WO2016118489A1 - Combined analysis of small molecules and proteins by mass spectrometry - Google Patents

Abstract

The present technology relates to quantitative assays for evaluation of small molecule analytes in complex samples, including clinical specimens such as human plasma and other proteinaceous samples (including for example tissues, secretions, and body fluids of all living things, as well as samples prepared from heterogeneous mixtures of these), and specifically to measurements made by mass spectrometry.

Description

COMBINED ANALYSIS OF SMALL MOLECULES AND PROTEINS BY MASS

SPECTROMETRY

SEQUENCE LISTING

[001] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 18, 2016, is named SIS_PAT2_PCT_SeqListing.txt and is 2 kilobytes in size.

CROSS-REFERENCE TO RELATED APPLICATIONS

[002] Incorporated by reference herein are the contents of each of the below patent applications, each in its entirety:

[003] 61/314, 149, entitled MS Internal Standards at Clinical Levels filed on March 15, 2010;

[004] 61/665,217, entitled Multipurpose Mass Spectrometric Assay Panels for Peptides filed on June 27, 2012

[005] 10/676,005, entitled High Sensitivity Quantitation of Peptides by Mass Spectrometry; filed 2 October 2003;

[006] 60/415,499, entitled Monitor Peptide Enrichment Using Anti-Peptide

Antibodies, filed 3 Oct 2002;

[007] 60/420,613, entitled Optimization of Monitor Peptide Enrichment Using Anti- Peptide Antibodies, filed 23 October 2002;

[008] 60/449,190, entitled High Sensitivity Quantitation of Peptides by Mass Spectrometry, filed 20 February 2003 ;

[009] 60/496,037, entitled Improved Quantitation of Peptides by Mass Spectrometry, filed 18 August 2003;

[0010] 60/557,261, entitled Selection of Antibodies and Peptides for Peptide

Enrichment, filed 29 March 2004;

[0011] 11/256,946, entitled Process For Treatment Of Protein Samples, filed 25 October 2005;

[0012] 12/042,931, entitled Magnetic Bead Trap and Mass Spectrometer Interface, filed 5 March, 2008;

[0013] 61/314,154 entitled Stable Isotope Labeled Peptides on Carriers, filed 15 March 2010; [0014] 61/314,149 entitled MS Internal Standards at Clinical Levels filed 15 March 2010;

[0015] PCT/US2011/028569, entitled Improved Mass Spectrometric Assays For Peptides filed 15 March 2011;

[0016] 61/665,217 entitled Multipurpose Mass Spectrometric Assay Panels For Peptides filed 27 June 2012;

[0017] 61/665,228 entitled Simultaneous Peptide And Metabolite Affinity Capture Mass Spectrometry filed 27 June 2012;

[0018] 61/670,493 entitled Proteolytic Digestion Kit With Dried Reagents filed 11 July 2012;

[0019] 61/720,386 entitled Peptide Fragments Of Human Protein C Inhibitor And Human Pigment Epithelium-Derived Factor And Use In Monitoring Of Prostate Cancer filed 30 October 2012; and

[0020] 62/105,134 entitled Combined Analysis of Small Molecules and Proteins by Mass Spectrometry filed 19 January 2015.

BACKGROUND

[0021] The art of employing mass spectrometry for the measurement of established and candidate biomarker molecules could benefit from improvements in methods for measuring so-called "small molecules" (such as thyroxine, the steroid hormones, cholesterol, triglycerides, neuropeptides, vitamin D, etc.) by mass spectrometry, and particularly from methods capable of measuring small molecules in multiple modes, in some cases together with large molecules (such as proteins) in a single integrated workflow or method. In a specific case, the art could benefit from the ability to measure small molecules in clinical samples such as blood, serum and plasma in concert with protein measurement using the SISCAPA technology disclosed in one or more of the patent filings referenced above, and a number of recent publications including, for instance, the following publications, each of which is incorporated by reference herein in its entirety:

[0022] Mass Spectrometric Quantitation of Peptides and Proteins Using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA). Anderson, N.L., Anderson, N.G., Haines, L.R., Hardie, D.B., Olafson. R.W., and Pearson, T.W. Journal of Proteome Research 3: 235-44 (2004); [0023] Antibody-based enrichment of peptides on magnetic beads for mass- spectrometry -based quantification of serum biomarkers. J. R. Whiteaker, L. Zhao, H. Y. Zhang, L-C Feng, B. D. Piening, L. Anderson and A. G. Paulo vich, Analytical

Biochemistry 362:44-54 (2007);

[0024] Anti-peptide antibody screening: selection of high affinity monoclonal reagents by a refined surface plasmon resonance technique. Matthew E. Pope, Martin V. Soste, Brett A. Eyford, N. Leigh Anderson and Terry W. Pearson, J Immunol Methods, 341(l-2):86-96 (2009);

[0025] SISCAPA Peptide Enrichment on Magnetic Beads Using an Inline Beadtrap Device. N. Leigh Anderson, Angela Jackson, Derek Smith, Darryl Hardie, Christoph Borchers, and Terry W. Pearson, Mol Cell Proteomics 8:995-1005 (2009);

[0026] An automated and multiplexed method for high throughput peptide

immunoaffinity enrichment and multiple reaction monitoring mass spectrometry-based quantification of protein biomarkers. Jeffrey R. Whiteaker, Lei Zhao, Leigh Anderson, and Amanda G. Paulovich, Mol. Cell. Proteomics 9: 184-196 (2010);

[0027] Proteomic-Based Multiplex Assay Mock Submissions: Supplementary Material to A Workshop Report by the NCI-FDA Interagency Oncology Task Force on Molecular Diagnostics, Fred E. Regnier, Steven J. Skates, Mehdi Mesri, Henry Rodriguez, Zivana Tezak, Marina V. Kondratovich, Michail A. Alterman, Joshua D. Levin, Donna Roscoe, Eugene Reilly, James Callaghan, Kellie Kelm, David Brown, Reena Philip, Steven A. Carr, Daniel C. Liebler, Susan J. Fisher, Paul Tempst, Tara Hiltke, Larry G. Kessler, Christopher R. Kinsinger, David F. Ransohoff, Elizabeth Mansfield4 and N. Leigh Anderson, Clin Chem. 56:2 165-171 (2010);

[0028] MALDI Immunoscreening (MiSCREEN): A Method for Selection of Anti- peptide Monoclonal Antibodies For Use in Immunoproteomics, Morteza Razavi, Matthew E. Pope, Martin V. Soste, Brett A. Eyford, N. Leigh Anderson and Terry W. Pearson, J. Immunol. Methods, 364 (2011) 50-64;

[0029] Evaluation of Large Scale Quantitative Proteomic Assay Development Using Peptide Affinity-based Mass Spectrometry. Jeffrey R. Whiteaker, Lei Zhao, Susan E. Abbatiello, Michael Burgess, Eric Kuhn, ChenWei Lin, Matthew E. Pope, Morteza Razavi, N. Leigh Anderson, Terry W. Pearson, Steven A. Carr, and Amanda G.

Paulovich, Mol Cell Proteomics (2011) 10: Ml 10.005645;

[0030] Inter-laboratory Evaluation of Automated, Multiplexed Peptide

Immunoaffinity Enrichment Multiple Reaction Monitoring Assay Performance for Quantifying Proteins in Plasma. Eric Kuhn, Jeffrey R Whiteaker, DR Mani, Angela M Jackson, Lei Zhao, Matthew Pope, Derek Smith, Keith D Rivera, N Leigh Anderson, Steven J. Skates, Terry W Pearson, Amanda G Paulo vich and Steven A Carr, Mol Cell Proteomics (2011).013854;

[0031] The Precision of Heavy -Light Peptide Ratios Measured by MALDI-TOF Mass Spectrometry. N. Leigh Anderson, Morteza Razavi, Terry W. Pearson, Gary Kruppa, Rainer Paape, and Detlev Suckau, J. Proteome Res (2011) DOI: 10.1021/pr201092v;

[0032] Ultra-fast quantitation of peptides from human plasma digests using SISCAPA and RapidFire high throughput mass spectrometry, Morteza Razavi, Lauren E Frick, William A LaMarr, Matthew E Pope, Christine A Miller, N Leigh Anderson and Terry W Pearson (2012), J. Proteome Res (2012) DOI: 10.1021/pr300652v

[0033] High Precision Quantification of Human Plasma Proteins Using the

Automated SISCAPA Immuno-MS Workflow. Razavi, M., Anderson, N.L., Pope, M. E., Yip, R. & Pearson, T. W., New Biotechnology (2016). doi: 10.1016/j.nbt.2015.12.008).

[0034] There are several examples in the art in which small molecule analytes have been enriched by capture on specific antibodies and subsequently eluted and detected by mass spectrometry. In these methods, small molecules are measured using a single mode of sample preparation, and proteins were not measured as part of the same method or workflow:

[0035] Quantification of 1 ,25-Dihydroxy Vitamin D by Immunoextraction and Liquid Chromatography -Tandem Mass Spectrometry. Strathmann, F. G., Laha, T. J. &

Hoofnagle, A. N. Clin Chem 57, 1279-1285 (2011).

[0036] Determination of drugs from urine by on-line immunoaffinity

chromatography-high-performance liquid chromatography -mass spectrometry. Rule, G. S. & Henion, J. D. Chromatogr 582, 103-112 (1992).

[0037] Quantitative multi-residue determination of beta-agonists in bovine urine using on-line immunoaffinity extraction-coupled column packed capillary liquid chromatography -tandem mass spectrometry. Cai, J. & Henion, J. Chromatogr B Biomed Sci Appl 691, 357-370 (1997).

[0038] Immunoaffinity chromatography combined on-line with high-performance liquid chromatography-mass spectrometry for the determination of corticosteroids. Creaser, C. S., Feely, S. J., Houghton, E. & Seymour, M. Chromatogr A 794, 37-43 (1998). [0039] Purification and analysis of drug residues in urine samples by on-line immunoaffinity chromatography/high-performance liquid chromatography/continuous- flowfast atom bombardment mass spectrometry. Davoli, E., Fanelli, R. & Bagnati, R. Anal Chem 65, 2679-2685 (1993).

