15 labeled amino acid.

[0080] In some embodiments,methods provided herein comprisemeasuring the amount of internal standard precursor ion having amass-to-charge ratio of 600.8± 0.5 and / or product ion having amass-to-charge ratio of 602.4± 0.5, 830.6± 0.5, or 958.7 ± 0.5.

[0081] In some embodiments,methods provided herein comprisemeasuring the amount of internal standard precursor ion having amass-to-charge ratio of 600.8± 0.5 and / or product ion having amass-to-charge ratio of 734.6± 0.5, 839.5± 0.5, or 997.6 ± 0.5.

[0082] In certain embodiments, the limit of quantitation of the methods is less than or equal to 100 ng / mL. In some embodiments, the limit of quantitation of themethods is less than or equal to 90 ng / mL. In some embodiments, the limit of quantitationof themethods is less thanor equal to 80ng / mL. In someembodiments, the limit of quantitationof themethods is less than or equal to 70 ng / mL. In some embodiments, the limit of quantitation of themethods is less than or equal to 60 ng / mL. In some embodiments, the limit of quantitation of the methods is less than or equal to 50 ng / mL.

[0083] In some embodiments, the limit of detection of the methods is less than or equal to 50 ng / mL. In some embodiments, the limit of detection of the methods is less than or equal to 40 ng / mL. In some embodiments, the limit of detection of the methods is less than or equal to 35.5 ng / mL.

[0084] In some embodiments, methods provided herein comprise linearity of quantitation across a range between 50 ng / mL to 50,000 ng / mL.

[0085] In some embodiments, methods provided herein comprise inter‑ and intra-assay reproducibility of CV ≤15%.

[0086] In some embodiments, CgA is not derivatized prior to mass spectrometry.

[0087] In certain embodiments, the sample is a body fluid. In some embodiments, the sample is cerebrospinal fluid (CSF). In some embodiments, the sample is plasma or serum. In some embodiments, the sample is whole blood. In some embodiments, the sample is saliva or urine.

[0088] In some embodiments, the methods may include adding an agent to the sample in an amount sufficient to deproteinate the sample.

[0089] In someembodiments, elevated levels of chromograninA as compared to a reference range indicates increased risk of neuroendocrine tumors (NET). In someembodiments, quantitated levels of chromograninA indicates the sizeof the neuroendocrine tumor. In some embodiments, quantitated levels of chromogranin A indicates the tumor burden of the neuroendocrine tumor. In someembodiments, quantitated levelsof chromograninA indicates the response to treatment of the neuroendocrine tumor. In some embodiments, quantitated levels of chromogranin A indicates the prognosis of the neuroendocrine tumor.

[0090] Suitable test samples include any test sample that may contain the analyte of interest. In some preferred embodiments, a sample is a biological sample; that is, a sample obtained fromany biological source, such as an animal, a cell culture, anorgan culture, etc. In certain preferredembodiments samplesareobtained fromamammaliananimal, such asadog, cat, horse, etc. Particularly preferredmammalian animals are primates,most preferablymale or female humans. Particularly preferred samples include blood, plasma, serum, hair, muscle, urine, saliva, tear, cerebrospinal fluid, or other tissue sample. Such samples may be obtained, for example, from a patient; that is, a living person, male or female, presenting oneself in a clinical setting for diagnosis, prognosis, or treatment of a disease or condition. The test sample is preferably obtained from a patient, for example, blood serum. 7 EP 4 653 862 A2 5 10 15 20 25 30 35 40 45 50 55 Sample Preparation for Mass Spectrometry

[0091] Methods that may be used to enrich in CgA relative to other components in the sample (e.g. protein) include for example, filtration, centrifugation, thin layer chromatography (TLC), electrophoresis including capillary electrophoresis, affinity separations including immunoaffinity separations, extraction methods including ethyl acetate extraction and methanol extraction, and the use of chaotropic agents or any combination of the above or the like.

[0092] Protein precipitation is one preferred method of preparing a test sample. Such protein purification methods are well known in the art, for example, Polson et al., Journal of Chromatography B 785:263‑275 (2003), describes protein precipitation techniques suitable for use in the methods. Protein precipitation may be used to remove most of the protein from the sample leaving CgA in the supernatant. The samplesmay be centrifuged to separate the liquid supernatant from the precipitated proteins. The resultant supernatant may then be applied to liquid chromatography and subsequent mass spectrometry analysis. In certain embodiments, the use of protein precipitation such as for example, acetonitrile protein precipitation, obviates theneed for high turbulence liquid chromatography (HTLC)or otheron-lineextractionprior toHPLC and mass spectrometry. Accordingly in such embodiments, the method involves (1) performing a protein precipitation of the sample of interest; and (2) loading the supernatant directly onto the HPLC-mass spectrometer without using on-line extraction or high turbulence liquid chromatography (HTLC).

