Method for analyte quantification

Abstract

The present invention relates to a method for determination or quantification of an analyte by mass spectrometry, a reagent kit, and a diagnostic system for performing the method.

Description

[0001] Method for Analyte Quantification

[0002] Field of the Invention

[0003] The present invention relates to a method for determination or quantification of an analyte by mass spectrometry. Further the invention relates to a kit and diagnostic systems related thereto.

[0004] Background of the Invention

[0005] Mass spectrometry (MS) is a widely used technique for the qualitative and quantitative analysis of chemical substances ranging from small molecules to macromolecules. In general, it is a very sensitive and specific method, allowing even for the analysis of complex biological, e.g. environmental or clinical samples. However, for several analytes, especially if analysed from complex biological matrices such as serum, the measurement remains an issue and the workflows used are incompatible with automation and limits their throughput.

[0006] For example, serum thyroglobulin (TG / Tg) levels serves as a post treatment biomarker (after surgery or radioiodone ablation) for differentiated thyroid cancer (DTC). Immunometric analysis is the method of choice for quantitative Thyroglobulin measurement from serum. However, 20-30 % of patients with DTC produces Tg autoantibodies (TgAb) which interferes with accurate immunometric anaylsis (Hoofhagle A. and M.H. Wener, Clin Lab Int 8: 12-14 (2006)). This causes falsely low measurement of Thyroglobulin amount in patient serum. To overcome autoantibody interference, a number of LC-MS / MS based workflows were developed (Hoofnagle et al. 2008, Clarke et al, 2012, Kushnir et al 2013, Netzel et al 2015, Schuford et al. 2020, Patents: US 7,807,172 B2, US 9,140,695 B2). These methods employ enzymatic digestion of serum proteins and then affinity capture of a Tg specific peptide followed by LC-MS / MS analysis. However, these LC-MS / MS methods have certain limitations, which makes these workflows incompatible with automation and limits their throughput.

[0007] There is, as demonstrated above, still a need of improving the MS analysis methods, particularly for the analysis of analytes with automation for improving the throughput. The present invention relates to a method for determination or quantification of an analyte by mass spectrometry. Further the invention relates to a kit and diagnostic systems related thereto.

[0008] It is an object of the present invention to provide a method, kit and a diagnostic system each of these are compatible with automation and improves the throughput of the detection of analytes by mass spectrometry.

[0009] This object is or these objects are solved by the subject matter of the independent claims. Further embodiments are subjected to the dependent claims.

[0010] Summary of the Invention

[0011] In a first aspect, the invention relates to a method for determination or quantification of an analyte by mass spectrometry comprising the steps of:

[0012] 1) providing a sample comprising proteins;

[0013] 2) optionally adding an internal standard of the analyte, preferably wherein the internal standard is stable isotope labeled;

[0014] 3) subjecting the sample from step 1) or 2) to a digestion step to provide peptide fragments of the proteins including the analyte, if present, wherein the digestion comprises i) adding a digestion buffer, and ii) adding a endopeptidase, preferably Trypsin and incubating less than about 60 min;

[0015] 4) inhibiting the endopeptidase, preferably Trypsin pH independent, wherein an inhibitor of the endopeptidase, preferably Trypsin is added to the mixture of 3);

[0016] 5) optionally enriching a peptide of interest, wherein the peptide of interest is a peptide of the analyte; and

[0017] 6) subjecting the mixture of 4) or 5) to an analysis by mass-spectrometry (MS), wherein the presence or concentration of the analyte is determined.

[0018] The second aspect of the present invention refers to a kit comprising

[0019] (i) a endopeptidase, preferably Trypsin,

[0020] (ii) a digestion buffer, comprising calcium chloride or an organic solvent, preferably 2-propanol, (ii) an inhibitor of the endopeptidase, preferably Trypsin,

[0021] (iv) optionally enrichment means for enriching peptides of interest, and

[0022] (v) optionally an elution medium for use in eluting sample components from the enrichment means of (iv).

[0023] The third aspect of the present invention refers to the use of the kit according to the invention in a method according to the invention.

[0024] The fourth aspect of the present invention refers to a diagnostic system for determining an analyte in a sample adapted for performing the method according to the invention.

[0025] The fifth aspect of the present invention refers to the use of the diagnostic system according to the invention in a method according to the invention.

[0026] The sixth aspect of the present invention refers to a method for determination or quantification of an analyte by mass spectrometry comprising the steps of:

[0027] 1) providing a sample comprising proteins;

[0028] 2) optionally adding an internal standard of the analyte, preferably wherein the internal standard is stable isotope labeled;

[0029] 3) subjecting the sample from step 1) or 2) to a digestion step to provide peptide fragments of the proteins including the analyte if present, wherein the digestion comprises i) adding a digestion buffer, comprising calcium chloride, and ii) adding a endopeptidase, preferably Trypsin, and incubating at a temperature range from about 50° to about 75°C less than about 60 min;

[0030] 4) inhibiting the endopeptidase, preferably Trypsin, pH independent, wherein an inhibitor of the endopeptidase, preferably Trypsin, is added to the mixture of 3);

[0031] 5) optionally enriching a peptide of interest, wherein the peptide of interest is a peptide of the analyte; and

[0032] 6) subjecting the mixture of 4) or 5) to an analysis by mass-spectrometry (MS), wherein the presence or concentration of the analyte is determined. In a seventh aspect, the invention relates to a method for determination or quantification of an analyte by mass spectrometry comprising the steps of:

[0033] 1) providing a sample comprising proteins;

[0034] 2) optionally adding an internal standard of the analyte, preferably wherein the internal standard is stable isotope labeled;

[0035] 3) subjecting the sample from step 1) or 2) to a digestion step to provide peptide fragments of the proteins including the analyte if present, wherein the digestion comprises i) adding a digestion buffer comprising an organic solvent, preferable acetronitrile, 2-propanol, methanol or ethanol, more preferably 2-propanol, and ii) adding a endopeptidase, preferably Trypsin, and incubating less than about 60 min;

[0036] 4) inhibiting the endopeptidase, preferably Trypsin, pH independent, wherein an inhibitor of the endopeptidase, preferably Trypsin, is added to the mixture of 3);

[0037] 5) optionally enriching a peptide of interest, wherein the peptide of interest is a peptide of the analyte; and

[0038] 6) subjecting the mixture of 4) or 5) to an analysis by mass-spectrometry (MS), wherein the presence or concentration of the analyte is determined.

[0039] The eight aspect of the present invention refers to the use of the kit kit according to the invention in a method according to the invention.

[0040] The ninth aspect of the present invention refers to a diagnostic system for determining an analyte in a sample adapted for performing the method according to the invention.

[0041] The tenth aspect of the present invention refers to the use of the diagnostic system according to the invention in a method according to the invention.

[0042] The eleventh aspect of the present invention relates to a method for determination or quantification of Thyroglobulin or ApoAl by mass spectrometry comprising the steps of:

[0043] 1) providing a sample comprising proteins; 2) optionally adding an internal standard of Thyroglobulin or ApoAl, preferably wherein the internal standard is isotope labeled, preferably wherein the internal standard is stable isotope labeled with13C and / or15N;

[0044] 3) subjecting the sample from step 1) or 2) to a digestion step to provide peptide fragments of the proteins including Thyroglobulin or ApoAl, if present, wherein the digestion comprises i) adding a digestion buffer comprising an organic solvent, preferable acetronitrile, 2-propanol, methanol or ethanol, more preferably 2-propanol, and ii) adding Trypsin and incubating less than about 60 min;

[0045] 4) inhibiting Trypsin pH independent, wherein an inhibitor of Trypsin is added to the mixture of 3);

[0046] 5) optionally enriching a peptide of interest, wherein the peptide of interest is a peptide of the Thyroglobulin or ApoAl; and

[0047] 6) subjecting the mixture of 4) or 5) to an analysis by mass-spectrometry (MS), wherein the presence or concentration of the Thyroglobulin or ApoAl is determined.