SUMMARY

[0040] The present technology relates to quantitative assays for evaluation of small molecule analytes in complex samples, including clinical specimens such as human plasma and other proteinaceous samples (including for example tissues, secretions, and body fluids of all living things, as well as samples prepared from heterogeneous mixtures of these), and specifically to measurements made by mass spectrometry.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 shows 5 samples processed according to variations of the basic conversion protocol so as to illustrate various features and capabilities of the combined SMA and peptide workflow as illustrated in Example 1 : A) a capture reaction in digested DBS that includes antibody to ApoB lOO peptide but no antibody to Ch, B) a capture reaction in digested DBS that includes antibody to Ch but no antibody to ApoB lOO peptide, C) a capture reaction in digested DBS that includes antibodies to both Ch and to ApoB lOO peptide, D) a capture reaction as in 'C but one in which 5

of 1 mg/mL (1 unit equivalent) of cholesterol esterase (Sigma; C1403-25UN) dissolved in 0.4 M potassium phosphate (pH 7.0) was added to the sample followed by shaking at 1000 rpm for 15 minutes at 40 °C before trypsin digestion of the DBS sample and E) a capture reaction as in 'C but one in which the cholesterol esterase was added after trypsin digestion and quenching, followed by shaking at 1000 rpm for 15 minutes at 40 °C.

DETAILED DESCRIPTION

[0042] The term "amount", "concentration" or "level" of an analyte or internal standard means the physical quantity of the substance referred to, either in terms of mass (or equivalently moles), in terms of concentration (the amount of mass or moles per volume of a solution or liquid sample), or in terms relative to another analysis carried out on a calibration sample (for example relative proportions compared to another sample).

[0043] The term "analyte", "pre-selected analyte" or "ligand" may be any of a variety of different molecules, or components, pieces, fragments or sections of different molecules that are to be measured or quantitated in a sample. In the present context analytes are typically selected in advance of the measurement so as to allow prior preparation of effective binding agents capable of capturing them from a sample. An analyte may thus be a protein, a peptide derived from a protein by digestion or other fragmentation technique, a small molecule (such as a hormone, metabolite, drug, drug metabolite), a nucleic acid (DNA, RNA, and fragments thereof produced by enzymatic, chemical or other fragmentation processes), a glycan structure, an atomic or diatomic ion, or any other atom or molecule of material substance that is measured by an analytical method.

[0044] The term "antibody" means a monoclonal or monospecific polyclonal immunoglobulin protein such as IgG or IgM. An antibody may be a whole antibody or antigen-binding antibody fragment derived from a species (e.g., rabbit or mouse) commonly employed to produce antibodies against a selected antigen, or may be derived from recombinant methods such as protein expression, and phage/virus display. See, e.g., U.S. Patent Nos.: 7,732,168; 7,575,896; and 7,431927, which describe preparation of rabbit monoclonal antibodies. Antibody fragments may be any antigen-binding fragment that can be prepared by conventional protein chemistry methods or may be engineered fragments such as scFv, diabodies, minibodies and the like. In the present context, antibodies are examples of binders.

[0045] The term "bind" or "react" or "capture" means any physical attachment or close association, which may be permanent or temporary. Generally, reversible binding includes aspects of charge interactions, hydrogen bonding, hydrophobic forces, van der Waals forces etc., that facilitate physical attachment between the molecule of interest and the analyte being measured. The "binding" interaction may be brief as in the situation where binding causes a chemical reaction to occur. Reactions resulting from contact between the binding agent and the analyte are also within the definition of binding for the purposes of the present technology, provided they can be later reversed to release a monitor fragment. A "capture" is a step in which molecules (generally analytes and/or SIS) are bound by a binder.

[0046] The term "binder" means a molecule or substance having an affinity for one or more analytes, and includes antibodies (for example polyclonal, monoclonal, single chain, and modifications thereof including fragments), aptamers (made of DNA, RNA, modified nucleotides, peptides, and other compounds), etc. "Specific binders" are those with particular affinity for a specific analyte molecule. It will be understood that other classes of molecules such as DNA and RNA aptamers configured as specific and high affinity binders may be used as alternatives to antibodies or antibody fragments in appropriate circumstances. An "anti-SMA binder" is a binder having specific affinity for a small molecule analyte, while an anti-peptide binder is a binder having specific affinity for a given peptide. "Contacting" a binder with its respective analyte (i.e., it ligand) refers to a situation in which the binder and the analyte (and in some cases its SIS version as internal standard) are mutually accessible, generally in solution, and capable of interacting so as to lead to formation of a binder: analyte complex (i.e., bound analyte).

[0047] The term "denaturant" includes a range of chaotropic and other chemical agents that act to disrupt or loosen the 3-D structure of proteins without breaking covalent bonds, thereby rendering them more susceptible to proteolytic treatment. Examples include urea, guanidine hydrochloride, ammonium thiocyanate, trifluoroethanol and deoxycholate, as well as solvents such as acetonitrile, methanol and the like. The concept of denaturant includes non-material influences capable of causing perturbation to protein structures, such as heat, microwave irradiation, ultrasound, and pressure fluctuations.

[0048] The term "electrospray ionization" (ESI) refers to a method for the transfer of analyte molecules in solution into the gas and ultimately vacuum phase through use of a combination of liquid delivery to a pointed exit and high local electric field.

[0049] The term "elution" means a process that separates a bound analyte from a binder, which can occur in solution (for example an acidic elution of peptide from an antibody by exposure to 5% acetic acid in water) or in an ionized gas phase (for example when a peptide separates from an antibody after laser- induced desorption on a MALDI target).

[0050] The term "immobilized enzyme" means any form of enzyme that is fixed to the matrix of a support by covalent or non-covalent interaction such that the majority of the enzyme remains attached to the support of the membrane.

[0051] The term "measured amount" used in reference to an amount of an analyte or internal standard means an amount that is either i) measured in relation to a known amount of an appropriate internal standard added to the sample to be measured (for example using a stable-isotope labeled version of an analyte, e.g., a SIS as defined below, that is spiked into a sample in an amount that is known or inferred based on external measurements); ii) measured to be in a specific quantitative relationship (e.g., a ratio) with an amount of analyte or internal standard present in a different sample or analysis (whether the ratio is measured before or after any specific event in a measurement procedure), an approach that includes the use of "calibrator" or "standard" samples separate from the sample to be measured; or iii) measured in terms of units established in a specific physical, chemical or biological measurement system, including for example inductively-coupled plasma-MS (ICP-MS) used to measure sulfur on an absolute molar scale in cysteine or methionine-containing peptides. It will be understood that the various steps of the actual determination, or computation, of the measured amount can take place before, during or after the physical analysis of any given sample.

[0052] The terms "magnetic particle" and "magnetic bead" are used interchangeably and mean particulate substances capable of carrying binding agents (whether attached covalently or non-covalently, permanently or temporarily) or serving other functions, and which can respond to the presence of a magnetic field gradient by movement. The term includes beads that are referred to as paramagnetic, superparamagnetic, and diamagnetic.

[0053] The terms "particle" or "bead" mean any kind of particle in the size range between lOnm and 1cm, and includes magnetic particles and beads.

[0054] The term "MALDI" means Matrix Assisted Laser Desorption Ionization and related techniques such as SELDI, and includes any technique that generates charged analyte ions from a solid analyte containing material on a solid support under the influence of a laser or other means of imparting a short energy pulse.

[0055] The term "Mass spectrometer" (or "MS") means an instrument capable of separating molecules on the basis of their mass m, or m/z where z is molecular charge, and then detecting them. In one embodiment, mass spectrometers detect molecules quantitatively. An MS may use one, two, or more stages of mass selection. In the case of multistage selection, some means of fragmenting the molecules is typically used between stages, so that later stages resolve fragments of molecules selected in earlier stages. Use of multiple stages typically affords improved overall specificity compared to a single stage device. Often, quantitation of molecules is performed in a triple-quadrupole mass spectrometer, but it will be understood herein that a variety of different MS

configurations may be used to analyze the molecules described, and specifically MALDI instruments including MALDI-TOF, MALDI-TOF/TOF, and MALDI-TQMS and electrospray instruments including ESI-TQMS and ESI-QTOF, in which TOF means time of flight, TQMS means triple quadrupole MS, and QTOF means quadrupole TOF.

"MRM" refers to a mode of detection in which a TQMS is used to quantitate a specific molecule by a two-stage sequential mass selection: selecting parent ion m/z in the first quadrupole, fragmenting the ion by collision with gas in a second quadrupole and finally selecting a specific fragment ion m/z in a third quadrupole, prior to ion detection. The prefix "LC-" means liquid chromatography when followed by an analytical measurement method such as MS (i.e., yielding the combined method LC-MS), MS/MS (yielding LC- MS/MS), or MRM (yielding LC-MRM), etc.

[0056] The term "monitor fragment" may mean any piece of an analyte up to and including the whole analyte that can be produced by a reproducible fragmentation process (or without a fragmentation if the monitor fragment is the whole analyte) and whose abundance or concentration can be used as a surrogate for the abundance or concentration of the analyte.

[0057] The term "monitor peptide" or "target peptide" means a peptide chosen as a monitor fragment of a protein or peptide.

[0058] The term "MS run" or MS analysis means an operation of measuring amounts of molecules in a mass spectrometer. In the case of analysis using LC-MS, a run typically consists of a cycle of the liquid chromatography system including loading sample onto an analytical column, elution of analytes (typically using a solvent gradient) into an electrospray ionization source in the MS, and re-equilibration of the column. In the case of flow-injection MS, a run typically consists of a direct injection of sample, or else very rapid elution of sample from a small "trap" column without an extensive gradient separation, into an electrospray ionization source in the MS. In the case of MALDI-TOF, a run typically consists of the collection of data from multiple laser flashes directed at a region of a target to which a sample has been applied. In each case, the results of a run are typically obtained by integrating the amount of analytes in peaks in a time dimension (when an LC step is used to introduce samples into the MS) and/or in a mass dimension (when a mass-resolving MS analyzer such a TOF or orbitrap MS analyzer is used).

[0059] The term "multimode" means an analytical method that combines a plurality of two or more sample preparation modes including binder-based enrichment of small molecules, peptides resulting from proteolytic digestion, small molecules freed from endogenous binders, small molecules converted from an endogenous form to another chemical form for measurement, and small molecules that participate in a coupled series of chemical reactions culminating in generation of a surrogate analyte. In particular the term includes the modes of sample preparation and analysis described in the embodiments of the present invention.