[0093] In some preferred embodiments, HPLC, alone or in combination with one or more purification methods, may be used to purify CgAprior tomass spectrometry. In such embodiments samplesmay be extracted using anHPLCextraction cartridge which captures the analyte, then eluted and chromatographed on a second HPLC 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 beminimized. This feature can result in savings of time and costs, and eliminate the opportunity for operator error.

[0094] It is believed that turbulent flow, such as that provided by HTLC columns and methods, may enhance the rate of mass transfer, improving separation characteristics. HTLC columns separate components by means of high chromato- graphic flow rates through a packed column containing rigid particles. By employing high flow rates (e.g., 3‑5 mL / min), turbulent flow occurs in the column that causes nearly complete interaction between the stationary phase and the analyte(s) of interest. An advantageof usingHTLCcolumns is that themacromolecular build-upassociatedwith biological fluid matrices is avoided since the high molecular weight species are not retained under the turbulent flow conditions. HTLC methods that combine multiple separations in one procedure lessen the need for lengthy sample preparation and operate at a significantly greater speed. Such methods also achieve a separation performance superior to laminar flow (HPLC) chromatography. HTLC allows for direct injection of biological samples (plasma, urine, etc.). Direct injection is difficult to achieve in traditional forms of chromatography because denatured proteins and other biological debris quickly block the separation columns. HTLC also allows for very low sample volume of less than 1mL, preferably less than .5mL, preferably less than .2 mL, preferably .1 mL.

[0095] Examples of HTLC applied to sample preparation prior to analysis by mass spectrometry have been described elsewhere.See,e.g.,Zimmeretal., J.Chromatogr.A854:23‑35 (1999); seealso,U.S.PatentsNos.5,968,367;5,919,368; 5,795,469; and 5,772,874. In certain embodiments of the method, samples are subjected to protein precipitation as described above prior to loading on the HTLC column; in alternative preferred embodiments, the samples may be loaded directly onto the HTLC without being subjected to protein precipitation. The HTLC extraction column is preferably a large particle column. In various embodiments, one of more steps of the methods may be performed in an on-line, automated fashion. For example, in one embodiment, steps (i)‑(v) are performed in an on-line, automated fashion. In another, the steps of ionization and detection are performed on-line following steps (i)‑(v).

[0096] Liquid chromatography (LC) including high-performance liquid chromatography (HPLC) relies on relatively slow, laminar flow technology. Traditional HPLCanalysis relies on column packings in which laminar flowof the sample through the column is the basis for separation of the analyte of interest from the sample. The skilled artisan will understand that separation in such columns is a diffusional process. HPLC has been successfully applied to the separation of compounds in biological samples but a significant amount of sample preparation is required prior to the separation and subsequent analysis with a mass spectrometer (MS), making this technique labor intensive. In addition, most HPLC systems do not utilize the mass spectrometer to its fullest potential, allowing only one HPLC system to be connected to a single MS instrument, resulting in lengthy time requirements for performing a large number of assays.

[0097] Variousmethods have been described for using HPLC for sample clean-up prior tomass spectrometry analysis. See,e.g.,Taylor et al., TherapeuticDrugMonitoring 22:608‑12 (2000); andSalmet al., Clin. Therapeutics 22Supl. B:B71- B85 (2000).

[0098] One of skill in the art may select HPLC instruments and columns that are suitable for use with CgA. The chromatographic column typically includes amedium (i.e.,apackingmaterial) to facilitate separation of chemicalmoieties (i.e., fractionation). The medium may include minute particles. The particles include a bonded surface that interacts with the various chemical moieties to facilitate separation of the chemical moieties. One suitable bonded surface is a hydrophobic bonded surface such as an alkyl bonded surface. Alkyl bonded surfaces may include C‑4, C‑8, C‑12, or 8 EP 4 653 862 A2 5 10 15 20 25 30 35 40 45 50 55 C‑18 bonded alkyl groups, preferably C‑18 bonded groups. The chromatographic column includes an inlet port for receivinga sample andanoutlet port for discharging aneffluent that includes the fractionated sample. In oneembodiment, the sample (or pre-purified sample) is 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 the analyte(s) of interest. For example, liquid chromatography may be performed using a gradient mode, an isocratic mode, or a polytyptic (i.e. mixed) mode. During chromatography, the separation of materials is effected by variables such as choice of eluent (also known as a "mobile phase"), elution mode, gradient conditions, temperature, etc.