[0048] In a twelfth aspect, the invention relates to a method for quantification of Thyroglobulin or ApoAl by mass spectrometry comprising the steps of:

[0049] 1) providing a sample comprising proteins;

[0050] 2) optionally adding an internal standard of Thyroglobulin or ApoAl, preferably wherein the internal standard is isotope labeled, preferably wherein the internal standard is stable isotope labeled with13C and / or15N;

[0051] 3) subjecting the sample from step 1) or 2) to a digestion step to provide peptide fragments of the proteins including Thyroglobulin or ApoAl, if present, wherein the digestion comprises i) adding a digestion buffer comprising the organic solvent 2-propanol, wherein the final 2-propanol concentration is about 15 to 40 %, and ii) adding Trypsin, wherein Trypsin is added at a ratio (w / w) protein in the sample :Trypsin 1: 15-1:40, and incubating less than about 60 min; 4) inhibiting Trypsin pH independent, wherein an inhibitor of Trypsin is added to the mixture of 3);

[0052] 5) optionally enriching a peptide of interest, wherein the peptide of interest is a peptide of Thyroglobulin or ApoAl, wherein the enrichment step 5) comprises a bound / free separation step i) comprising contacting the sample with a solid phase having a surface for the binding of the peptide of interest under conditions wherein the peptide of interest binds to the solid phase, wherein the enrichment step 5) further comprises ii) washing of the solid phase to which the peptide of interest, if present in the sample, is bound, preferably under conditions which do not elute the peptide of interest; and / or iii) eluting the bound peptides from the solid phase under conditions suitable to allow the elution of the peptide of interest,, and iv) a chromatographic step such as a HPLC of the sample; and

[0053] 6) subjecting the mixture of 5) or 4) to an analysis by mass-spectrometry (MS), wherein the concentration of Thyroglobulin or ApoAl is determined, wherein the ratio of the internal standard of Thyroglobulin or ApoAl to Thyroglobulin or ApoAl, respectively, is determined and the concentration of Thyroglobulin or ApoAl is quantified; wherein step 6) comprises an MS / MS analysis; and wherein steps 1 to 6 are carried out in about 2.5 to 3.5 hours.

[0054] The thirteenth aspect of the present invention refers to the use of the kit according to second aspect of the present invention or embodiments thereof in a method according to the invention.

[0055] The fourteenth aspect of the present invention refers to a diagnostic system for determining an analyte in a sample adapted for performing the method according to the invention.

[0056] The fifteenth aspect of the present invention refers to the use of the diagnostic system according to the invention in a method according to the invention. List of Figures

[0057] Fig. 1: Graphical illustration of conventional peptide-centric, immunoenrichment based absolute quantification of a protein from complex matrixes by LC-MS / MS.

[0058] Fig. 2: Graphical illustration of developed peptide -centric, immunoenrichment based absolute quantification of a protein from complex matrixes by LC-MS / MS.

[0059] Fig. 3: SDS-PAGE image of time-dependent tryptic serum digest with conventional digestion buffer (50 mM NH4HCO3, pH 7.8, 5 mM CaCL) without using denaturing agent.

[0060] Fig. 4: SDS-PAGE image of temperature-dependent serum digestion with trypsin with conventional digestion buffer.

[0061] Fig. 5: SDS-PAGE image of calcium concentration screening upon serum digestion with trypsin using conventional digestion buffer at 60°C.

[0062] Fig. 6: SDS-PAGE image of temperature-dependent serum digestion with trypsin using heat-assisted digestion buffer.

[0063] Fig. 7: SDS-PAGE image of protease inhibitor concentration screening for heat based trypsin digestion at 70°C.

[0064] Fig. 8: Histogram plot showing the effect of the buffer pH on Thyroglobulin peptide recovery efficiency from immunoprecipitation.

[0065] Fig. 9: Comparison between Tg quantification results obtained from Elecsys TG II assay versus LC-MS / MS method based on heat-assisted digestion and Tg peptide IP. Serum samples (n=16) with different Tg amount (2.0 - 66.6 ng / mL, quantitatively measured by Elecsys Tg II assay) were analysed by Elecsys Tg II and LC-MS / MS Tg Peptide IP workflow.

[0066] Fig. 10: SDS-PAGE image of tryptic serum digest with organic solvent based buffers for 9 minutes digestion at 37°C. Digestion with A) Acetonitrile, B) 2- Propanol, C) Methanol, D) Ethanol based buffer.

[0067] Fig. 11: Comparison between Tg detection results obtained from Elecsys TG II versus LC- MS / MS method based on organic-solvent assisted digestion and Tg peptide IP. Serum samples (n=16) with different Tg amount (2.0 - 66.6 ng / mL, quantitatively measured by Elecsys Tg II assay) were analysed by Elecsys Tg II and LC-MS / MS Tg Peptide IP workflow.

[0068] Fig. 12: Effect of the spiking of two Elecsys Tg II capture antibodies (non-biotinylated) to mimic the presence of auto-Tg antibodies on Tg quantification by Elecsys TG II assay and LC-MS / MS Tg Pep IP workflow.

[0069] Fig. 13: Effect of the spiking of increasing amounts of stable isotope labelled Tg into serum (Tg 0.095 ng / mL) with very high Anti-Tg amount (1998 lU / mL). A) shows the measured values B) is an enlarged representation of the values in A) in the red box.

[0070] Fig. 14: Tg organic solvent assisted LC-MS / MS Tg Peptide IP method vs Elecsys Tg II assay with serum high anti-Tg autoantibodies (32 -1998 lU / mL). Serum samples (n=8) with different Tg amount (0 - 0.45 ng / mL, quantitatively measured by Elecsys Tg II assay) were analysed by Elecsys Tg II and LC-MS / MS Tg Peptide IP workflow.

[0071] Fig. 15: SDS-PAGE image of organic solvent elution profile of peptides from carboxylate- modified magnetic beads.

[0072] Fig. 16: CV estimation of the ApoAl quantification with spiking of full length stable isotope labeled ApoAl into serum

[0073] Fig. 17: Effect of IP time on CV estimation of LC-MS / MS Tg Peptide IP with organic solvent based digestion.

[0074] Detailed Description of the Invention

[0075] Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular embodiments and examples described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0076] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings recited in the present specification, the text of the present specification takes precedence.

[0077] In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0078] Definitions

[0079] The word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0080] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents, unless the content clearly dictates otherwise.

[0081] Percentages, concentrations, amounts, and other numerical data may be expressed or presented herein in a “range” format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of "4% to 20 %" should be interpreted to include not only the explicitly recited values of 4 % to 20 %, but to also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 4, 5, 6, 7, 8, 9, 10, ... 18, 19, 20 % and sub-ranges such as from 4-10 %, 5-15 %, 10-20%, etc. This same principle applies to ranges reciting minimal or maximal values. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described. The term “about” when used in connection with a numerical value is meant to encompass numerical values within a range having a lower limit that is 5% smaller than the indicated numerical value and having an upper limit that is 5% larger than the indicated numerical value.

[0082] The term “Mass Spectrometry” (“Mass Spec” or “MS”) or “mass spectrometric determination” or “mass spectrometric analysis“ relates to an analytical technology used to identify compounds by their mass. MS is a method of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "m / z". MS technology generally includes (1) ionizing the compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating a mass-to-charge ratio. The compounds may be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionizer and an ion detector. In general, one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrographic instrument where, due to a combination of magnetic and electric fields, the ions follow a path in space that is dependent upon mass ("m") and charge ("z"). The term "ionization" or "ionizing" refers to the process of generating an analyte ion having a net electrical charge equal to one or more electron units. Negative ions are those having a net negative charge of one or more electron units, while positive ions are those having a net positive charge of one or more electron units. The MS method may be performed either in "negative ion mode", wherein negative ions are generated and detected, or in "positive ion mode" wherein positive ions are generated and detected.