[0060] The term "Natural" or "Nat" means the form of such a peptide that is derived from a natural biological sample by proteolytic digestion, and thus, contains

approximately natural abundances of elemental isotopes. Nat peptides typically do not contain appreciable amounts of a stable isotope label such as is intentionally incorporated in SIS internal standards.

[0061] The term "panel" means a set of two or more analytes measured together. In general the components of a panel are measured together because the combination of individual results provides superior information to the end user compared to a single analyte. In some cases panels consist of several analytes each of which is known to be associated with a disease process (e.g., Apo A-I and Apo B lipoproteins with CRP as contributors to cardiovascular risk), or the combination may together provide a statistically meaningful result where none of the components appear individually significant.

[0062] The term "patient" or subject refers to any person, animal or biological entity from which a sample is obtained for analysis. Patients can be persons under medical care, athletes, persons judged to be at risk for disease, or anyone who has an interest for whatever reason in learning or using the results of analytical tests pertaining to

themselves or someone else.

[0063] The term "proteolytic enzyme cleavage site" refers to a site within an extended SIS peptide sequence at which the chosen proteolytic treatment (typically an enzyme such as trypsin) cleaves the extended SIS sequence, releasing peptides fragments (typically two) of which one is the SIS peptide sequence (identical to the analyte, or Nat, sequence for which the SIS serves as an internal standard).

[0064] The term "proteolytic treatment" or "enzyme" may refer any of a large number of different enzymes, including trypsin, chymotrypsin, lys-C, v8 and the like, as well as chemicals, such as cyanogen bromide. In this context, a proteolytic treatment acts to cleave peptide bonds in a protein or peptide in a sequence- specific manner, generating a collection of shorter peptides (a digest).

[0065] The term "proteotypic peptide" means a peptide whose sequence is unique to a specific protein or restricted group of closely-related proteins in an organism, and therefore may be used as a stoichiometric surrogate for the protein (or family), or at least for one or more forms of the protein in the case of a protein with splice variants.

[0066] The term "sample" means any complex biologically-generated sample derived from humans, other animals, plants or microorganisms, or any combinations of these sources. "Complex digest" means a proteolytic digest of any of these samples resulting from use of a proteolytic treatment. [0067] The terms "SIS", "stable isotope standard" and "stable isotope labeled version of an analyte" mean a molecule that is identical or substantially identical to that of a selected analyte, and includes a label of some kind (e.g., a stable isotope) that allows its use as an internal standard for mass spectrometric quantitation of the natural (unlabeled, typically biologically generated) version of the analyte (see US Patent No. 7,632,686 "High Sensitivity Quantitation of Peptides by Mass Spectrometry"). Any molecule that contains the SIS, and from which the SIS is liberated by one or more treatment steps of the workflow or method, can be used as a SIS. Hence a SIS is 1) recognized as equivalent to the analyte in a pre-analytical workflow, and is not appreciably

differentially enriched or depleted compared to the analyte prior to mass spectrometric analysis, and 2) differs from it in a manner that can be distinguished by a mass spectrometer, either through direct measurement of molecular mass or through mass measurement of fragments (e.g., through MS/MS analysis), or by another equivalent means. SIS molecules typically include sites at which stable isotope (e.g., 13C, 15N, 180 or 2H) are present a high level of substitution (> 95%, > 96%, > 97%, > 98% or > 99%) at the specific sites within the SIS structure where the isotope(s) is/are incorporated (i.e., those sites that depart significantly from the natural un-enriched isotope

distribution). The term SIS is also used herein to indicate stable isotope labeled versions of peptide, protein, and non-peptide small molecule analytes (hormones, metabolites, drugs, etc.) of the same or similar structure as a target analyte.

[0068] The terms "SIS-Peptide", "labeled reference peptide", "peptide stable isotope standard" and "stable isotope labeled version of a peptide or protein analyte" mean a peptide or protein, such as a peptide or protein having a unique sequence that is identical or substantially identical to that of a selected peptide or protein analyte, and including a label of some kind (e.g., a stable isotope) that allows its use as an internal standard for mass spectrometric quantitation of the natural (unlabeled, typically biologically generated) version of the analyte (see US Patent No. 7,632,686 "High Sensitivity

Quantitation of Peptides by Mass Spectrometry"). In one embodiment, a SIS peptide or protein comprises a peptide sequence that has a structure that is chemically identical to that of the molecule for which it will serve as a standard, except that it has isotopic labels at one or more positions that alter its mass. A SIS can be a peptide of the same structure as the selected target signature peptide (made for example by chemical synthesis), or it can be a larger molecule including additional amino acid residues on either (or both) n- or c-terminal ends such that it is cleaved during the workflow to yield the target peptide sequence in labeled form, or it can be a subsequence of a protein of any size, including a labeled version of the intact parent protein from which the target peptide is derived during the workflow. Any molecule that contains the target peptide sequence, wherein that peptide sequence includes sites substituted with a stable isotope label at high occupancy, and from which the target sequence is liberated by one or more treatment steps of the workflow or method, can be used as a SIS. Hence a SIS is 1) recognized as equivalent to the analyte in a pre-analytical workflow, and is not appreciably differentially enriched or depleted compared to the analyte prior to mass spectrometric analysis, and 2) differs from it in a manner that can be distinguished by a mass spectrometer, either through direct measurement of molecular mass or through mass measurement of fragments (e.g., through MS/MS analysis), or by another equivalent means. Stable isotope standards include peptides having non-material modifications of this sequence, such as a single amino acid substitution (as may occur in natural genetic polymorphisms), substitutions (including covalent conjugations of cysteine or other specific residues), or chemical modifications (including glycosylation, phosphorylation, and other well-known post- translational modifications) that do not materially affect enrichment or depletion compared to the analyte prior to mass spectrometric analysis. In one embodiment, SIS peptides are generated by chemical synthesis or by in vitro or in vivo biosynthesis so as to produce a high level of substitution (> 95%, > 96%, > 97%, > 98% or > 99%) of each stable isotope (e.g., 13C, 15N, 180 or 2H) at the specific sites within the peptide structure where the isotope(s) is/are incorporated (i.e., those sites that depart significantly from the natural un-enriched isotope distribution). When used as a quantitative internal standard in the methods described herein, it is typically important to establish the quantity of SIS- Peptide that is added to standardize a test sample. This quantity can be established, e.g., by amino acid analysis, prior to addition of a known molar amount of SIS-Peptide to a sample, or it can be established later using measurements carried out on in parallel on standard samples that contain a known, or calibrating, amount of the peptide.

[0069] The terms "SIS-SMA", "labeled reference SMA", "stable isotope standard small molecule" and "stable isotope labeled version of a small molecule analyte" mean a small molecule, such as a metabolite, drug, hormone, and the like having a unique structure that is identical or substantially identical to that of a selected small molecule analyte, and including a label of some kind (e.g., a stable isotope) that allows its use as an internal standard for mass spectrometric quantitation of the natural (unlabeled, typically biologically generated) version of the analyte. In one embodiment, a SIS-SMA comprises a structure that is chemically identical to that of the molecule for which it will serve as a standard, except that it has isotopic labels at one or more positions that alter its mass. A SIS-SMA can be a molecule of the same structure as the selected target small molecule (made for example by chemical synthesis), or it can be a larger molecule including additional atoms that it is modified during the workflow to yield the target small molecule in labeled form, or it can be a component of a larger molecule from which the SIS-SMA is derived during the workflow. Any molecule that contains the SIS-SMA, wherein that SIS-SMA includes sites substituted with a stable isotope label at high occupancy, and from which the SIS-SMA is liberated by one or more treatment steps of the workflow or method, can be used as a SIS-SMA. Hence a SIS-SMA is 1) recognized as equivalent to the analyte in a pre-analytical workflow, and is not appreciably differentially enriched or depleted compared to the analyte prior to mass spectrometric analysis, and 2) differs from it in a manner that can be distinguished by a mass spectrometer, either through direct measurement of molecular mass or through mass measurement of fragments (e.g., through MS/MS analysis), or by another equivalent means. Stable isotope standards include molecules having non-material structural modifications that do not materially affect enrichment or depletion compared to the analyte prior to mass spectrometric analysis. In one embodiment, SIS-SMA are generated by chemical synthesis or by in vitro or in vivo biosynthesis so as to produce a high level of substitution (> 95%, > 96%, > 97%, > 98% or > 99%) of each stable isotope (e.g., 13C, 15N, 180 or 2H) at the specific sites within the SIS-SMA where the isotope(s) is/are incorporated (i.e., those sites that depart significantly from the natural un-enriched isotope distribution). When used as a quantitative internal standard in the methods described herein, it is typically important to establish the quantity of SIS-SMA that is added to standardize a test sample. This quantity can be established, e.g., by gravimetric or elemental analysis, prior to addition of a known molar amount of SIS-SMA to a sample, or it can be established later using measurements carried out on in parallel on standard samples that contain a known, or calibrating, amount of the SIS-SMA.

[0070] The term "SISCAPA" means the method described in US Patent No.

7,632,686, entitled High Sensitivity Quantitation of Peptides by Mass Spectrometry, and in the publication Mass Spectrometric Quantitation of Peptides and Proteins Using Stable Isotope Standards and Capture by Anti- Peptide Antibodies (SISCAPA). Anderson, N.L., Anderson, N.G., Haines, L.R., Hardie, D.B., Olafson. R.W., and Pearson, T.W, Journal of Proteome Research 3: 235-44 (2004). [0071] The term "small molecule" means a multi-atom molecule other than a polymer of amino acids or nucleotides (i.e., composed of protein, DNA or RNA). The term "small molecule" thus includes but is not limited to small hormones (such as steroids and thyroxine), nutrients, vitamins, products of metabolism, amino acids, bile acids, glucose, heparin, metabolic intermediate compounds, drugs, drug metabolites, toxicants and their metabolites, and fragments of larger biomolecules.

[0072] The abbreviation "SMA" means a small molecule analyte that is the intended analyte of a measurement procedure.

[0073] The term "coupled SMA" means an SMA whose amount is measured in a sample indirectly, by means of one or a linked series of chemical, often enzymatic, reactions beginning with the coupled SMA (typically in the role of a substrate, coenzyme or catalyst) and generating, through the linked reactions, a different SMA that is then measured (e.g., by MS) as a quantitative surrogate of the coupled SMA. Reactions are linked when, for example, the product of one reaction serves as a substrate, coenzyme or catalyst in the next reaction. Such a chain of linked reactions converts the amount of the coupled SMA into an amount of a different, easily measured SMA that may contain no atoms from the coupled SMA and yet accurately reflect its amount in the sample. An example is the biochemical measurement of total serum triglycerides, in which

lipoprotein lipasejiydrolyzes triglycerides to glycerol and free fatty acids; glycerol kinase converts glycerol to G-l-P; glycerol phosphate oxidase converts this G-l-P to DAP and H202; and finally peroxidase catalyzes the coupling of H202 with Amp lex Red to yield resorufin, which can be detected colorimetrically or by MS.