[0099] In certain embodiments, an analytemaybepurified byapplying a sample to a columnunder conditionswhere the analyte of interest is reversibly retainedby the columnpackingmaterial,while oneormoreothermaterials arenot retained. In these embodiments, a first mobile phase condition can be employed where the analyte of interest is retained by the column, and a second mobile phase condition can subsequently be employed to remove retained material from the column, once the non-retained materials are washed through. Alternatively, an analyte may be purified by applying a sample to a column under mobile phase conditions where the analyte of interest elutes at a differential rate in comparison to one ormore othermaterials. Such proceduresmay enrich the amount of one ormore analytes of interest relative to one or more other components of the sample.

[0100] In one preferred embodiment, the HTLC may be followed by HPLC on a hydrophobic column chromatographic system. In certain preferred embodiments, a TurboFlowCyclone P® polymer-based column fromCohesive Technologies (60 µm particle size, 50 x 1.0 mm column dimensions, 100Å pore size) is used. In related preferred embodiments, a Synergi Polar-RP®ether-linkedphenyl, analytical column fromPhenomenex Inc (4µmparticle size, 150 x 2.0mmcolumn dimensions, 80Å pore size) with hydrophilic endcapping is used. In certain preferred embodiments, HTLC and HPLC are performed using HPLC Grade Ultra Pure Water and 100% methanol as the mobile phases.

[0101] By careful selection of valves and connector plumbing, two ormore chromatography columnsmaybe connected as needed such that material is passed from one to the next without the need for any manual steps. In preferred embodiments, the selectionof valvesandplumbing is controlledbyacomputer pre-programmed toperform thenecessary steps. Most preferably, the chromatography system is also connected in such an on-line fashion to the detector system, e.g., an MS system. Thus, an operator may place a tray of samples in an autosampler, and the remaining operations are performed under computer control, resulting in purification and analysis of all samples selected.

[0102] In certain preferred embodiments, CgA or fragments thereof in a sample may be purified prior to ionization. In particularly preferred embodiments the chromatography is not gas chromatography. Detection and Quantitation by Mass Spectrometry

[0103] In various embodiments, CgA or fragments thereof may be ionized by any method known to the skilled artisan. Mass spectrometry is performed using a mass spectrometer, which includes an ion source for ionizing the fractionated sample and creating charged molecules for further analysis. For example ionization of the sample may be performed by electron ionization, chemical ionization, electrospray ionization (ESI), photon ionization, atmospheric pressure chemical ionization (APCI), photoionization, atmospheric pressure photoionization (APPI), fast atom bombardment (FAB), liquid secondary ionization (LSI), matrix assisted laser desorption ionization (MALDI), field ionization, field desorption, thermospray / plasmaspray ionization, surface enhanced laser desorption ionization (SELDI), inductively coupled plasma (ICP) and particle beam ionization. The skilled artisan will understand that the choice of ionization method may be determined based on the analyte to be measured, type of sample, the type of detector, the choice of positive versus negative mode, etc.

[0104] In preferred embodiments, CgA or a fragment thereof is ionized by electrospray ionization (ESI) in positive or negative mode.

[0105] After the sample has been ionized, the positively charged or negatively charged ions thereby created may be analyzed to determine a mass-to-charge ratio. Suitable analyzers for determining mass-to-charge ratios include quadru- poleanalyzers, ion trapsanalyzers, and time-of-flight analyzers. The ionsmaybedetectedusingseveral detectionmodes. For example, selected ionsmaybedetected i.e.,using a selective ionmonitoringmode (SIM), or alternatively, ionsmaybe detected using a scanning mode, e.g., multiple reaction monitoring (MRM) or selected reaction monitoring (SRM). Preferably, the mass-to-charge ratio is determined using a quadrupole analyzer. For example, in a "quadrupole" or "quadrupole ion trap" instrument, ions in an oscillating radio frequency field experience a force proportional to the DC potential applied between electrodes, the amplitude of the RF signal, and the mass / charge ratio. The voltage and amplitudemaybeselectedso that only ionshavingaparticularmass / charge ratio travel the lengthof thequadrupole,while all other ions are deflected. Thus, quadrupole instrumentsmay act as both a "mass filter" and as a "mass detector" for the ions injected into the instrument.

[0106] Onemayenhance the resolution of theMS techniquebyemploying "tandemmass spectrometry," or "MS / MS". In this technique, a precursor ion (also called a parent ion) generated from a molecule of interest can be filtered in an MS instrument, and theprecursor ion is subsequently fragmented to yieldoneormore fragment ions (alsocalleddaughter ions 9 EP 4 653 862 A2 5 10 15 20 25 30 35 40 45 50 55 or product ions) that are then analyzed in a second MS procedure. By careful selection of precursor ions, only ions produced by certain analytes are passed to the fragmentation chamber, where collisions with atoms of an inert gas produce the fragment ions. Because both the precursor and fragment ions are produced in a reproducible fashion under a given set of ionization / fragmentation conditions, theMS / MS techniquemay provide an extremely powerful analytical tool. For example, the combination of filtration / fragmentation may be used to eliminate interfering substances, and may be particularly useful in complex samples, such as biological samples.