[0083] “Tandem mass spectrometry” or “MS / MS” involves multiple steps of mass spectrometry selection, wherein fragmentation of the analyte occurs in between the stages. In a tandem mass spectrometer, ions are formed in the ion source and separated by mass-to-charge ratio in the first stage of mass spectrometry (MSI). Ions of a particular mass-to-charge ratio (precursor ions or parent ion) are selected and fragment ions (or daughter ions) are created by collision-induced dissociation, ion-molecule reaction, or photodissociation. The resulting ions are then separated and detected in a second stage of mass spectrometry (MS2).

[0084] Since a mass spectrometer separates and detects ions of slightly different masses, it easily distinguishes different isotopes of a given element. Mass spectrometry is thus, an important method for the accurate mass determination and characterization of analytes, including but not limited to low-molecular weight analytes, peptides, polypeptides or proteins. Its applications include the identification of proteins and their post-translational modifications, the elucidation of protein complexes, their subunits and functional interactions, as well as the global measurement of proteins in proteomics. De novo sequencing of peptides or proteins by mass spectrometry can typically be performed without prior knowledge of the amino acid sequence.

[0085] Most sample workflows in MS further include sample preparation and / or enrichment steps, wherein e.g. the analyte(s) of interest are separated from the matrix using e.g. gas or liquid chromatography. Typically, for the mass spectrometry measurement, the following three steps are performed:

[0086] 1. a sample comprising an analyte of interest is ionized, usually by complex formation with cations, often by protonation to cations. Ionization source include but are not limited to electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI).

[0087] 2. the ions are sorted and separated according to their mass and charge. High-field asymmetric-waveform ion-mobility spectrometry (FAIMS) may be used as ion fdter.

[0088] 3. the separated ions are then detected, e.g. in multiple reaction mode (MRM), and the results are displayed on a chart.

[0089] Mass spectrometric determination may be combined with additional analytical methods including chromatographic methods such as gas chromatography (GC), liquid chromatography (LC), particularly HPLC, and / or ion mobility -based separation techniques.

[0090] In the context of the present disclosure, the term “analyte”, “analyte molecule” or “analyte(s) of interest” are used interchangeably referring the chemical species to be analysed via mass spectrometry. Chemical species suitable to be analysed via mass spectrometry, i.e. analytes, can be any kind of molecule present in a living organism, include but are not limited to nucleic acid (e.g. DNA, mRNA, miRNA, rRNA etc.), amino acids, peptides, proteins (e.g. cell surface receptor, cytosolic protein etc.), metabolite or hormones (e.g. testosterone, estrogen, estradiol, etc.), fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids (e.g. Vitamin D), molecules characteristic of a certain modification of another molecule (e.g. sugar moieties or phosphoryl residues on proteins, methyl residues on genomic DNA) or a substance that has been internalized by the organism (e.g. therapeutic drugs, drugs of abuse, toxins, etc.) or a metabolite of such a substance. In the embodiments of the present invention, the analyte comprises peptide bonds. These peptide bonds could be digested by the endopeptidase used in the method according of the invention. In specific embodiments of the present invention, the analyte is a protein or a peptide. Such analyte may serve as a biomarker. In the context of present invention, the term “biomarker” refers to a substance within a biological system that is used as an indicator of a biological state of said system. In preferred embodiments the analyte is an endogenous analyte.

[0091] The term '"limit of detection" or "LOD" is the lowest concentration of an analyte that the bioanalytical procedure can reliably differentiate the analyte from background noise.

[0092] Analytes may be present in a sample of interest, e.g. a biological or clinical sample. The term "sample" or "sample of interest" are used interchangeably herein, referring to a part or piece of a tissue, organ or individual, typically being smaller than such tissue, organ or individual, intended to represent the whole of the tissue, organ or individual. Upon analysis a sample provides information about the tissue status or the health or diseased status of an organ or individual. Examples of samples include but are not limited to fluid samples such as blood, serum, plasma, synovial fluid, spinal fluid, urine, saliva, and lymphatic fluid, or solid samples such as dried blood spots and tissue extracts. Further examples of samples are cell cultures or tissue cultures.

[0093] In the context of the present disclosure, the sample may be derived from an “individual” or “subject”. Typically, the subject is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0094] Typically, an “internal standard“ (ISTD) is a known amount of a substance which exhibits similar properties as the analyte of interest when subjected to the mass spectrometric detection workflow (i.e. including any pre-treatment, enrichment and actual detection step). Although the ISTD exhibits similar properties as the analyte of interest, it is still clearly distinguishable from the analyte of interest. Exemplified, during chromatographic separation, such as gas or liquid chromatography, the ISTD has about the same retention time as the analyte of interest from the sample. Thus, both the analyte and the ISTD enter the mass spectrometer at the same time. The ISTD however, exhibits a different molecular mass than the analyte of interest from the sample. This allows a mass spectrometric distinction between ions from the ISTD and ions from the analyte by means of their different mass / charge (m / z) ratios. Both are subject to fragmentation and provide daughter ions. These daughter ions can be distinguished by means of their m / z ratios from each other and from the respective parent ions. Consequently, a separate determination and quantification of the signals from the ISTD and the analyte can be performed. Since the ISTD has been added in known amounts, the signal intensity of the analyte from the sample can be attributed to a specific quantitative amount of the analyte. Thus, the addition of an ISTD allows for a relative comparison of the amount of analyte detected, and enables unambiguous identification and quantification of the analyte(s) of interest present in the sample when the analyte(s) reach the mass spectrometer. Typically, but not necessarily, the ISTD is an isotopically labeled variant (comprising e.g.2H,13C, or15N etc. label) of the analyte of interest.

[0095] The sample may also be subjected to one or more enrichment steps. In the context of the present disclosure, the term “enriching a peptide of interest” or “enrichment step” refers to an enrichment process which occurs subsequent to the digestion step of the sample and provides a sample comprising an enriched analyte or peptides of the analyte relative to the initial sample. Well-known enrichment methods include but are not limited to chemical precipitation, the use of a solid phase, and chromatographic methods. The first enrichment step may comprise chemical precipitation (e.g. using acetonitrile) or the use of a solid phase. Chemical precipitation refers to the addition of chemical components to the sample, which cause certain constituents of the sample to precipitate. Exemplified, a well-known precipitation method is the addition of acetonitrile to the sample.

[0096] Suitable solid phases include but are not limited to Solid Phase Extraction (SPE) cartridges, and beads. Beads may be non-magnetic, magnetic, or paramagnetic. Beads may be coated differently to be e.g. specific for the analyte of interest. Beads may also bind e.g. several peptides and thus are not specific for the analyte. The coating may differ depending on the use intended, i.e. on the intended capture molecule. It is well-known to the skilled person which coating is suitable for which analyte. The beads may be made of various different materials. The beads may have various sizes and comprise a surface with or without pores.

[0097] The term "chromatography" refers to a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of the chemical entities as they flow around or over a stationary liquid or solid phase.

[0098] The term “liquid chromatographic” or "LC" refers to a process of selective retardation of one or more components of a fluid solution as the fluid uniformly percolates through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid, (i.e., mobile phase), as this fluid moves relative to the stationary phase (s). Methods in which the stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase, silica as the stationary phase) are termed normal phase liquid chromatography (NPLC) and methods in which the stationary phase is less polar than the mobile phase (e.g., water-methanol mixture as the mobile phase and Cl 8 (octadecylsilyl) as the stationary phase) is termed reversed phase liquid chromatography (RPLC).