[0074] The term "stable isotope" means an isotope of an element naturally occurring or capable of substitution in proteins or peptides that is stable (does not decay by radioactive mechanisms) over a period of a day or more. The primary examples of interest in this context are C, N, H, and O, of which the most commonly used are 2H, 13C, 180 and 15N.

[0075] The term "standardized sample" means a sample in which one or more internal standard substances (including SIS, SIS-Peptide and/or SIS-SMA) are present at levels that are known at the time of addition, or subsequently determined by some means (including comparison before or after sample analysis with results obtained from analysis of other standard or calibrator samples) and thus serve as internal standards.

[0076] The term "undigested analyte" or "UA" means a molecule that is present in a sample but that is not the product of a proteolytic digestion of a sample protein. UA's include, but are not limited to, small molecules, metabolites, drugs and their metabolites, compounds absorbed or ingested from the environment, and nucleic acids (including micro RNA's and fragments of DNA, rRNA and mRNA).

[0077] The following embodiments of the present technology make use of a series of concepts described in this specification. These concepts provide perspective as to the need for improved methods in the art and specific embodiments of the technology described herein.

[0078] The SISCAPA Method. SISCAPA assays combine affinity enrichment of specific peptides with quantitative measurement of those peptides by mass spectrometry. In order to detect and quantitatively measure protein analytes, the SISCAPA technology makes use of anti-peptide antibodies (or any other binder that can reversibly bind a specific peptide sequence of about 5-20 residues) to capture specific peptides from a mixture of peptides arising from the specific cleavage of a protein mixture (like human serum or a tissue lysate) by a proteolytic enzyme such as trypsin or a chemical reagent such as cyanogen bromide. The selected peptides are generally chosen to be proteotypic (i.e. to uniquely represent a specific protein by having a sequence that does not appear in any other protein of the organism) and thus to be usable as quantitative surrogates for the parent protein or one of its forms. In many preferred embodiments the proteolysis process involves denaturation of the sample proteins (typically using urea, deoxycholate, heat, etc.), reduction and alkylation of protein cysteine residues, and digestion with trypsin (after dilution, if necessary to reduce the concentration of denaturant to a level compatible with activity of the trypsin). At the conclusion of the digestion step of the SISCAPA method (which may include addition of a trypsin inhibitor to stop protein digestion), the sample still contains its original non-protein constituents (including small molecules, DNA, etc.), while the proteins have been destroyed and converted to peptides. In addition, the composition of the digest, and its pH, remain compatible with the subsequent activity of both i) antibodies (and other binders) needed to capture sample molecules for analysis, and ii) other enzymes that may be used to accomplish chemical conversion of peptides, DNA or small molecules into forms amenable to subsequent analysis.

[0079] By capturing a specific peptide through binding to a binder (the binder being typically coupled to a solid support either before or after peptide binding), followed by washing of the bindenpeptide complex to remove unbound peptides (thereby achieving a separation of the bound and unbound peptides), and finally elution of the bound peptide into a small volume (typically achieved by an acid solution such as 5% acetic acid or 1% formic acid), the SISCAPA technology makes it possible to enrich specific peptides that may be present at low concentrations in the whole digest, and therefore undetectable in simple mass spectrometry (MS) or liquid chromatography- MS (LC/MS) systems against the background of more abundant peptides present in the mixture. It also provides a sample that is much less complex, and therefore exhibits reduced 'matrix effects' and fewer analytical interferences, than the starting digest, which in turn enables use of shorter (or no) additional separation processes to introduce samples into a suitable mass spectrometer. This enrichment step is intended to capture peptides of high, medium or low abundance and present them for MS analysis: it therefore discards information as to the relative abundance of a peptide in the starting mixture in order to boost detection sensitivity. This abundance information, which is of great value in diagnostics and in the field of proteomics, can be recovered, however, through the use of isotope dilution methods: the SISCAPA technology makes use of such methods (e.g. , by using stable isotope labeled versions of target peptides as internal standards) in combination with specific peptide enrichment, to provide a method for quantitative analysis of peptides, including low-abundance peptides.

[0080] The capture of analytes by specific binders followed by removal of unbound species (for example by washing magnetic beads to which the binders are attached) effectively enriches the analytes and separates them from the other components of a sample. Once this specific separation has been achieved, release of the bound analytes (for example by exposure to an elution solvent like 1% formic acid in water) from the binders yields very highly purified analytes. Such purified analytes, having already been separated from most other sample components by this specific affinity capture and washing, require less additional separation by chromatography, mass analysis or other separations than would be needed for unfractionated samples, which allows more sensitive and precise quantitative measurement.

[0081] It is an object of the present invention to extend the advantages of the SISCAPA method to the analysis of small molecule analytes (SMA) and to do this in a manner that is compatible with simultaneous SISCAPA analysis so as to allow measurement of both protein/peptide and SMA bio markers in the same workflow and ideally at the same time.

[0082] Small Molecule Measurement by Mass Spectrometry. Numerous published methods exist for the measurement of small molecules by mass spectrometry, including clinical biomarkers (such as thyroxine, vitamin D, testosterone), drugs and their metabolites, environmental pollutants, and food adulterants. An increasing variety of clinical MS tests are offered by leading clinical reference laboratories and a large contract research (CRO) industry has grown up providing MS-based analytical services to the pharmaceutical industry and to government regulators.

[0083] In several instances, investigators have used antibody-based specific affinity enrichment to facilitate small molecule measurement by MS. For example Strathmann et al (Quantification of 1,25-Dihydroxy Vitamin D by Immunoextraction and Liquid Chromatography-Tandem Mass Spectrometry. Clin Chem 57, 1279-1285 (2011)) enriched 1,25-dihydroxyvitamin D from human samples that had been fractionated by precipitation to remove proteins. In this case the capture of vitamin D by a specific antibody served to enrich the analyte from a large volume of supernatant containing only small molecules (i.e., human serum or plasma after an organic protein crash step, and removal of the soluble supernatant).

[0084] A number of published reports described the enrichment of small molecules from urine. Rule (Determination of drugs from urine by on-line immunoaffinity chromatography-high-performance liquid chromatography-mass spectrometry. Rule, G. S. & Henion, J. D. J Chromatogr 582, 103-112 (1992)), Cai (Quantitative multi-residue determination of beta-agonists in bovine urine using on-line immunoaffinity extraction- coupled column packed capillary liquid chromatography-tandem mass spectrometry. Cai, J. & Henion, J. J Chromatogr B Biomed Sci Appl 691, 357-370 (1997)), and Davoli (Purification and analysis of drug residues in urine samples by on-line immunoaffinity chromatography/high-performance liquid chromatography/continuous-flow fast atom bombardment mass spectrometry. Davoli, E., Fanelli, R. & Bagnati, R. Anal Chem 65, 2679-2685 (1993)) enriched drugs from urine, while Creaser (Immunoaffinity

chromatography combined on-line with high-performance liquid chromatography-mass spectrometry for the determination of corticosteroids. Creaser, C. S., Feely, S. J., Houghton, E. & Seymour, M. J Chromatogr A 794, 37-43 (1998)) enriched

corticosteroids from urine. Urine has a very low protein concentration compared to blood or tissues, and thus resembles crashed plasma in presenting small molecules in a matrix with very low protein concentration. In each of these methods the analytes measured by MS were limited to a single class of small molecules, and in each case any proteins that were present in the samples analyzed were large compared to the SMA's. [0085] What is Lacking in Current Technology. Available methods for MS measurement of bio markers focus on either peptides (including peptides derived from digestion of proteins) or on small molecules, but not both in the same method. Small molecules in the sample have typically been considered interferents (i.e., interfering substances) in conventionally protein assays: heme (present in hemolyzed serum) and bilirubin (present in jaundiced samples) present challenges for correct operation of colorimetric immunoassays because of their optical absorbance. Conversely, proteins frequently interfere with measurement of small molecules by binding or sequestering an unknown fraction of the SMA (rendering the bound SMA's unavailable for detection): serum albumin for example binds variable amounts of many drugs and metabolite SMA's in blood, causing the total amount of SMA to be different from the amount available free in solution. For these reasons most current methods for small molecule analysis by MS utilize a "protein crash" step (typically addition of an amount of organic solvent that precipitates the proteins) to remove essentially all protein from the sample before MS analysis for small molecules. The advantage of this approach is that, by removing proteins, most of the dissolved mass in samples like serum or plasma is removed, effectively enriching the small molecules very significantly, while the solvent also tends to dissociate small molecules from protein carriers to which they may have been bound. The disadvantage, however, is that by such an approach, proteins have been removed from the sample and rendered unsuitable (as precipitates) for many protein analytical methods such as immunoassay.

[0086] Numerous clinical situations require measurement of both protein and small molecule analytes, and therefore it would be advantageous to have a single method capable of dealing with both. In particular, when a limited amount of sample is available it would be beneficial to measure multiple analytes in the same volume of sample rather than, as is current practice, to measure different analytes in different aliquots of the sample, each of which is of necessity only a fraction of the small volume available and thus contains a reduced amount of analyte available for detection. The small volumes of pediatric blood samples, or dried blood spot samples, thus present major challenges for measurement of multiple analytes when each assay requires a separate volume of sample.

[0087] Many of the available small molecule measurement methods require dissociation of a small molecule from one or more binding protein(s) to make it accessible to measurement (e.g., dissociation of thyroxine from the thyroxine binding globulin and other proteins to measure its total amount in serum; dissociation of vitamin D from vitamin D binding protein; or the dissociation of steroid hormones from the sex hormone binding globulin). Many SMAs bind to multiple, often unidentified, proteins in blood. In many cases SMA measurement thus requires an inconvenient extraction step in the workflow (e.g., protein precipitation with organic or acidic extraction to liberate the analyte) that effectively partitions the sample into a portion that is used and a discarded portion that may still contain valuable analytes. In general these extraction methods are not directly compatible with methods used to measure proteins in the same sample.