[0107] The mass spectrometer typically provides the user with an ion scan; that is, the relative abundance of each ion with a particular mass / charge over a given range (e.g., 100 to 1000 amu). The results of an analyte assay, that is, a mass spectrum, may be related to the amount of the analyte in the original sample by numerous methods known in the art. For example, given that sampling and analysis parameters are carefully controlled, the relative abundance of a given ionmay be compared to a table that converts that relative abundance to an absolute amount of the originalmolecule. Alternatively, molecular standardsmay be runwith the samples, and a standard curve constructed based on ions generated from those standards. Using such a standard curve, the relative abundance of a given ionmay be converted into an absolute amount of the original molecule. In certain preferred embodiments, an internal standard is used to generate a standard curve for calculating thequantity ofCgA.Methodsof generating andusing such standard curves arewell known in theart andoneof ordinary skill is capable of selecting an appropriate internal standard. For example, an isotope of CgAmay be used as an internal standard. Numerous other methods for relating the amount of an ion to the amount of the original molecule will be well known to those of ordinary skill in the art.

[0108] Oneormore stepsof themethodsmaybeperformedusingautomatedmachines. In certainembodiments, oneor morepurification stepsareperformedon-line, andmorepreferably all of thepurification andmass spectrometry stepsmay be performed in an on-line fashion.

[0109] In certain embodiments, such as MS / MS, where precursor ions are isolated for further fragmentation, collision activation dissociation is oftenused togenerate the fragment ions for further detection. InCAD, precursor ions gain energy through collisionswith an inert gas, and subsequently fragment by a process referred to as "unimolecular decomposition". Sufficient energymustbedeposited in theprecursor ion so that certainbondswithin the ioncanbebrokendue to increased vibrational energy.

[0110] In particularly preferred embodiments, CgA is detected and / or quantified usingMS / MS as follows. The samples are subjected to liquid chromatography, preferably HPLC, the flow of liquid solvent from the chromatographic column enters the heated nebulizer interface of an MS / MS analyzer and the solvent / analyte mixture is converted to vapor in the heated tubingof the interface. Theanalyte is ionizedby theselected ionizer. The ions, e.g. precursor ions, pass through the orifice of the instrument and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters, allowing selection of ions (i.e., "precursor" and "fragment" ions) based on their mass to charge ratio (m / z). Quadrupole 2 (Q2) is the collisioncell,where ionsare fragmented.Thefirst quadrupoleof themassspectrometer (Q1)selects formoleculeswith the mass to charge ratios of CgA. Precursor ions with the correct mass / charge ratios of CgA are allowed to pass into the collision chamber (Q2), while unwanted ionswith any othermass / charge ratio collide with the sides of the quadrupole and are eliminated. Precursor ions entering Q2 collide with neutral argon gas molecules and fragment. This process is called collision activated dissociation (CAD). The fragment ions generated are passed into quadrupole 3 (Q3), where the fragment ions of CgA are selected while other ions are eliminated.

[0111] The methods may involve MS / MS performed in either positive or negative ion mode. Using standard methods well known in the art, one of ordinary skill is capable of identifying one ormore fragment ions of a particular precursor ion of CgA that may be used for selection in quadrupole 3 (Q3).

[0112] As ions collide with the detector they produce a pulse of electrons that are converted to a digital signal. The acquired data is relayed to a computer, which plots counts of the ions collected versus time. The resulting mass chromatograms are similar to chromatograms generated in traditional HPLC methods. The areas under the peaks corresponding to particular ions, or the amplitude of such peaks, are measured and the area or amplitude is correlated to the amount of the analyte of interest. In certain embodiments, the area under the curves, or amplitude of the peaks, for fragment ion(s) and / or precursor ions are measured to determine the amount of CgA. As described above, the relative abundance of a given ion may be converted into an absolute amount of the original analyte, using calibration standard curves based on peaks of one or more ions of an internal molecular standard.