[0099] "High performance liquid chromatography" or "HPLC" refers to a method of liquid chromatography in which the degree of separation is increased by forcing the mobile phase under pressure through a stationary phase, typically a densely packed column. Typically, the column is packed with a stationary phase composed of irregularly or spherically shaped particles, a porous monolithic layer, or a porous membrane. HPLC is historically divided into two different sub-classes based on the polarity of the mobile and stationary phases. Methods in which the stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase, silica as the stationary phase) are termed normal phase liquid chromatography (NPLC) and the opposite (e.g., water-methanol mixture as the mobile phase and C18 (octadecylsilyl) as the stationary phase) is termed reversed phase liquid chromatography (RPLC).

[0100] Micro LC refers to a HPLC method using a column having a narrow inner column diameter, typically below 1 mm, e.g. about 0.5 mm. “Ultra high performance liquid chromatography" or “UHPLC” refers to a HPLC method using a pressure of 120 MPa (17,405 lbf / in2), or about 1200 atmospheres.

[0101] Rapid LC refers to an LC method using a column having an inner diameter as mentioned above, with a short length <2 cm, e.g. 1 cm, applying a flow rate as mentioned above and with a pressure as mentioned above (Micro LC, UHPLC). The short Rapid LC protocol includes a trapping / wash / elution step using a single analytical column and realizes LC in a very short time <1 min.

[0102] Nano LC refers to methods typically using flow rates below 1 pL / min either in static or dynamic mode. Preferably, nano LC uses a flow rate of 50 to 500 nl / min, e.g. 500 nl / min. 500 nl / min is equal to 0.5 pl / min.

[0103] Further well-known LC methods include “Hydrophilic interaction chromatography' (HILIC), size-exclusion LC, ion exchange LC, and affinity LC. LC separation may be single-channel LC or multi-channel LC comprising a plurality of LC channels arranged in parallel. In LC analytes may be separated according to their polarity or log P value, size or affinity, as generally known to the skilled person.

[0104] A "kit" is any manufacture (e.g., a package or container) comprising at least one reagent. The kit is preferably promoted, distributed, or sold as a unit for performing the methods of the present invention. Typically, a kit may further comprise carrier means being compartmentalised to receive in close confinement one or more container means such as vials, tubes, and the like. In particular, each of the container means comprises one of the separate elements to be used in the method of the first aspect. Kits may further comprise one or more other reagents including but not limited to reaction catalyst. Kits may further comprise one or more other containers comprising further materials including but not limited to buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. A label may be present on the container to indicate that the composition is used for a specific application, and may also indicate directions for either in vivo or in vitro use. The computer program code may be provided on a data storage medium or device such as a optical storage medium (e.g., a Compact Disc) or directly on a computer or data processing device. Moreover, the kit may, comprise standard amounts for the biomarkers as described elsewhere herein for calibration purposes.

[0105] A “diagnostics system” is a laboratory automated apparatus dedicated to the analysis of samples for in vitro diagnostics. The clinical diagnostics system may have different configurations according to the need and / or according to the desired laboratory workflow. Additional configurations may be obtained by coupling a plurality of apparatuses and / or modules together. A “module” is a work cell, typically smaller in size than the entire diagnostics system, which has a dedicated function. This function can be analytical but can be also pre -analytical or post analytical or it can be an auxiliary function to any of the pre- analytical function, analytical function or post-analytical function. In particular, a module can be configured to cooperate with one or more other modules for carrying out dedicated tasks of a sample processing workflow, e.g. by performing one or more pre -analytical and / or analytical and / or post-analytical steps. In particular, the clinical diagnostics system can comprise one or more analytical apparatuses, designed to execute respective workflows that are optimized for certain types of analysis, e.g. clinical chemistry, immunochemistry, coagulation, hematology, liquid chromatography separation, mass spectrometry, etc. Thus the diagnostic system may comprise one analytical apparatus or a combination of any of such analytical apparatuses with respective workflows, where pre-analytical and / or post analytical modules may be coupled to individual analytical apparatuses or be shared by a plurality of analytical apparatuses. In alternative pre -analytical and / or post-analytical functions may be performed by units integrated in an analytical apparatus. The diagnostics system can comprise functional units such as liquid handling units for pipetting and / or pumping and / or mixing of samples and / or reagents and / or system fluids, and also functional units for sorting, storing, transporting, identifying, separating, detecting. The diagnostic system can comprise a sample preparation station for the automated preparation of samples comprising analytes of interest, a liquid chromatography (LC) separation station comprising a plurality of LC channels and / or a sample preparation / LC interface for inputting prepared samples into any one of the LC channels. The diagnostic system can further comprise a controller programmed to assign samples to pre-defined sample preparation workflows each comprising a pre-defined sequence of sample preparation steps and requiring a pre-defined time for completion depending on the analytes of interest. The diagnostic system can further comprise a mass spectrometer (MS) and an LC / MS interface for connecting the LC separation station to the mass spectrometer. The term “automatically” or “automated” as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to a process which is performed completely by means of at least one computer and / or computer network and / or machine, in particular without manual action and / or interaction with a user.

[0106] The diagnostic system makes use of LC coupled to mass spectrometry more convenient and more reliable and therefore suitable for clinical diagnostics. In particular, high-throughput, e.g. up to 100 samples / hour or more with random access sample preparation and LC separation can be obtained while enabling online coupling to mass spectrometry. Moreover the process can be fully automated increasing the walk-away time and decreasing the level of skills required.

[0107] “Peptide fragment” or “peptide fragments” as used herein refers to any portion of a protein, such as thyroglobulin, which can be produced by a reproducible fragmentation process, such as a digestion with a protease such as trypsin.

[0108] As used herein the term “antibody” refers herein to include both polyclonal and monoclonal antibodies, as well as fragments thereof, such as Fv, Fab, and F(ab)2 fragments that are capable of binding the antigen. Moreover, encompassed are single chain antibodies and nanobodies. As used herein the term “Trypsin” refers to a serine endopeptidase, which could be an active or proenzyme form. It catalyzes hydrolysis of peptide bonds in polypeptides at the C Terminal side of arginine and lysine. Trypsin definition also includes proteoforms of trypsin, which can be chemically and biologically modified e.g. methylation of Lysine residues.

[0109] As used herein the term “ApoAl” refers to human Apolipoprotein A-l. ApoAl may be homologues to the amino acid sequence as shown in SEQ ID NO: 3.

[0110] As used herein the term “Thyroglobulin”, “TG” or “Tg” refers to human Thyroglobulin. Thyroglobulin may be homologues to the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0111] Embodiments

[0112] In further embodiments, the present invention relates to the following aspects:

[0113] In a first aspect, the invention relates to a method for determination or quantification of an analyte by mass spectrometry comprising the steps of:

[0114] 1) providing a sample comprising proteins;

[0115] 2) optionally adding an internal standard of the analyte, preferably wherein the internal standard is stable isotope labeled;

[0116] 3) subjecting the sample from step 1) or 2) to a digestion step to provide peptide fragments of the proteins including the analyte, if present, wherein the digestion comprises i) adding a digestion buffer, and ii) adding a endopeptidase, preferably Trypsin and incubating less than about 60 min;

[0117] 4) inhibiting the endopeptidase, preferably Trypsin pH independent, wherein an inhibitor of the endopeptidase, preferably Trypsin is added to the mixture of 3);

[0118] 5) optionally enriching a peptide of interest, wherein the peptide of interest is a peptide of the analyte; and

[0119] 6) subjecting the mixture of 4) or 5) to an analysis by mass-spectrometry (MS), wherein the presence or concentration of the analyte is determined.