[0088] Many important small molecule measurement methods require enzymatic conversion of the small molecule into a modified form prior to measurement (e.g., the conversion of cholesterol esters to cholesterol prior to total cholesterol measurement). In some cases an extended series of "coupled" chemical reactions is used to convert the small molecule analyte amount into an amount of a completely different, easily detected analyte (e.g., total serum triglyceride measurements in which lipoprotein lipase hydrolyzes triglycerides to glycerol and free fatty acids; glycerol kinase converts glycerol to G-l-P; glycerol phosphate oxidase converts this to DAP and H₂O₂; and finally peroxidase catalyzes the coupling of H₂O₂ with Amp lex Red to yield resorufin, which can be detected colorimetrically). While such biochemical (enzymatic) assays are considered robust and very inexpensive, they are not directly compatible with methods used to measure proteins in the same sample: this is why such enzymatic SMA assays are not conducted in the same sample volume as protein assays (either conventional

immunoassays or MS assays).

[0089] Current practice therefore teaches away from a combination of protein/peptide and SMA assays in a single sample. In contrast, the method presented here offers a series of specific advances over the current state of the art, namely:

[0090] 1) measurement of proteins (which can be of any size up to and including more than lmillion Dalton mass, e.g., mucins such as CA125) and SMAs (which can have masses down to the level of single amino acids or below) in a single workflow, which is to say, in a series of stepwise manipulations. The ability to measure one or more proteins and one or more SMAs in a unified workflow based on the same concepts of specific binder enrichment (implemented for example using antibodies as binders on easily-manipulated magnetic beads) with mass spectrometric detection substantially simplifies the technology platform required carry out a broad menu of analyses. This combination makes possible a "general purpose" biomarker platform capable of detecting and measuring most of the clinically-important analytes other than nucleic acids (which are generally detected by qualitative next-generation sequencing techniques, rather than by quantitative methods).

[0091] 2) measurement of proteins and SMAs on the same sample. The ability to measure this wide variety of analytes in a single sample (as opposed to current methods that generally measure each analyte in a separate volume, or aliquot, of a sample) reduces the total amount of sample required. Two important consequences arise from this capability. First, it allows measurement of more analytes in the limited amounts of sample obtainable from pediatric patients, small animals, or in dried blood spots obtained by fingerprick. Second, it improves analytical sensitivity by allowing all of an analyte present in the whole sample to be presented to the MS for detection, rather than the smaller amount of analyte present in one aliquot of a similar sample divided between separate assays. Since sensitivity is critical in many clinical applications, for example in the accurate measurement of vitamin D or of testosterone in women, this advantage of access to a larger sample volume enables better clinical results.

[0092] 3) measurement of proteins and SMA's in a single MS operation, rather than separate runs, thereby decreasing analysis time. It has been common in the art to measure SMAs by LC-MS methods that are similar but operationally distinct: there appear to be no published precedents for MS detection and measurement of a peptide and a non- peptide SMA in the same 'injection' (or 'run') of an LC-MS system. Thus accomplishing measurement of both classes of analytes in a single injection can, in the general case, provide both results in less time (e.g., half the time for a simple case) that would be required for separate analysis. Published methods described analysis of various SMAs in ~3min LC-MS operations, and other publications describe measurement of a multiplex panel of peptides also in a ~3min LC-MS operation. If the two can be combined, both results can be obtained in ~3min, thus reducing by half the time (and cost) of the measurements.

[0093] The capture of the SMA and peptide analytes can be carried out at separate times (e.g., through serial capture events in which a sample is exposed first to one binder, which then removed, followed by exposure to a second binder) or at one time (e.g., using a combination of the binders). Likewise the elution of the SMA and peptide analytes from their respective binders can be carried out separately (if they have been captured separately), or together (if they have been captured in one combined binding reaction, or if separately captured binders are pooled before elution). And finally the SMA and peptide analytes can be measured by MS separately, if they have been captured and eluted separately, or they can be analyzed together in one MS run. In each case a user can decide whether to carry out actions for different analytes separately or in combination based on factors such as cost (e.g., to economize on reagents and analysis time) and assay performance required.

[0094] A surprising feature observed in combining the SISCAPA method with SMA measurement by this approach is that SMAs can be effectively enriched by available binders from extremely complex proteolytic digests of samples such as body fluids. While it is known in the art that SMAs can be detected by MS directly in fractionated body fluids depleted of proteins (e.g., by precipitation with organic solvents), or after enrichment from whole body fluids by capture on a specific binder, the enrichment of SMAs from the much more complex mixture resulting from proteolytic digestion of a sample like a body fluid has not been demonstrated or anticipated. This is because a proteolytic digest of an unfractionated biological sample contains vastly more molecules of small and intermediate size (i.e., the proteolytic peptides derived from the digested proteins) than the original sample, and thus contains many more potentially- interfering substances capable of disrupting either the specific binding (and capture) of an SMA or the specific MS detection of that SMA. The method and results presented here demonstrate, however, that a binder for an SMA can in fact capture that SMA from a proteolytic digest of whole blood that contains hundreds of thousands of tryptic peptides not present in the original blood sample.

[0095] It is an object of the present invention to enable various small molecule measurement approaches to be carried out in concert with each other and with protein measurements on the same sample.

[0096] It is a further object of the present invention to enable combinations of small molecule and protein measurement methods that measure both total and protein-bound small molecules.

[0097] It is a further object of the present invention to enable measurement approaches that employ one or more chemical or enzymatic transformations to be carried out on one or more analytes prior to MS measurement, and to do so in such a way that other measurement modes (e.g., of proteins) are unaffected.

[0098] It is a further object of the invention to enable measurement of small molecule biomarkers in a method compatible with measurement of protein biomarkers using the SISCAPA technology.

[0099] Embodiments [00100] The following paragraphs summarize various components of the embodiments described below and combined in various groups in a single integrated workflow.

[00101] 1. In a first embodiment, a "free" version of a small molecule analyte (SMA) is captured from a biological sample such as human blood serum using a binder (which may be a specific antibody, many of which have been developed to provide specific immunoassays for many SMA's) having specific affinity for the SMA. The binder can be immobilized on a chromatographic column, on magnetic or non-magnetic beads, or by other equivalent methods that allow the binder to be removed from contact with the sample (or the sample removed from contact with the binder) after it has been incubated with the sample long enough to allow capture of the SMA.

[00102] An example of this embodiment is the measurement of free thyroxine (T4) in human serum. The measurement process can be standardized by the addition of an internal standard (e.g., a SIS-SMA) in a measured amount prior to the capture of T4 by the binder, where the SIS-SMA is a molecule very similar to the T4 and is captured by the binder with equal efficiency. The most preferred internal standard is one whose chemical structure is identical to the T4 but whose mass is different (hence rendering it

distinguishable by a mass spectrometer, but not by the binder) as a result of the substitution of one or more T4 atoms with a stable isotope such as deuterium (2H) or 13C (allowing use of the well-known isotope dilution method of quantitation). It is preferred that the stable isotope substitution occurs with high efficiency (>95%) at specific sites in the molecule, so that only a very small amount of unlabeled version contaminates the internal standard. Here a preferred standard is deuterated L-thyroxine (T4-d5) commercially available from IsoSciences.

[00103] A specific binder to T4 (many are commercially available, including Abeam ab30833 antibody) is attached to magnetic beads (Dynal Dynabeads G), and these beads are added to a serum sample and incubated to allow the binder to capture T4 and SIS- SMA (T4-d5) that is free in solution (i.e., not bound by an endogenous binding protein). Then the beads, carrying the binder and its cargo of captured T4 and T4-d5, are removed from the serum sample and washed to remove any no n- specifically bound materials (this washing reduces so-called matrix interference in later MS detection). The now-isolated T4 and T4-d5 are dissociated from the binder using acidic solvent and then measured using liquid chromatography mass spectrometry (LC-MS/MS) following the approach of Soldin (The measurement of free thyroxine by isotope dilution tandem mass

spectrometry. Clin Chim Acta 358, 113-118 (2005).). In this approach the measured MS peak area observed by LC-MS/MS for T4 is divided by the measured MS peak area observed by LC-MS/MS for T4-d5 (for which the MS signal is observed at 5 dalton higher mass) and multiplied by the known amount or concentration of the T4-d5 SIS- SMA to obtain the amount of free T4 in the sample.

[00104] The remaining serum sample, containing all its original components other than the free T4 removed on the antibody-coated beads, may be further analyzed, for example using the SISCAPA procedure for measurement of proteins (e.g., thyroxin-binding globulin measured using the peptide SILFLGK (SEQ ID No. 1) and thyroid stimulating hormone measured using peptide YALSQDVCTYR; SEQ ID No. 2). In this case, labeled SIS internal standard signature peptides are added in measured amounts to the sample, either before (preferred) or after the addition, incubation and removal of the anti-T4 binder. Once the SISCAPA workflow, including protein denaturation, sulfhydral reduction, alkylation, tryptic digestion, and signature peptide recovery using specific antipeptide antibodies has been completed, the captured peptides are eluted and subjected to MS measurement, yielding the ratio between endogenous target and SIS peptide versions for each target peptide and thus providing a standardized quantitative measurement of the target peptide amount.

[00105] In a preferred case, the magnetic beads carrying the anti-T4 binder (set aside prior to the SISCAPA steps) are added to the beads carrying the various anti-peptide antibodies resulting from the SISCAPA workflow, so that all the analytes (T4 and peptides) can be eluted and then subjected to LC-MS/MS analysis together. This combination of separate affinity eluates into one sample for MS analysis can improve sample throughput, since one liquid chromatography run is required rather than 2 or more.

[00106] "Free" levels of other analytes can also be measured by this approach, including particularly SMA's that bind to proteins in blood, either because of a physiologically useful carrier protein or apparently non-specific binding to proteins like serum albumin. A notable example is vitamin D, much of which is bound by vitamin D binding protein in blood.