[0113] The followingexamples serve to illustrate the invention. Theseexamplesare in noway intended to limit the scope of the methods. EXAMPLES Example 1: CgA quantitation by mass spectrometry 10 EP 4 653 862 A2 5 10 15 20 25 30 35 40 45 50 55 Reagent summary:

[0114] Reagents Supplier & Catalog Number Quantity Chromogranin-A Abcam, ab85486 250 ug Chromogranin-A IS (See CgA Internal Standard Prep below for sequence) New England Peptide, Custom Synthesis ≥95% purity 6 mg Biocell Charcoal Stripped and Delipidated Serum Biocell, 1101‑00 1 L Ammonium Bicarbonate (AmBic) Sigma, A Millipore, FX0440‑56141‑500G 500 g Triethylammonium bicarbonate, 1.0M Sigma, T7408‑500mL 500 mL Dithiothreitol Sigma, 43819‑25G 25g Iodoacetamide Sigma, I1149‑25G 25g Trypsin Sigma, T1426‑500MG 0.5 g Formic Acid, 98% Millipore, FX0440‑5 0.5 L Ammonium hydroxide, 28‑30% Sigma, 221228‑500ML-A 500 mL HPLC Water Burdick & Jackson, 365‑4 4 L Acetonitrile Burdick & Jackson, 015‑4 4 L Methanol Fisher Scientific, A454‑4 4 L Bovine Serum Albumin Sigma, A2153‑500G 500 g MicroAmp Clear Adhesive Film Thermo Fisher, 4306311 100 films Thermo Scientific Accucore C18 100 x 2.1 mm, 2.6u Themro, 17126‑102130 1 column Oasis MAX 96-well 30mg SPE Plate Waters, 186000373 1 plate

[0115] Sciex 6500+ QTrap Mass Spectrometer, Thermo Fisher Aria Cohesive TLX4 with Agilent Pumps, Hamilton Microlab Star, SPEware IP8 were used to detect CgA.

[0116] Patient serum (100 µL) was added to 600 µL of 120 mM ammonium bicarbonate, and the entire volume was extracted using a mixed-mode anion exchange plate. (Waters Oasis® MAX 30mg). Samples are then washed twice with 3%ammoniumhydroxideandwater, respectively.Next,CgAwaseluted, internal standard (IS) added,and thesamplewas evaporated under heated nitrogen.

[0117] After dry-down, samples were reconstituted, reduced, alkylated, and tryptically digested for 2 hours in a rapid enzyme digestion microwave system (Hudson Technology).

[0118] Post-digestion, sampleswereacidifiedandanalyzedbyLC-MS / MSonanAriaTLX‑4TranscendUPLC.Peptides were resolvedusingaThermoAccucoreC18100×2.1mm,2.6micronHPLCcolumnusingwater / acetonitrile / 0.1% formic acid gradients. TheMSdetector was aSciex 6500+Qtrap. Quantitation of CgAwas based on the chromatographic peaks of fragment ions produced during MS / MS corresponding to a representative tryptic peptide and its corresponding heavy- labeled IS. The analytical workflow is summarized in Figure 1.

[0119] Multiple reaction monitoring was used for detecting both the analyte and the internal standard (IS).

[0120] Concentrations of Chromogranin A (CgA) were determined using peak area ratio and a calibration curve. In assays detecting ELQDLALQGAK (SEQ ID NO: 1), a labeled winged peptide internal standard was used: IL- SILRHQNLLKELQDLAL*QGAK*ERAHQQK (SEQ IDNO:2): *C13N15 labeledaminoacids.After digestion, the resulting peptide was ELQDLAL*QGAK* (SEQ ID NO: 3). In assays detecting RRPEDQELESLSAIEAELEK (SEQ ID NO: 4), a labeled winged peptide internal standard was used: EGSANRRPEDQELESL*SAIEAELEK*VAHQL (SEQ ID NO: 5); *C13N15 labeled amino acids. After digestion, the resulting peptide was RRPEDQELESL*SAIEAELEK* (SEQ ID NO: 6). 11 EP 4 653 862 A2 5 10 15 20 25 30 35 40 45 50 55 Table 1: CgA fragment used to quantitate CgA levels in the sample and precursor and product ions (mass-to-charge m / z ratios) Analyte Precursor Ion (m / z) Product Ion (m / z) CgA: ELQDLALQGAK 593.2 516.3, 815.5 CgA IS: ELQDLAL*QGAK* 600.8 602.4, 830.6, 958.7 Table 2: CgA fragment used to quantitate CgA levels in the sample and precursor and product ions (mass-to-charge m / z ratios) Analyte Precursor Ion (m / z) Product Ion (m / z) CgA: RRPEDQELESLSAIEAELEK 729.6 831.5, 989.5 CgA: RRPEDQELESL*SAIEAELEK* 734.6 839.5, 997.6 Table 3: The values obtained were compared to the values quantitated by ELISA immunoassay. See also Figure 4. Sample ID# Mayo Result ARUP Result Dawn Result Average (Mayo, ARUP, Dawn) LCMS Ref Ranges <93 0‑95 N / A N / A N / A Units ng / mL ng / mL ng / mL ng / mL ng / mL 1 1654 2286 3070 1753 1402 2 872 2172 1724 1192 2084 3 1286 1603 1680 1142 1774 4 793 1151 1220 791 730 5 499 1077 1285 715 827 6 588 639 657 471 4021 7 42 479 482 251 237 8 207 302 297 202 589 9 277 284 285 212 677 10 156 212 187 139 412 11 114 181 182 119 94 12 120 145 136 101 342 13 82 120 130 83 115 14 64 105 101 68 50 15 63 96 101 65 62 16 57 88 86 58 <50 17 62 80 80 56 <50 18 47 74 76 49 <50 19 41 60 60 40 <50 20 <20 36 45 40 <50