[0120] The immunometric quantification of analytes can face challenges. For instance, Thyroglobulin (Tg) faces the challenge of Tg autoantibody (aAB) interference, which is present in roughly 20-30% of the patients with differentiated thyroid cancer (DTC). LC- MS / MS based methodologies overcome this issue by protein denaturation and enzymatic digestion. However, there are certain shortcomings of the prior art.

[0121] One of the shortcomings is the high number of used chemicals and thus the need for instruments, e.g. centrifuges, which are incompatible with automation. In the prior art two types of workflows are described: Precipitation based workflow and chemical denaturationbased workflow. The precipitation-based workflow often use ammonium sulfate for precipitation, centrifugation, denaturation and renaturation (Clarke et al 2012, Netzel et al 2015). Chemical denaturation-based workflows use denaturation, reduction and / or alkylation before digesting the proteins (Hoofnagle et al. 2008, Kushnir et al 2013, Schuford et al. 2020, Patents: US 7,807,172 B2). A further shortcoming of the prior art is the very low throughput. Due to the long protein digestion times of about 2.5 to 22 hours the total workflow in the prior art lasts for about 4.5 hours to 22 hours.

[0122] The present inventors have developed a method for determination and / or quantification of an analyte, which overcome the above described shortcomings. The method i.a. comprise a very fast digestion and allow a sensitive peptide immunoenrichment, if needed. The method eliminates the use of chemical denaturation agent, which partially inactivates proteases such as trypsin, prolongs digestion time, requires clean-up steps, and potentially impairs the immunoenrichment step.

[0123] Further the method comprise a chemical inactivation of the protease to enable the seamless connection with downstream process steps like immunocapture. Thus, the inventive method is a fully integrated workflow that bridges very fast protein digestion with sensitive immune- enrichment of a peptide from complex protein digest for accurate protein quantification.

[0124] In one embodiment of the first aspect of the present invention, the sample is whole blood, serum, plasma, bronchioalveolar lavage (BAL), epithelial lining fluid (ELF), urine, sputum or sweat, preferably serum or plasma.

[0125] A blood sample may be a whole blood sample, or a processed blood sample e.g., serum, plasma etc. Methods for obtaining biological fluid samples (e.g., whole blood, serum, plasma, etc) from a subject are well known in the art. For example, methods for obtaining blood samples from a subject are well known and include established techniques used in phlebotomy. The obtained blood samples may be further processed using standard techniques to obtain e.g., a serum sample, or a plasma sample. Advantageously, methods for obtaining biological fluid samples from a subject are typically low-invasive or non-invasive. A whole blood sample is defined as a blood sample drawn from the body and from which (substantially) no constituents (such as platelets or plasma) have been removed. In other words, the relative ratio of constituents in a whole blood sample is substantially the same as a blood in the body. In this context, “substantially the same” allows for a very small change in the relative ratio of the constituents of whole blood e.g., a change of up to 5%, up to 4%, up to 3%, up to 2%, up to 1% etc. Whole blood contains both the cell and fluid portions of blood. A whole blood sample may therefore also be defined as a blood sample with (substantially) all of its cellular components in plasma, wherein the cellular components (i.e., at least comprising the requisite white blood cells, red blood cells, platelets of blood) are intact.

[0126] In further embodiments of the present invention, the endopeptidase is a serine endopeptidase.

[0127] In embodiments of the present invention, the analyte is a protein or a peptide. In embodiments of the present invention, the analyte is a protein.

[0128] In a further embodiment of the first aspect of the present invention, the analyte is Thyroglobulin or ApoAl. In a specific embodiment the analyte is Thyroglobulin.

[0129] In embodiments of the first aspect of the present invention, Thyroglobulin is homologues to the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2 or ApoAl is homologous to the amino acid sequence as shown in SEQ ID NO: 3. In embodiments of the first aspect of the present invention, ApoAl comprises SEQ ID NO: 3. In embodiments Thyroglobulin comprises SEQ ID NO: 1 or SEQ ID NO: 2.

[0130] In a further embodiment of the first aspect of the present invention, the sample is not subjected to a denaturation step using denaturating agents, reduction, alkylation and / or precipitation before adding the digestion buffer.

[0131] Methods using denaturating steps using denaturating agents, reduction, alkylation and precipitation are well known in the art. For example, ammonium sulfate is often used for precipitation and urea is often used as denaturating agent. Further dithiothreitol is often used for reduction and iodoacetamide for alkylation in the art. Also methods for precipitation are well known in the art.

[0132] In an embodiment of the first aspect of the present invention, step 3) ii) is incubated for less than about 45 min, preferably less than about 30 min, more preferably less than about 15 min. In a further specific embodiment of the first aspect of the present invention step 3) ii) is incubated for at least about 60 sec to less than 45 min, preferably for at least about 90 sec to less than about 30 min, more preferably for at least about 2 min to less than about 20 min, most preferably for at least about 5 min to less than about 20 min.

[0133] In a further embodiment of the first aspect of the present invention, the endopeptidase, preferably Trypsin in step 3) ii) is added at a ratio of protein in the sample: endopeptidase, preferably Trypsin of 100: 1 to 5: 1 (w / w).

[0134] In a further embodiment of the first aspect of the present invention the endopeptidase, preferably Trypsin is inhibited by adding an inhibitor. In a specific embodiment of the first aspect of the present invention the endopeptidase, preferably Trypsin is inhibited by adding an inhibitor of the endopeptidase, preferably Trypsin.

[0135] In embodiments of the first aspect of the present invention, the inhibitor is an inhibitor of endopeptidases. In embodiments of the first aspect of the present invention the inhibitor is 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid. In embodiments of the first aspect of the present invention, 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid is added at a final concentration of 0.1-100 mM, preferably of 1 mM-40 mM, more preferably of 5-20 mM, most preferably of 5- 15 mM.

[0136] In embodiments of the first aspect of the present invention, the enrichment step 5) may include one or more enrichment methods, in particular a first and / or a second enrichment step. Enrichment methods are well-known in the art and include but are not limited to chemical enrichment methods including but not limited to chemical precipitation, and enrichment methods using solid phases including but not limited to solid phase extraction methods, bead workflows, and chromatographic methods (e.g. gas or liquid chromatography). Accordingly, in particular embodiments, enrichment step 5) comprise one or more enrichment methods selected from the group consisting of chemical precipitation, methods using solid phase extraction methods, bead workflows, and chromatographic methods.

[0137] In embodiments of the first aspect of the present invention, the enrichment step 5) comprises a bound / free separation step 5) i) comprising contacting the sample with a solid phase having a surface for the binding of the peptide of interest under conditions wherein the peptide of interest binds to the solid phase. The solid phase may be comprised of solid particles or of a non-particular solid phase, e.g. a coated surface within a vessel or a well. In particular embodiments, the solid phase is comprised of magnetic or paramagnetic particles, in particular particles having a magnetic or paramagnetic core. In embodiments, said magnetic or paramagnetic core comprises a metal oxide and / or a metal carbide. In an especially particular embodiment, the core comprises FC3O4.

[0138] The surface of the solid phase, in particular the magnetic or paramagnetic beads, may be a hydrophobic surface, in particular comprising hydrophobic organic groups such as C3-C18 alkyl groups, more particularly C4 alkyl groups. Further, the hydrophobic surface of the solid phase, in particular the surface of the magnetic or superparamagnetic beads, comprises pores. The pore size may be in the range of from 1 nm to 200 nm, in particular less than about 100 nm, in particular less than about 10 nm. Suitable hydrophobic surfaces may e.g. be found in “The HPLC Expert: Possibilities and Limitations of Modem High Performance Liquid Chromatography” DOI: 10.1002 / 9783527677610.