[00107] 2. In a second embodiment, an SMA is captured from the sample using the same approach as used above for T4, but in this case after the digestion stage of the SISCAPA process - i.e., carried out on a serum sample following proteolytic digestion of its proteins to peptides, a step which generally destroys the sample proteins' ability to bind small molecules but does not affect the structure or amount of other components such as small molecules. This elimination of the proteins' ability to interact in a stereo specific manner is the key to a major advantage of the SISCAPA method, and has been used to successfully eliminate assay interferences caused by protek protein interactions in conventional immunoassays for thyroglobulin (Tg), where some patients have endogenous autoantibodies to Tg, and insulin-like growth factor 1 (IGF-1), where most IGF-1 in the blood is tightly bound to IGF binding proteins. Thus, after digestion, all the small molecules are expected to be 'free' in solution, available for binding by an immobilized SMA-specific binder. That proportion of an SMA (e.g., T4) that was "protein bound" in the initial sample is now free because there are no more intact proteins present. The use of proteolytic digestion to accomplish this liberation of SMA is independent of whether digested peptides are also recovered for analysis (as would be the case when using the SISCAPA method). Free (embodiment 1) and total (the present embodiment) SMA measurements may be equivalent if none of the SMA is bound to protein in the sample.

[00108] In some cases, it is convenient to capture only a small proportion of the SMA (e.g., in order to economize on the amount of binder required) while still obtaining an accurate measurement of the total amount of the SMA. This is made possible by the addition, prior to the addition of binder to capture the SMA, of a measured amount of labeled internal standard version of the SMA (for example the deuterated T4 referenced above when total T4 is the target analyte) to serve as internal standard.

[00109] Using this approach, it is possible to measure total T4 in the sample (if the free T4 was not removed by a prior capture as described in the first embodiment) or else to measure bound T4 (if most or all of the free T4 was previously removed from the sample).

[00110] 3. In a third embodiment, the methods of the first and second embodiments are combined to provide measurements of both free and total SMA through the use of two differentially labeled SIS-SMAs. For example, free T4 is measured as in the first embodiment using L-thyroxine (tyrosine ring 13C6) as SIS-SMA-1 internal standard, followed by protein digestion and SISCAPA measurement of peptides from relevant proteins, followed by a second measurement of T4 (consisting of remaining free T4 not captured and removed in the first T4 measurement plus the liberated previously protein- bound T4 present in the now-digested sample) using a separately added SIS-SMA-2 such as L-thyroxine (tyrosine ring 13C12). In this second T4 measurement, three molecules are separately measured by LC-MS/MS for completeness: the T4 analyte, SIS-SMA-1 and SIS-SMA-2 (both SIS-SMAs having been added in measured amounts, SIS-SMA- 1 having been added before the initial T4 capture, and SIS-SMA-2 added after the initial T4 capture but before the second T4 capture. The ratio of T4 to SIS-SMA-2 multiplied by the amount of SIS-SMA-2 added gives the amount of T4 in the sample at the time of the second measurement. Since this amount would include any of the free T4 that was not removed by the first T4 capture, it is useful to also measure the remaining SIS-SMA- 1 and by comparing this with the SIS-SMA-2 (added after the first capture and therefore still present in a known amount) to determine the fraction of SIS-SMA- 1 (equal to the fraction of free T4) removed in the first capture. Knowing the amount of free T4 in the sample (from the first T4 measurement) and the fraction removed in the first capture, the amount of free T4 remaining in the sample after the first capture can be computed. This remaining free T4 can then be subtracted from the total T4 measured in the second capture (using the SIS-SMA-2 internal standard) to yield the total bound T4. Total T4 can be calculated as the free T4 plus bound T4.

[00111] 4. In a fourth "conversion" embodiment, an SMA is converted from one chemical form to another, either enzymatically or as a result of chemical reactions, prior to capture by a binder. Cholesterol, for example, appears in human serum both as the parent compound (cholesterol) and as a series of cholesteryl esters. In order to measure the clinically important amount of total cholesterol, it is desirable to convert the cholesteryl esters into their constituents (cholesterol and various fatty acids) so as to be able to measure all the cholesterol as a single molecular entity. This can be accomplished by addition of an enzyme (e.g., a cholesteryl esterase, available from many commercial sources) that liberates the free cholesterol. Following this enzymatic conversion of the cholesterol esters to cholesterol, a binder can be used to specifically capture and enrich the cholesterol. A measured amount of a SIS-SMA internal standard (in this case, for example, a d6 labeled version of cholesterol, such as item 488577 from Aldrich) can be added to the sample prior to the capture so that the amount of sample-derived cholesterol can be compared with the amount of the cholesterol standard using a mass spectrometer, yielding a ratio measure of the amount or concentration of cholesterol in the sample. When this internal standardization approach is used, and the result of the measurement is the ratio between endogenous and added SIS-SMA standard forms of cholesterol, it is not necessary to capture all of the cholesterol using a binder: since the binder does not distinguish between the labeled and unlabeled cholesterol forms, the fraction of total cholesterol and SIS-SMA captured on the binder preserves the cholesterol:SIS-SMA ratio present in the sample. Hence the amount of binder can be adjusted so as to capture that amount of the combined forms of cholesterol needed to provide the desired measurement precision in the MS analysis (this is frequently much less than the total amount of the cholesterol forms present in the sample).

[00112] The conversion step described in this embodiment may be included either before or after a proteolytic digestion step, or both (repeating the conversion). In a preferred case described here for cholesterol measurement, addition of the d6-cholesterol SIS-SMA occurs at the beginning of the workflow, thus providing an internal standard for all subsequent steps. The enzymatic conversion of cholesteryl esters to cholesterol and subsequent capture on anti-cholesterol antibodies is, however, preferably carried out after digestion, thereby ensuring that no intact sample proteins (e.g., the apolipoproteins) remain that could interfere with the availability of cholesterol esters for enzymatic conversion or capture.

[00113] A further elaboration of this embodiment allows measurement of both free and total cholesterol by including two separate Ch captures, one before and one after addition of the converting enzyme (e.g., the esterase) and both occurring after addition of the Ch- SIS, in a scheme that parallels the third embodiment.

[00114] 5. In a fifth "coupled" embodiment, an SMA analyte is transformed chemically (either enzymatically or by chemical reaction, through one or more sequential steps) in such a way as to generate, through coupled reactions, a stoichiometrically- related amount of a different compound that is amenable to direct measurement by MS. Taking the example of serum triglycerides, a conventional clinical assay modeled on classical methods (such as that described by Bucolo, G. & David, H. Quantitative determination of serum triglycerides by the use of enzymes. Clin Chem 19, 476-482 (1973)) follows an approach similar to that described for the Sigma- Aldrich TR0100 assay kit: "Triglycerides are first hydrolyzed by lipoprotein lipase to glycerol and free fatty acids. Glycerol is then phosphorylated by adenosine-5'-triphosphate (ATP) forming glycerol- 1-phosphate (G-l-P) and adenosine- 5 '-diphosphate (ADP) in the reaction catalyzed by glycerol kinase (GK). G-l-P is then oxidized by glycerol phosphate oxidase (GPO) to dihydroxyacetone phosphate (DAP) and hydrogen peroxide (H202). Peroxidase (POD) catalyzes the coupling of H202 with 4-aminoantipyrine (4-AAP) and sodium N- ethyl-N-(3-sulfopropyl) m-anisidine (ESPA) to produce a quinoneimine dye that shows an absorbance maximum at 540 nm. The increase in absorbance at 540 nm is directly proportional to triglyceride concentration of the sample." The final detection analyte in this case does not contain any material derived from the target analyte to be measured in the sample, but the enzymatic conversions are sufficiently reproducible or complete as to preserve a stoichiometric relationship between the initial target the final detection analyte. Analysis of calibration samples alongside test samples allows calibration of the assay and delivery of results in the desired measurement system: the efficiencies of the various enzymatic steps do not need to be 100%, merely consistent between test samples and standard "calibrator" samples. In the present embodiment, the last step can be modified so as to utilize peroxidase and the H₂O₂ created by the coupled reactions to convert the dye Amplex Red (Life Technologies) to the dye Resorufin with a loss of 44 amu mass. Commercially available antibodies (e.g., Life Technologies Fluorescein regon Green® Rabbit IgG Antibody Fraction Catalog number: A-889) can serve as a binder for the resulting resorufin (the coupled surrogate analyte), allowing it to be captured from the sample. As shown by Dierks (Dierks, E. A. et al. Drug Metabolism and Disposition 29, 23-29 (2001)), the resulting resorufin can be measured by LC-MS/MS, and the mass difference between Amplex Red and resorufin, together with differences in

chromatographic behavior, prevent any Amplex Red that is bound by the binder from interfering in the resorufin measurement. Stable isotope labeled d6-resorufin (Medical Isotopes, Inc. Cat. No. D18016) can be added at any stage prior to the antibody capture as a SIS-SMA internal standard in the MS measurement, providing a ratio measurement (enzymatically-generated unlabeled resorufin compared to a measured amount of labeled resorufin added as an internal standard spike). Measurement of this ratio by MS avoids the requirement to capture all of the generated resorufin using the binder, since only the ratio of two forms need be measured.

[00115] 6. In a sixth embodiment any two of the above embodiments are combined in a multimode method and executed on the same sample to yield multiple analytical results.

[00116] 7. In a seventh embodiment, the methods of the first (including SISCAPA peptide capture), third, fourth and fifth embodiments are combined into a multimode method capable of measuring a variety of analytes in one sample. For example, a method could include the following: 1) free T4, using pre-digestion capture of T4 from the sample; 2) a series of proteins (including e.g., thyroxin-binding globulin measured using the peptide SILFLGK (SEQ ID NO: 1); thyroid stimulating hormone measured using peptide YALSQDVCTYR (SEQ ID NO: 2); Apo A-I lipoprotein using peptide

ATEHLSTLSEK (SEQ ID NO: 3); and Apo B lipoprotein using peptide FPEVDVLTK (SEQ ID NO: 4)) measured after digestion using the SISCAPA method to capture the four indicated proteotypic peptides; 3) bound T4 using the method of the third embodiment; 4) total cholesterol using the "conversion" method of the fourth embodiment; and 5) total triglycerides by the "coupled" method of the fifth embodiment. In a preferred

embodiment, the binders for each of these analytes is immobilized on magnetic beads which are added to the sample, incubated and removed at the appropriate times in a continuous multi-stage workflow. At the end of the workflow, all the beads, with their binders and analyte cargoes, are preferably pooled and eluted together, resulting in an enriched sample containing T4, various peptides, cholesterol and resorufin (together with their respective internal standards). This analyte mixture is preferably analyzed in a single injection into a suitably optimized liquid chromatography tandem mass

spectrometry (LC-MS/MS) system, in which the analytes are separated (e.g. using reversed phase chromatography on C8 or C12 columns) prior to delivery into a triple quadrupole MS for quantitation using selected reaction monitoring methods specific for each analyte and internal standard.