[0121] Samples from 308 patients were analyzed to compare CgA serum values measured by the Cisbio CGA-ELISA- US immunoassay (Codolet, France) with values from our LC-MS / MS assay.

[0122] Afternatural logarithm-transformationof themeasurements, anormal distributionwasseen in the308patients.A paired t-test was performed on these data and the concordance between the assays was measured by the Pearson correlation coefficient and Passing & Bablok curve fitting. All analyses were performed in Analyse-it v2.30.

[0123] The assay was validated. Performance characteristics are presented in Table 4: 12 EP 4 653 862 A2 5 10 15 20 25 30 35 40 45 50 55 Characteristics Values Intra-assay precision 5.2‑15.7% Inter-assay precision 7.4‑10.1% Recovery of CgA spiked into patient samples 90‑110% Analytical sensitivity (Limit of Detection) 35.5 ng / mL Analytical sensitivity (Limit of Quantitation) 50 ng / mL Linearity 50‑50,000 ng / mL Reference Range <140 ng / mL (n=160)

[0124] Typical results for patient samples with low and high concentrations of circulating CgA are shown in Figure 3.

[0125] Measured CgA levels of the LC-MS / MS were on average comparable to the CisBio assay, although there was substantial scatter (Pearson’s correlation 0.76) (Figure 5).

[0126] Conclusion: We have developed and validated a fully automated LC-MS / MS assay for CgA from serum

[0127] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information fromany such articles, patents, patent applications, or other physical and electronic documents.

[0128] Themethods illustratively described hereinmaysuitably bepracticed in theabsenceof anyelement or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including," contain- ing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have beenusedas termsof descriptionandnot of limitation, and there is no intention in theuseof such termsandexpressionsof excluding any equivalents of the features shown and described or portions thereof. It is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the invention embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0129] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the methods. This includes the generic description of the methodswith a proviso or negative limitation removing any subjectmatter from the genus, regardless ofwhether or not the excised material is specifically recited herein.

[0130] Other embodiments are within the following claims. In addition, where features or aspects of the methods are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. SUMMARY PARAGRAPHS:

[0131] 1. A method for determining the amount of chromogranin A (CgA) in a sample, said method comprising: (a) purifying CgA in the sample; (b) ionizing CgA to produce one or more ion(s) of CgA detectable by mass spectrometry; (c) determining the amount of the ion(s) fromstep (b) bymass spectrometry, wherein the amount of ions is related to the amount of CgA in the sample. 2. The method of paragraph 1, wherein said purifying comprises extraction by solid phase extraction (SPE). 3. The method of paragraph 2, wherein the SPE is an anion exchange solid phase extraction. 4. The method of paragraph 2, wherein the SPE is a mixed-mode anion exchange solid phase extraction. 5. The method of paragraph 2, wherein extracted samples are enzymatically digested. 13 EP 4 653 862 A2 5 10 15 20 25 30 35 40 45 50 55 6. The method of paragraph 5, wherein the digestion comprises trypsin digestion. 7. The method of paragraph 1, wherein said purifying comprises liquid chromatography. 8. The method of paragraph 7, wherein said liquid chromatography comprises high performance liquid chromato- graphy (HPLC). 9. Themethod of paragraph 7,wherein said liquid chromatography comprises high turbulence liquid chromatography (HTLC). 10. The method of paragraph 1, wherein said ionization comprises electrospray ionization (ESI). 11. The method of paragraph 1, further comprising adding an internal standard. 12. The method of paragraph 11, wherein said internal standard is isotopically labeled. 13. The method of paragraph 1, wherein the sample is serum. 14. The method of paragraph 1, wherein the sample is cerebrospinal fluid (CSF). 15.Themethodofparagraph1,wherein themethodcomprisesmeasuring theamountof precursor ionhavingamass- to-charge ratio of 729.6 ± 0.5. 16. The method of paragraph 1, wherein the method comprises measuring the amount of a fragment of CgA. 17. The method of paragraph 16, wherein the CgA fragment measured comprises a sequence RRPEDQELESL- SAIEAELEK (SEQ ID NO: 4). 18. Themethod of paragraph 1, wherein themethod comprisemeasuring the amount of fragment ion having amass- to-charge ratio of 831.5 ± 0.5 or 989.5 ± 0.5. 19. The method of paragraph 1, wherein the method comprises adding an internal standard. 20. The method of paragraph 19, wherein the internal standard is isotopically labeled. 21. The method of paragraph 19, wherein the internal standard comprises a C13N15 labeled amino acid. 22. The method of paragraph 19, wherein the internal standard is labeled on a leucine (L) or lysine (K). 23. The method of paragraph 19, wherein the internal standard comprises a sequence EGSANRRPEDQELESL*- SAIEAELEK*VAHQL (SEQ ID NO: 5), wherein L* and K* is each a C13N15 labeled amino acid. 23. Themethodof paragraph19,wherein themethodcomprisesmeasuring theamount of internal standardprecursor ion having amass-to-charge ratio of 734.6± 0.5 or product ion having amass-to-charge ratio of 839.5± 0.5 or 997.6 ± 0.5. 24. The method of paragraph 1, wherein the limit of quantitation of the methods is less than or equal to 50 ng / mL. 25. The method of paragraph 1, wherein the limit of detection of the methods is less than or equal to 35.5 ng / mL. 26.Themethodof paragraph1,wherein themethodhasa linearity of quantitationacrossa rangebetween50ng / mL to 50,000 ng / mL. 27. The method of paragraph 1, wherein the method has an inter‑ and intra-assay reproducibility of CV ≤15%. 28. The method of paragraph 1, wherein the mass spectrometry is selected reaction monitoring (SRM) mass spectrometry. 14 EP 4 653 862 A2 5 10 15 20 25 30 35 40 45 50 55 29. The method of paragraph 1, wherein the method is fully automated. 30. The method of paragraph 1, wherein the method is antibody-free. Claims 1. A method for determining an amount of chromogranin A (CgA) in a sample, the method comprising: (a) purifying CgA in the sample; (b) adding an internal standard; (c) ionizing CgA and the internal standard to produce one or more CgA ion(s) and one or more internal standard ion(s) detectable by mass spectrometry; (d) determining an amount of the ion(s) from step (c) by mass spectrometry; and (e) correlating the amount of CgA in the sample to the amount of ion(s) measured in step (d). 2. The method of claim 1, wherein the purifying comprises extraction by solid phase extraction (SPE). 3. The method of claim 2, wherein the extracted sample is enzymatically digested. 4. The method of claim 3, wherein the digestion comprises trypsin digestion. 5. The method of claim 1, wherein the purifying comprises liquid chromatography. 6. The method of claim 1, wherein the ionization comprises electrospray ionization. 7. The method of claim 1, wherein the internal standard is isotopically labeled. 8. The method of claim 1, wherein the internal standard comprises a sequence of EGSANRRPEDQELESL*SAIEAE- LEK*VAHQL (SEQ ID NO: 5), wherein L* and K* is each a C13N15 labeled amino acid. 9. The method of claim 1, wherein the method further comprises measuring an amount of internal standard precursor ion(s) having a mass-to-charge ratio of 734.6± 0.5 or product ion(s) having a mass-to-charge ratio of 839.5± 0.5 or 997.6 ± 0.5. 10. Themethodofclaim1,wherein themethod further comprisesmeasuringanamountof aCgAprecursor ion(s) havinga mass-to-charge ratio of 729. 6± 0. 5 or product ion(s) having a mass-to-charge ratio of 831.5± 0.5 or 989.5± 0.5. 11. The method of claim 1, wherein the sample is serum or cerebrospinal fluid. 12. The method of claim 1, wherein the method further comprises measuring an amount of a fragment of CgA. 13. The method of claim 12, wherein the fragment of CgA comprises a sequence of RRPEDQELESLSAIEAELEK (SEQ ID NO: 4). 14. The method of claim 1, wherein the limit of quantitation of the method is less than or equal to 50 ng / mL. 15. The method of claim 1, wherein the method is antibody-free. 15 EP 4 653 862 A2 5 10 15 20 25 30 35 40 45 50 55 16 EP 4 653 862 A2 17 EP 4 653 862 A2 18 EP 4 653 862 A2 19 EP 4 653 862 A2 20 EP 4 653 862 A2 21 EP 4 653 862 A2 22 EP 4 653 862 A2 23 EP 4 653 862 A2 REFERENCES CITED IN THE DESCRIPTION This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard. Patent documents cited in the description • US 62644210