[0139] In specific embodiments, the binding is a selective binding. Abundant plasma proteins might be quantified without enrichment step. Furthermore these proteins can be quantified with an enrichment step comprising a bound / free separation step without a selective binding.

[0140] In embodiments of the first aspect of the present invention, the solid phase comprises an antibody selectively binding to the peptide of interest coupled to the surface. Specific antibodies, for instance, may be obtained using the analyte as antigen by methods well known in the art. Antibodies as referred to herein include both polyclonal and monoclonal antibodies, as well as fragments thereof, such as Fv, Fab, and F(ab)2 fragments that are capable of binding the antigen. Moreover, encompassed are single chain antibodies and nanobodies.

[0141] In embodiments of the first aspect of the present invention the enrichment step 5) further comprises

[0142] 5) ii) washing of the solid phase to which the peptide of interest, if present in the sample, is bound, preferably under conditions which do not elute the peptide of interest; and / or

[0143] 5) iii) eluting the bound peptides from the solid phase under conditions suitable to allow the elution of the peptide of interest.

[0144] In embodiments, the method comprises a washing step (Wl) after incubation with solid phase, preferably magnetic beads. Depending on the analyte(s) one or more additional washing steps (W2) are performed. One washing step (Wl, W2) comprises a series of steps including magnetic bead separation by a magnetic bead handling unit comprising magnets or electromagnets, aspiration of liquid, addition of a washing buffer, resuspension of the magnetic beads, another magnetic bead separation step and another aspiration of the liquid. Moreover, washing steps may differ in terms of type of solvent (water / organic / salt / pH), apart from volume and number or combination of washing cycles. It is well-known to the skilled person how to choose the respective parameters. The last of the washing steps (Wl, W2) is followed by the addition of an elution reagent followed by resuspension of the magnetic beads and a pre-defined incubation period for releasing the peptides of the analyte(s) of interest from the magnetic beads. The bound-free magnetic beads are then separated and the supernatant containing the peptides of the analyte is captured.

[0145] In embodiments of the first aspect of the present invention, step 5) comprises a chromatographic step iv), in particular liquid chromatography such as a HPLC, micro LC, nano LC or rapid LC, of the sample. In embodiments, the chromatographic step is gas or liquid chromatography. Both methods are well known to the skilled person. In embodiments, the liquid chromatography is selected from the group consisting of HPLC, rapid LC, micro- LC, nano LC, flow injection, and trap and elute. In particular embodiments, the chromatographic separation comprises the use of a single chromatic column, or the use of two or more chromatic columns. In particular embodiments, wherein two or more chromatic columns are used, the columns are positioned downstream of each other, i.e. a second column is positioned downstream of a first column, and an optional third column is position downstream of the second column, etc.. In embodiments wherein two or more columns are used, these columns may be identical or may differ from each other depending on the desired function. It is well-known to the skilled person to choose the correct columns and set up.

[0146] In embodiments of the first aspect of the present invention, the enrichment step 5) comprises i) contacting the sample with a solid phase having a surface for the binding of the peptide of interest under conditions wherein the peptide of interest binds to the solid phase, preferably wherein the binding is a selective binding of the peptide of interest, ii) washing of the solid phase to which the peptide of interest, if present in the sample, is bound, preferably under conditions which do not elute the peptide of interest, iii) eluting the bound peptides from the solid phase, and / or iv) a chromatographic step, in particular liquid chromatography such as a HPLC, micro LC, nano LC or rapid LC, of the sample.

[0147] In this embodiment the enrichment step 5) comprises a first enrichment step 5) i) and a second enrichment step 5) iv), wherein the second enrichment step 5) iv) is performed subsequent to the first enrichment step 5) i). In particular embodiments, the first enrichment step 5) i) comprises bead workflow as described in detail above, and the second enrichment step 5) iv) comprises liquid chromatography, in particular selected from the group consisting of HPLC, rapid LC, micro-LC, nano LC, flow injection, and / or trap and elute.

[0148] In embodiments of the first aspect of the present invention, the solid phase comprising an antibody is incubated in step 5) i) for up to about 2 hours, preferably around 0. 1 to around 2 hours.

[0149] In embodiments of the first aspect of the present invention, step 6) comprises an MS / MS analysis, preferably a triple quadrupole-MS / MS analysis.

[0150] In embodiments of the first aspect of the present invention, the ratio of the internal standard of the analyte to the analyte is determined and the analyte is quantified.

[0151] In embodiments of the first aspect of the present invention, the digestion buffer in step 3) i) is a buffer comprising calcium chloride.

[0152] In embodiments of the first aspect of the present invention, the digestion buffer in step 3) i) comprises calcium chloride and wherein the incubation in step 3) ii) is carried out at a temperature range from about 50°C to about 75°C, preferably at about 60°C to about 70°C. The inventors surprisingly discovered a workflow that complex matrix like serum is digested on a minute scale without using chemical detergent agents and thereafter accurately quantified absolute abundance of protein analyte by designing a calcium dichloride based heat-assisted digestion system or organic solvent assisted digestion system.

[0153] In embodiments of the first aspect of the present invention, the digestion buffer in step 3) i) is a buffer comprising at least about 250 mM calcium chloride, preferable at least about 300 mM, more preferable at least about 500 mM.

[0154] In embodiments of the first aspect of the present invention, the digestion buffer in step 3) i) comprises calcium chloride and has a pH of about 7 to about 10, preferably a pH of about 8 to about 10. In embodiments of the first aspect of the present invention, the digestion buffer comprises 100 mM Tris HC1, preferably pH about 9.5, and 250-800 mM calcium chloride.

[0155] In embodiments of the first aspect of the present invention, the digestion of step 3) is carried out with Trypsin, particularly in a ratio of protein in the sample: Trypsin of 1: 100 to 1:5 (w / w) in a digestion buffer comprising of about 650 mM calcium chloride at a pH of about 9.5, for a time period of about 5 to 15 min at a temperature of about 60°C, and the digestion is stopped by adding 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid up to a final concentration of 5- 50 mM.

[0156] In embodiments of the first aspect of the present invention, the digestion buffer comprises an organic solvent, preferable acetonitrile, 2-propanol, methanol or ethanol, more preferably 2 -propanol. The inventors surprisingly discovered that organic solvents in the buffer could strongly aid in endopeptidase digestion efficiency (see Example 8, FIG. 10 A-D).

[0157] In embodiments of the first aspect of the present invention, the digestion buffer comprises an organic solvent and wherein the final organic solvent concentration is about 15 to 40 %.

[0158] In embodiments of the first aspect of the present invention, the digestion buffer comprises an organic solvent and wherein the digestion buffer has a pH of about 7 to about 10

[0159] In embodiments of the first aspect of the present invention, the digestion buffer comprises an organic solvent and wherein the incubation in step 3) ii) is carried out at a temperature range from about 35 °C to about 40°C, preferably at about 37°C.

[0160] In embodiments of the first aspect of the present invention, the digestion buffer comprises an organic solvent and wherein the digestion of step 3) is carried out with Trypsin, particularly in a ratio of protein in the sample: Trypsin of 1: 100 to 1:5 (w / w) in a digestion buffer comprising of about 15 to 40% 2-propanol at a pH of about 8.5, for a time period of about 5 to 15 min at a temperature of about 37°C, and the digestion is stopped by adding 4- (2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid up to a final concentration of 5- 50 mM.

[0161] In embodiments of the first aspect of the present invention, the internal standard of the analyte is stable isotope labeled with13C and / or15N.