[00117] Taken together this collection of analytes, including (or stoichiometrically representing) small molecule and protein biomarkers, provides a valuable panel for assessment of cardiovascular risk in human patients. Using the methods disclosed here, this panel may be measured less expensively than is currently the case when each analyte is measured in a separate assay using different methodologies. In addition, by measuring the panel in a single small aliquot of a patient's body fluid (e.g., serum, plasma, or whole blood) the analysis requires substantially less sample than required by conventional methods.

[00118] 8. In an eighth embodiment, the method of the seventh embodiment is carried out on a dried specimen such as a dried blood spot (DBS). Each of the analytes disclosed in the examples is stable in DBS and can be recovered from DBS samples for accurate measurement. Proteins in particular are well-measured by SISCAPA in DBS samples because the method is insensitive to protein denaturation, aggregation or unfolding due to drying the DBS (since the proteins are finally unfolded and digested during SISCAPA analysis).

[00119] It will be understood by those skilled in the art that, while capture of proteolytic peptides is done after a proteolysis step, the capture of SMAs can be carried out either before or after a digestions step (yielding measurements described here as "free" or "total" SMA measurements respectively), and with or without a chemical or enzymatic conversion step, and with or without a series of coupling reactions. It will also be understood that the various SMA and peptide analytes can be captured together at one time (e.g., using a mixture of appropriate binding agents) or separately (e.g., using individual capture agents to sequentially remove the respective analytes from a sample). Likewise the various SMA and peptide analytes can be eluted from their respective binding agents together at one time (e.g., using a mixture of appropriate binding agents) or separately (e.g., in the case where individual capture agents are used to sequentially remove the respective analytes from a sample). Finally it will be clear that the various SMA and peptide analytes can be analyzed by MS together at one time (e.g., using LC- MS/MS or MALDI-TOF to analyze a mixture of analytes in one MS run) or separately (e.g., by performing MS analysis on analytes eluted from individual capture agents eluted separately).

[00120] Table 1. Peptide sequences

[00121] Examples

[00122] Example 1: Measurement of Cholesterol (Ch; a small molecule) and Apolipoprotein B (ApoB; a very large protein) in a unified workflow. In the example a unified workflow is used to measure cholesterol, a prototypical small molecule of medical importance, and a proteotypic peptide of ApoB 100, both present in a sample of dried human blood (a dried blood spot; DBS). This workflow is modified (where indicated below) from a published protocol (High Precision Quantification of Human Plasma Proteins Using the Automated SISCAPA Immuno-MS Workflow. Razavi, M., Anderson, N.L., Pope, M. E., Yip, R. & Pearson, T. W., New Biotechnology (2016). doi: 10.1016/j.nbt.2015.12.008) to include SMA measurement.

[00123] Digestion protocol: A 903 Protein Saver Card (Whatman) containing dried human blood was punched two times using a DBS puncher with a diameter of 1/16" to provide a single sample in this protocol (each test sample contained two replicate DBS punches made using the same human whole blood sample, equivalent to a total of approximately 5 μΐ_^ of plasma). The two punches constituting a sample were placed in a well of a 96-well plate (Axygen; Deep Well P-DW- 11-C) and 67 μL· of the denaturation solution (9M urea, 0.2M Trizma pH 8.1, 0.03M tris-(2-carboxyehtyl)phosphine-TCEP in water/0.1% 3-[(3-Cholamidopropyl)dimethylammonio]-l-propanesulfonate-CHAPS) was added, followed by shaking at 1000 rpm for 30 minutes at room temperature. After the 30 minute shake, an aluminum microseal (BioRad; MSFIOOI) was placed on the plate and it was transferred to a heater/shaker incubator, which was set to shake (1000 rpm) at 40°C for 30 minutes. Iodoacetamide (40 μΐ_^ of 9.9 mg/mL solution in water) was then added to the well followed by 30 seconds of shaking at 1000 rpm. The plate was incubated in the dark for 10 minutes before the addition of 460 μΐ_^ of 0.2 Trizma (pH 8.1). The content of the well was mixed by shaking for 30 seconds at 1000 rpm. L-(tosylamido-2- phenyl)ethyl chloromethyl ketone (TPCK)-treated trypsin (Worthington; TRTPCK) was dissolved in 10 mM HC1 to a final concentration of 3.65 mg/mL. Forty microliters of the trypsin solution was added to the reaction followed by a 30 second shake at 1000 rpm. A fresh aluminum microseal was placed on the plate before transferring it an incubator at 37°C for 18 hours.

[00124] After the digestion, 40 μΐ_^ of 0.11 mg/mL tosyl-L- lysine chloromethyl ketone (TLCK, a trypsin inhibitor) in 10 mM HC1 was added to the reaction to stop trypsin activity.

[00125] All samples were spiked (after digestion and optional esterase addition) with 50 nmol of the stable isotope standard (SIS) version of Ch (cholesterol-[2,2,3,4,4,6-D6]; IsoSciences; S9196), whose precursor ion has a mass shift of +6 amu compared to unlabeled cholesterol.

[00126] All samples were spiked (after digestion and optional esterase addition) with 30 pmol of the stable isotope standard (SIS) version of the ApoB lOO peptide which contained a heavy labeled (U 13 C/U 15 N) lysine residue at the C terminus causing a mass shift of +8 amu compared to the endogenous ApoB lOO peptide.

[00127] Enrichment protocol: Commercially available polyclonal antibodies to cholesterol (antibodies-online.com; Product No. ABIN2120139 ) were used to capture Ch from the sample digest, while rabbit monoclonal antibodies to peptide FPEVDVLTK (SEQ ID NO: 4; proteotypic for ApoB; a product of SISCAPA Assay Technologies, Inc.) were used to simultaneously capture that peptide from the same sample digest. In each case, the antibodies were bound to protein G-coated magnetic beads (Dynabeads G;

ThermoFisher) prior to being exposed to the digest, then washed once (in phosphate- buffered saline/0.03% CHAPS) and finally the bound molecules were eluted (in 0.5% formic acid/0.03 %CHAPS in water) prior to analysis via LC-MRM mass spectrometry. In the test of the combination workflow, the Ch and ApoB antibodies were added to the sample, incubated and removed together (except as noted below).

[00128] MS analysis protocol: The LC-MS/MS platform consisted of an Agilent 1290 Infinity UHPLC coupled to an Agilent 6490 triple-quadrupole tandem mass spectrometer. A 20 μΐ_^ aliquot of the final eluate was separated on a 2.1 x 50 mm 1.8-micron Zorbax 300 SB-C8 column (Agilent Technologies; Part No. 857750-906). The flow rate was set at 600 μί/ητίη with 0.1% formic acid (FA) in HPLC-grade water as solvent A and 90% acetonitrile (ACN)/0.1% FA in HPLC-grade water as solvent B. A 10 minute gradient was optimized such that from the initial conditions of 3% B, a gradient was developed to 8% at 0.43 min, 13% at 1 min, 13% at 1.6 min, 13% at 2.1 min, 16% at 2.8 min, 16% at 3.1 min, 18% at 3.6 min, 90% at 4.5 min 90% at 6.5 min and then back to 3% until the end of the gradient (to re-equilibrate the column for the next run). The LC was configured for overlapping injections with a needle wash (70% ACN/0.1% FA in HPLC-grade water) in between injections. MS source conditions included drying gas at 200 °C, sheath gas at 250 °C and 11 L/min flow for both drying and sheath gases. Ions were isolated in Ql using 0.7 full width half maximum resolution followed by a Q3 separation with a 1.2 full width half maximum resolution. MassHunter Workstation Software (Agilent) was used for data acquisitions as well as data interpretation. Peptide FPEVDVLTK (SEQ ID NO: 4) and its SIS-Peptide version containing stable isotope labeled c-terminal lysine were measured by MRM using parent > fragment m/z parameters of 524.3 > 803.5 and 528.3 > 811.5, respectively. Cholesterol and its SIS-SMA Ch-d6 were measured using parent > fragment m/z parameters of 369.4 > 95.2 and 375.4 > 95.2, respectively.

[00129] Example 1 consists of 5 samples processed according to variations of the basic conversion protocol so as to illustrate (as shown in Figure 1) various features and capabilities of the combined SMA and peptide workflow: A) a capture reaction in digested DBS that includes antibody to ApoB 100 peptide but no antibody to Ch, B) a capture reaction in digested DBS that includes antibody to Ch but no antibody to

ApoB 100 peptide, C) a capture reaction in digested DBS that includes antibodies to both Ch and to ApoB 100 peptide, D) a capture reaction as in 'C but one in which 5 μΐ_^ of 1 mg/mL (1 unit equivalent) of cholesterol esterase (Sigma; C1403-25UN) dissolved in 0.4 M potassium phosphate (pH 7.0) was added to the sample followed by shaking at 1000 rpm for 15 minutes at 40 °C before trypsin digestion of the DBS sample and E) a capture reaction as in 'C but one in which the cholesterol esterase was added after trypsin digestion and quenching, followed by shaking at 1000 rpm for 15 minutes at 40 °C.

[00130] Panel A shows results of measuring three MRM transitions for the ApoB lOO peptide that clearly demonstrate its specific detection without significant interferences (i.e., any peaks other than the main peak), while no signal is detected for Ch in absence of relevant antibody (when the anti-Ch antibody is omitted from the protocol, Ch is not captured).

[00131] Panel B shows results of measuring two transitions for Ch that clearly demonstrate its specific detection without significant interferences (i.e., any peaks other than the main peak), while no signal is detected for ApoB lOO in absence of relevant antibody (when the anti-peptide antibody is omitted from the protocol, the peptide is not captured). These results clearly demonstrate that the SMA and peptide analyte peaks measured at the expected masses are as expected only observed when the respective antibody is used in the capture enrichment step.

[00132] Panel C shows results of measuring both ApoB 100 peptide and Ch when antibodies against both molecules are used simultaneously, capturing both. Taken together, the results shown in panels A, B and C demonstrate that both Ch and the ApoB peptide were captured by their respective antibodies from the same aliquot of processed sample, recovered in purified form, and separately measured in a single LC-MRM run (e.g., in panel C).