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[0097] (19) *EP004653862A3* (11) EP 4 653 862 A3 (12) EUROPEAN PATENT APPLICATION (88) Date of publication A3: 14.01.2026 Bulletin 2026 / 03 (43) Date of publication A2: 26.11.2025 Bulletin 2025 / 48 (21) Application number: 25203365.9 (22) Date of filing: 15.03.2019 (51) International Patent Classification (IPC): G01N 33 / 68 (2006.01) G01N 30 / 72 (2006.01) G01N 30 / 88 (2006.01) G01N 30 / 04 (2006.01) (52) Cooperative Patent Classification (CPC): G01N 33 / 6848; G01N 30 / 88; G01N 30 / 7233; G01N 2030 / 009; G01N 2030 / 045; G01N 2030 / 8831 (84) Designated Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR (30) Priority: 16.03.2018 US 201862644210 P (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: 19767849.3 / 3 765 858 (71) Applicant: Quest Diagnostics Investments LLC Secaucus, NJ 07094 (US) (72) Inventors: • WEBER, Darren Laguna Niguel, CA 92677 (US) • CAULFIELD, Michael P. Oceanside, CA 92057 (US) • MCPHAUL, Michael J. Capistrano Beach, CA 92624 (US) • GOLDMAN, Scott Laguna Niguel, CA 92677 (US) • CLARKE, Nigel San Clemente, CA 92673 (US) (74) Representative: Harrison IP Limited Mereside, Alderley Park Congleton Road Nether Alderley Macclesfield, Cheshire SK10 4TG (GB) (54) METHODS FOR DETECTING CHROMOGRANIN A BY MASS SPECTROMETRY (57) Provided are methods for detecting chromogra- nin A by mass spectrometry. In another aspect, provided herein are methods for quantitating chromogranin A by mass spectrometry. In another aspect, provided herein are methods for prognosis of or measuring the size of neuroendocrine tumors by mass spectrometry. EP 4 65 3 86 2 A 3 Processed by Luminess, 75001 PARIS (FR) 2 EP 4 653 862 A3 5 10 15 20 25 30 35 40 45 50 55 3 EP 4 653 862 A3 5 10 15 20 25 30 35 40 45 50 55 摘 要 提供了利用質譜法檢測嗜鉻粒蛋白 A的方法。在另一方面，本文提 供了利用質譜法測定嗜鉻粒蛋白 A的數量的方法。在另一方面，本文提供 了利用質譜法預測或測量神經內分泌腫瘤的大小的方法。

Claims

1. A method for determining an amount of chromogranin A (CgA) in a sample, the method comprising: (a) purifying CgA in the sample; (b) adding an internal standard; (c) ionizing CgA and the internal standard to produce one or more CgA ion(s) and one or more internal standard ion(s) detectable by mass spectrometry; (d) determining an amount of the ion(s) from step (c) by mass spectrometry; and (e) correlating the amount of CgA in the sample to the amount of ion(s) measured in step (d).

2. The method of claim 1, wherein the purifying comprises extraction by solid phase extraction (SPE).

3. The method of claim 2, wherein the extracted sample is enzymatically digested.

4. The method of claim 3, wherein the digestion comprises trypsin digestion.

5. The method of claim 1, wherein the purifying comprises liquid chromatography.

6. The method of claim 1, wherein the ionization comprises electrospray ionization.

7. The method of claim 1, wherein the internal standard is isotopically labeled.

8. The method of claim 1, wherein the internal standard comprises a sequence of EGSANRRPEDQELESL*SAIEAELEK*VAHQL (SEQ ID NO: 5), wherein L* and K* is each a C13N15 labeled amino acid.

9. The method of claim 1, wherein the method further comprises measuring an amount of internal standard precursor ion(s) having a mass-to-charge ratio of 734.6 ± 0.5 or product ion(s) having a mass-to-charge ratio of 839.5 ± 0.5 or 997.6 ± 0.5.

10. The method of claim 1, wherein the method further comprises measuring an amount of a CgA precursor ion(s) having a mass-to-charge ratio of 729. 6 ± 0. 5 or product ion(s) having a mass-to-charge ratio of 831.5 ± 0.5 or 989.5 ± 0.5.

11. The method of claim 1, wherein the sample is serum or cerebrospinal fluid.

12. The method of claim 1, wherein the method further comprises measuring an amount of a fragment of CgA.

13. The method of claim 12, wherein the fragment of CgA comprises a sequence of RRPEDQELESLSAIEAELEK (SEQ ID NO: 4).

14. The method of claim 1, wherein the limit of quantitation of the method is less than or equal to 50 ng / mL.

15. The method of claim 1, wherein the method is antibody-free.