[0162] The second aspect of the present invention refers to a kit comprising

[0163] (iii) a endopeptidase, preferably Trypsin, (ii) a digestion buffer, comprising calcium chloride or an organic solvent, preferably 2-propanol,

[0164] (iv) an inhibitor of the endopeptidase, preferably Trypsin,

[0165] (iv) optionally enrichment means for enriching peptides of interest, and

[0166] (v) optionally an elution medium for use in eluting sample components from the enrichment means of (iv).

[0167] With regard to particular embodiments of the second aspect of the present invention, reference is made to all embodiments described above in detail in context with the first aspect.

[0168] In embodiments of the second aspect of the present invention, the inhibitor of (iii) is 4-(2- Aminoethyl)-benzylsulfonylfluorid hydrochlorid.

[0169] In embodiments of the second aspect of the present invention, the digestion buffer comprising calcium chloride comprises at least about 250 mM calcium chloride, preferable at least about 300 mM, more preferable at least about 500 mM.

[0170] In embodiments of the second aspect of the present invention, the digestion buffer comprising calcium chloride has a pH of about 7 to about 10, preferably a pH of about 9.5.

[0171] In embodiments of the second aspect of the present invention, the digestion buffer comprising calcium chloride comprises 100 mM Tris-HCl, pH about 9.5 and 250-800 mM calcium chloride.

[0172] In embodiments of the second aspect of the present invention, the digestion buffer comprises an organic solvent and wherein the final organic solvent concentration is about 15 to 40 %.

[0173] In embodiments of the second aspect of the present invention, the digestion buffer comprises an organic solvent and wherein the digestion buffer has a pH of about 7 to about 10, preferably about 8.5.

[0174] In embodiments, the kit comprises (iv) enrichment means such as a solid phase, e.g. particles such as particles having a chemically modified surface and (v) an elution medium comprising an organic water-miscible solvent such as a acetonitrile in an amount of 20 to 60% (v / v), in particular in an amount of about 20, 30, 40, or 50% (v / v) for use in eluting sample components from the solid phase (iv). In embodiments, the kit comprises enrichment means adapted for carrying out at least one enrichment step, in particular a bound / free separation step and / or a chromatographic step. In embodiments, the enrichment means comprises a solid phase which may be comprised of solid particles, e.g. magnetic or paramagnetic particles, or of a non-particular solid phase.

[0175] In further particular embodiments, the enrichment means may comprise a chromatographic column, in particular a chromatographic column for performing a chromatographic step.

[0176] In embodiments, the elution medium optionally comprised in the kit comprises an aqueous solution comprising an organic water-miscible solvent, in particular acetonitrile (ACN). The organic water-miscible solvent, in particular the ACN is present in an amount of about 20 to 60% (v / v), in particular in an amount of about 20, 30, 40, or 50% (v / v).

[0177] The kit may be provided as a single package comprising containers of the individual components or as a set of several packages each comprising a container of the individual components.

[0178] In embodiments, the kit further comprises a package insert. In embodiments, the package insert comprises information on the indications, usage, performance, and / or warnings concerning the kit.

[0179] The third aspect of the present invention refers to the use of the kit according to the invention in a method according to the invention.

[0180] The fourth aspect of the present invention refers to a diagnostic system for determining an analyte in a sample adapted for performing the method according to the invention. In embodiments of the fourth aspect of the invention the diagnostic system quantifies the analyte in the sample.

[0181] The fifth aspect of the present invention refers to the use of the diagnostic system according to the invention in a method according to the invention.

[0182] The sixth aspect of the present invention refers to a method for determination or quantification of an analyte by mass spectrometry comprising the steps of:

[0183] 1) providing a sample comprising proteins;

[0184] 2) optionally adding an internal standard of the analyte, preferably wherein the internal standard is stable isotope labeled; 3) subjecting the sample from step 1) or 2) to a digestion step to provide peptide fragments of the proteins including the analyte, if present, wherein the digestion comprises i) adding a digestion buffer, comprising calcium chloride, and ii) adding a endopeptidase, preferably Trypsin, and incubating at a temperature range from about 50° to about 75°C less than about 60 min;

[0185] 4) inhibiting the endopeptidase, preferably Trypsin, pH independent, wherein an inhibitor of the endopeptidase, preferably Trypsin, is added to the mixture of 3);

[0186] 5) optionally enriching a peptide of interest, wherein the peptide of interest is a peptide of the analyte; and

[0187] 6) subjecting the mixture of 4) or 5) to an analysis by mass-spectrometry (MS), wherein the presence or concentration of the analyte is determined.

[0188] The immunometric quantification of analytes can face challenges. For instance, Tg faces the challenge of Tg autoantibody (aAB) interference, which is present in roughly 20-30% of the patients with differentiated thyroid cancer (DTC). LC-MS / MS based methodologies overcome this issue by protein denaturation and enzymatic digestion. However, there are certain shortcomings of the prior art.

[0189] One of the shortcomings is the high number of used chemicals and thus the need for instruments, e.g. centrifuges, which are incompatible with automation. In the prior art two types of workflows are described: Precipitation based workflow and chemical denaturationbased workflow. The precipitation-based workflow often use ammonium sulfate for precipitation, centrifugation, denaturation and renaturation (Clarke et al 2012, Netzel et al 2015). Chemical denaturation-based workflows use denaturation, reduction and / or alkylation before digesting the proteins (Hoofnagle et al. 2008, Kushnir et al 2013, Schuford et al. 2020, Patents: US 7,807,172 B2). A further shortcoming of the prior art is the very low throughput. Due to the long protein digestion times of about 2.5 to 22 hours the total workflow in the prior art lasts for about 4.5 hours to 22 hours.

[0190] The present inventors have developed a method for determination and / or quantification of an analyte, which overcomes the above described shortcomings. The method i.a. comprise a very fast digestion and allow a sensitive peptide immunoenrichment, if needed. The method eliminates the use of chemical denaturation agent, which partially inactivates proteases such as trypsin, prolongs digestion time, requires clean-up steps, and potentially impairs the immune enrichment step. The inventors surprisingly discovered that endopeptidases such as trypsin are highly active at high temperatures when the digestion buffer comprises calcium chloride and thus the digestion was extremely fast (see e.g. Examples 3 and 4, Figs. 5 and 6).

[0191] Further the method comprises a chemical inactivation of the protease to enable the seamless connection with downstream process steps like immunocapture. Thus, the inventive method is a fully integrated workflow that bridges very fast protein digestion with sensitive immune- enrichment of a peptide from complex protein digest for accurate protein quantification.

[0192] In embodiments of the sixth aspect of the invention the incubation in step 3) ii) is carried out at a temperature range from about 60°C to about 70°C. In embodiments the digestion buffer in step 3) i) is a buffer comprising at least about 250 mM calcium chloride, preferable at least about 400 mM, more preferable at least about 600 mM.

[0193] In embodiments, the digestion buffer in step 3) i) has a pH of about 7 to about 10, preferably a pH of about 8 to about 10.

[0194] In embodiments, the digestion buffer comprises 100 mM Tris-HCl, pH about 9.5 and 250- 800 mM calcium chloride.

[0195] In embodiments, the digestion buffer further comprises glycerol, preferably 1.5-5% glycerol. The glycerol is added to aid the solubilization of protein at elevated temperatures and aids to prevent the precipitation of proteins.

[0196] In embodiments, the digestion of step 3) is carried out with Trypsin, particularly in a ratio of protein in the sample: Trypsin of 1: 100 to 1:5 (w / w), in a digestion buffer comprising of about 400- 750 mM calcium chloride at a pH of about 9.5, for a time period of about 5 to 15 min at a temperature of about 60°C, and the digestion is stopped by adding 4-(2- Aminoethyl)-benzylsulfonylfluorid hydrochlorid up to a final concentration of 5- 50 mM.

[0197] In embodiments, the digestion buffer comprises 100 mM Tris-HCl, pH about 9.5.