[00133] By measuring these two analytes in the same workflow, both were measured in the same small aliquot of sample (in the case of the DBS sample shown here, equivalent to approximately 5 μΐ of plasma), whereas conventional clinical analyses of these two analytes would require 20-200μ1 plasma. This economy of sample is important when sample volume is limited, as occurs with pediatric samples, with irreplaceable stored samples from large clinical studies, or with dried blood specimens that are often generated from single drops of blood. Both analytes were also measured in the same MS run, rather than requiring two separate analytical runs for independent methods. Addition of a second analyte to such an LC-MS/MS method often requires no increase in the LC cycle time, and thus adds essentially nothing to the LC-MS/MS analytical cost. Hence the combined SMA and protein method decreases both the sample volume requirement and the analytical time (and hence cost) compared to a conventional separate approach. [00134] In Panels B and C, the Ch that has been captured by the anti-Ch antibody and detected by the MS in that fraction of the sample Ch that was present in the 'free' form, i.e., not combined with fatty acids as cholesterol esters: the anti-Ch antibody is unlikely to efficiently bind cholesteryl esters, and even if it did, the MS detection parameters (parent and fragment masses) used would not detect these larger molecules.

[00135] To measure total Ch, the cholesteryl esters are converted to free Ch and fatty acids, for example using a cholesterol esterase enzyme, after which it is possible to recover the combined initially- free and initially-esterified Ch: i.e., the total Ch.

[00136] Addition of the esterase enzyme before digestion (Panel D), while the lipoproteins are in denatured state but not digested to peptides by trypsin, increases the free Ch in solution by approximately 3 fold in comparison to a situation where no esterase is added (Panel C).

[00137] Addition of the esterase enzyme after tryptic digestion (Panel E), i.e., after the lipoproteins are fully digested to peptides, increases the free Ch in solution by

approximately 4 fold in comparison to the circumstance where no esterase is added (Panel C). The esterase (a protein enzyme) is active after trypsin digestion because the trypsin's activity was eliminated by addition of trypsin inhibitor prior to addition of the esterase.

[00138] The results (Figure 1) demonstrate that the ratio of unlabeled Ch to Ch-SIS increases in the presence (D and E) of cholesterol esterase (compared to the case where esterase is absent in panel C), as expected due to the conversion of cholesteryl esters to cholesterol: addition of cholesterol esterase liberates fatty acid chains from cholesteryl esters hence increasing the amount of free cholesterol. The results shown in Panel E indicate that approximately 75% of the total cholesterol was initially present in the sample in esterified form (a level consistent with clinical expectations of 60-80% esterified cholesterol in normal human subjects). The slightly greater yield of Ch when esterase is added after tryptic digestion is likely to be due to slightly greater accessibility of the esterified Ch (to attack by the esterase) when the lipoprotein particles (in which Ch and the esters are carried in plasma) are effectively disrupted by destruction of the intact proteins (including ApoB) that determine their structures.

[00139] In this example the molar amount of Ch binding sites on the anti-Ch antibodies is a limiting factor, and these sites are effectively saturated in (B), (C), (D) and (E): hence the total amount of Ch + Ch-D6 captured by the antibody remains relatively constant in these samples while the ratio of Ch:Ch-D6 increases in presence of the cholesterol esterase (as expected in this case using the isotope dilution method for quantitation).

[00140] Example 2: Measuring total thyroxine and thyroxine-binding globulin together. Using the methodology described above, thyroxine (T4; an SMA) and thyroxine-binding globulin (TBG; the corresponding SMA binding protein) are measured in a sample of human plasma for the purpose of characterizing the level of thyroxine hormone available to tissues of the patient (which is influenced by both the total amount of SMA present and the amount of a binding protein that sequesters part of the SMA, buffering its available concentration). TBG is measured by LC-MRM quantitation of proteotypic peptide SILFLGK (SEQ ID NO: 1) in relation to a measured amount of a stable isotope labeled version (SIS) having the same peptide sequence but incorporating U13C-U15N-lysine, both labeled and unlabeled peptides being enriched from the digest by an antibody specific for the peptide sequence (as described in the general SISCAPA method). T4 is measured by LC-MRM quantitation of T4 in relation to a measured amount of a stable isotope labeled version of T4 incorporating 5 deuterium atoms (T4- d5), both SMAs being enriched from the digest by an antibody specific for T4.

Enrichment of both peptide and SMA are carried out at the same time using a mixture of the two antibodies on magnetic beads, after which the peptide and SMA are eluted from the corresponding antibodies and subjected to reversed phase chromatography followed by MRM quantitation in a triple-quadrupole MS (MRM detection parameters shown in Table 2.

[00141] Example 3: Measuring total estradiol and sex hormone binding globulin together. Using the methodology described above, estradiol (E2; an SMA) and sex hormone-binding globulin (SHBG; the corresponding SMA binding protein) are measured in a sample of human plasma for the purpose of characterizing the level of estradiol hormone available to tissues of the patient (which is influenced by both the total amount of SMA present and the amount of a binding protein that sequesters part of the SMA, buffering its available concentration). SHBG is measured by LC-MRM

quantitation of proteotypic peptide IALGGLLFPASNLR (SEQ ID NO: 6) in relation to a measured amount of a stable isotope labeled version (SIS) having the same peptide sequence but incorporating U13C-U15N-arginine, both labeled and unlabeled peptides being enriched from the digest by an antibody specific for the peptide sequence (as described in the general SISCAPA method). E2 is measured by LC-MRM quantitation of E2 in relation to a measured amount of a stable isotope labeled version of E2 incorporating 5 deuterium atoms (E2-d5), both SMAs being enriched from the digest by an antibody specific for E2. Enrichment of both peptide and SMA are carried out at the same time using a mixture of the two antibodies on magnetic beads, after which the peptide and SMA are eluted from the corresponding antibodies and subjected to reversed phase chromatography followed by MRM quantitation in a triple-quadrupole MS (MRM detection parameters shown in Table 2.

[00142] Example 4: Measuring total vitamin D and vitamin D binding globulin together. Using the methodology described above, l,25(OH)2D3 (an active form of vitamin D; an SMA) and vitamin D-binding globulin (VitDBG; the corresponding SMA binding protein) are measured in a sample of human plasma for the purpose of characterizing the level of l,25(OH)2D3 available to tissues of the patient (which is influenced by both the total amount of SMA present and the amount of a binding protein that sequesters part of the SMA, buffering its available concentration). VitDBG is measured by LC-MRM quantitation of proteotypic peptide HLSLLTTLSNR (SEQ ID NO: 5) in relation to a measured amount of a stable isotope labeled version (SIS) having the same peptide sequence but incorporating U13C-U15N-arginine, both labeled and unlabeled peptides being enriched from the digest by an antibody specific for the peptide sequence (as described in the general SISCAPA method). l,25(OH)2D3 is measured by LC-MRM quantitation of l,25(OH)2D3 in relation to a measured amount of a stable isotope labeled version of l,25(OH)2D3 incorporating 5 deuterium atoms (E2-d5), both SMAs being enriched from the digest by an antibody specific for l,25(OH)2D3.

Enrichment of both peptide and SMA are carried out at the same time using a mixture of the two antibodies on magnetic beads, after which the peptide and SMA are eluted from the corresponding antibodies and subjected to reversed phase chromatography followed by MRM quantitation in a triple-quadrupole MS (MRM detection parameters shown in Table 2.

Table 2.

Claims

What is claimed is:

1. A method for measuring in a single aliquot the amount of a small molecule

analyte (SMA) and a protein in a sample collected from a subject, comprising, in a single aliquot of sample:

contacting a standardized mixture comprising said sample, or a proteolytic digest of said aliquot, and a labeled reference SIS-SMA with an anti-SMA binder, wherein said anti-SMA binder specifically binds a preselected SMA in said digest and said SIS-SMA; separating SMA and SIS-SMA bound by said anti-SMA binder from unbound molecules, eluting SMA and SIS-SMA bound by said binder from said binder; measuring by mass spectrometry the amount of said preselected SMA and said SIS-SMA separated from said binder; and calculating the amount of said SMA in said sample, and

contacting a standardized mixture comprising a proteolytic digest of said aliquot and a labeled reference SIS-Peptide with an anti-peptide binder, wherein said anti-peptide binder specifically binds a preselected peptide in said digest and said SIS-Peptide; separating peptides bound by said binder from unbound peptides, eluting peptides bound by said binder from said binder; measuring by mass spectrometry the amount of said preselected peptide and said SIS-Peptide separated from said binder; and calculating the amount of said protein in said sample.

2. The method of claim 1, wherein the amounts are measured in a single MS run.

3. The method of claim 1, wherein one or more of the binders are antibodies.

4. The method of claim 1 wherein one or more of the binders are antibody

fragments.

5. The method of Claim 1, wherein said eluted SMA and SIS-SMA, and said peptide and SIS-Peptide are combined prior to mass spectrometric measurement.

6. The method of Claim 1, wherein said bound SMA and SIS-SMA, and said bound peptide and SIS-Peptide are combined prior to elution.

7. The method of Claim 1, wherein said SMA and SIS-SMA, and said peptide and SIS-Peptide are contacted with anti-SMA and anti-peptide binders in the same aliquot.

8. The method of Claim 1, wherein said SMA and SIS-SMA are captured by said anti-SMA binder prior to proteolytic digestion.

9. The method of Claim 1, wherein said SMA and SIS-SMA are captured by said anti-SMA binder after proteolytic digestion.

10. The method of Claim 1, wherein said SMA and peptide analytes are components of a single biomarker panel.

11. The method of Claim 1, wherein the protein is an SMA binding protein.

12. The method of claim 1, further comprising: converting a precursor of said SMA in the sample aliquot into said SMA by means of a chemical or enzymatic process.

13. The method of Claim 1, further comprising: generating said SMA in said sample through one or more coupled chemical or enzymatic reaction steps dependent on presence and amount of a different coupled SMA in said sample, and calculating the amount of said coupled SMA.

14. The method of Claim 1, further comprising a second SMA measurement, wherein the SMA and its SIS-SMA are captured by said anti-SMA binder prior to proteolytic digestion and then the SMA and an SIS-SMA are captured a second time by anti-SMA binder after proteolytic digestion, and wherein the SMA and SIS-SMA captured in each capture are analyzed separately.

15. The method of Claim 14, wherein two versions of SIS-SMA differing in mass are employed as internal standards in said first and second captures, and wherein the second SIS-SMA is added to the aliquot after the first capture has occurred and the associated anti-SMA binder has been separated from the aliquot.