[0198] In embodiments, the sample is whole blood, serum, plasma, bronchioalveolar lavage (BAL), epithelial lining fluid (ELF), urine, sputum or sweat, preferably serum or plasma.

[0199] A blood sample may be a whole blood sample, or a processed blood sample e.g., serum, plasma etc. Methods for obtaining biological fluid samples (e.g., whole blood, serum, plasma, etc) from a subject are well known in the art. For example, methods for obtaining blood samples from a subject are well known and include established techniques used in phlebotomy. The obtained blood samples may be further processed using standard techniques to obtain e.g., a serum sample, or a plasma sample. Advantageously, methods for obtaining biological fluid samples from a subject are typically low-invasive or non-invasive.

[0200] A whole blood sample is defined as a blood sample drawn from the body and from which (substantially) no constituents (such as platelets or plasma) have been removed. In other words, the relative ratio of constituents in a whole blood sample is substantially the same as a blood in the body. In this context, “substantially the same” allows for a very small change in the relative ratio of the constituents of whole blood e.g., a change of up to 5%, up to 4%, up to 3%, up to 2%, up to 1% etc. Whole blood contains both the cell and fluid portions of blood. A whole blood sample may therefore also be defined as a blood sample with (substantially) all of its cellular components in plasma, wherein the cellular components (i.e., at least comprising the requisite white blood cells, red blood cells, platelets of blood) are intact.

[0201] In further embodiments of the present invention, the endopeptidase is a serine endopeptidase.

[0202] In embodiments of the present invention, the analyte is a protein or a peptide. In embodiments of the present invention, the analyte is a protein.

[0203] In a further embodiment of the sixth aspect of the present invention, the analyte is Thyroglobulin or ApoAl. In a specific embodiment the analyte is Thyroglobulin.

[0204] In embodiments of the sixth aspect of the present invention, Thyroglobulin is homologues to the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2 or ApoAl is homologous to the amino acid sequence as shown in SEQ ID NO: 3. In embodiments of the first aspect of the present invention, ApoAl comprises SEQ ID NO: 3. In embodiments Thyroglobulin comprises SEQ ID NO: 1 or SEQ ID NO: 2.

[0205] In a further embodiment of the sixth aspect of the present invention, the sample is not subjected to a denaturation step using denaturating agents, reduction, alkylation and / or precipitation before adding the digestion buffer.

[0206] Methods using denaturating steps using denaturating agents, reduction, alkylation and precipitation are well known in the art. For example, ammonium sulfate is often used for precipitation and urea is often used as denaturating agent. Further dithiothreitol is often used for reduction and iodoacetamide for alkylation in the art. Also methods for precipitation are well known in the art.

[0207] In an embodiment of the sixth aspect of the present invention, step 3) ii) is incubated for less than about 45 min, preferably less than about 30 min, more preferably less than about 15 min. In a further specific embodiment of the first aspect of the present invention step 3) ii) is incubated for at least about 60 sec to less than 45 min, preferably for at least about 90 sec to less than about 30 min, more preferably for at least about 2 min to less than about 20 min, most preferably for at least about 5 min to less than about 20 min.

[0208] In a further embodiment of the sixth aspect of the present invention, the endopeptidase, preferably Trypsin in step 3) ii) is added at a ratio of protein in the sample: endopeptidase, preferably Trypsin of 100: 1 to 5: 1 (w / w).

[0209] In a further embodiment of the sixth aspect of the present invention the endopeptidase, preferably Trypsin is inhibited by adding an inhibitor. In a specific embodiment of the sixth aspect of the present invention the endopeptidase, preferably Trypsin is inhibited by adding an inhibitor of the endopeptidase, preferably Trypsin.

[0210] In embodiments of the sixth aspect of the present invention, the inhibitor is an inhibitor of endopeptidases. In embodiments of the sixth aspect of the present invention the inhibitor is 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid. In embodiments of the sixth aspect of the present invention, 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid is added at a final concentration of 0.1-100 mM, preferably of 1 mM-40 mM, more preferably of 5-20 mM, most preferably of 5 - 15 mM.

[0211] In embodiments of the sixth aspect of the present invention, the enrichment step 5) may include one or more enrichment methods, in particular a first and / or a second enrichment step. Enrichment methods are well-known in the art and include but are not limited to chemical enrichment methods including but not limited to chemical precipitation, and enrichment methods using solid phases including but not limited to solid phase extraction methods, bead workflows, and chromatographic methods (e.g. gas or liquid chromatography). Accordingly, in particular embodiments, enrichment step 5) comprise one or more enrichment methods selected from the group consisting of chemical precipitation, methods using solid phase extraction methods, bead workflows, and chromatographic methods. In embodiments of the sixth aspect of the present invention, the enrichment step 5) comprises a bound / free separation step 5) i) comprising contacting the sample with a solid phase having a surface for the binding of the peptide of interest under conditions wherein the peptide of interest binds to the solid phase. The solid phase may be comprised of solid particles or of a non-particular solid phase, e.g. a coated surface within a vessel or a well. In particular embodiments, the solid phase is comprised of magnetic or paramagnetic particles, in particular particles having a magnetic or paramagnetic core. In embodiments, said magnetic or paramagnetic core comprises a metal oxide and / or a metal carbide. In an especially particular embodiment, the core comprises FC3O4.

[0212] The surface of the solid phase, in particular the magnetic or paramagnetic beads, may be a hydrophobic surface, in particular comprising hydrophobic organic groups such as C3-C18 alkyl groups, more particularly C4 alkyl groups. Further, the hydrophobic surface of the solid phase, in particular the surface of the magnetic or superparamagnetic beads, comprises pores. The pore size may be in the range of from 1 nm to 200 nm, in particular less than about 100 nm, in particular less than about 10 nm. Suitable hydrophobic surfaces may e.g. be found in “The HPLC Expert: Possibilities and Limitations of Modem High Performance Liquid Chromatography” DOI: 10.1002 / 9783527677610.

[0213] In specific embodiments, the binding is a selective binding. Abundant plasma proteins might be quantified without enrichment step. Furthermore these proteins can be quantified with an enrichment step comprising a bound / free separation step without a selective binding.

[0214] In embodiments of the sixth aspect of the present invention, the solid phase comprises an antibody selectively binding to the peptide of interest coupled to the surface. Specific antibodies, for instance, may be obtained using the analyte as antigen by methods well known in the art. Antibodies as referred to herein include both polyclonal and monoclonal antibodies, as well as fragments thereof, such as Fv, Fab, and F(ab)2 fragments that are capable of binding the antigen. Moreover, encompassed are single chain antibodies and nanobodies.

[0215] In embodiments of the sixth aspect of the present invention, the enrichment step 5) further comprises 5) ii) washing of the solid phase to which the peptide of interest, if present in the sample, is bound, preferably under conditions which do not elute the peptide of interest; and / or

[0216] 5) iii) eluting the bound peptides from the solid phase under conditions suitable to allow the elution of the peptide of interest.

[0217] In embodiments, the method comprises a washing step (Wl) after incubation with solid phase, preferably magnetic beads. Depending on the analyte(s) one or more additional washing steps (W2) are performed. One washing step (Wl, W2) comprises a series of steps including magnetic bead separation by a magnetic bead handling unit comprising magnets or electromagnets, aspiration of liquid, addition of a washing buffer, resuspension of the magnetic beads, another magnetic bead separation step and another aspiration of the liquid. Moreover, washing steps may differ in terms of type of solvent (water / organic / salt / pH), apart from volume and number or combination of washing cycles. It is well-known to the skilled person how to choose the respective parameters. The last of the washing steps (Wl, W2) is followed by the addition of an elution reagent followed by resuspension of the magnetic beads and a pre-defined incubation period for releasing the peptides of the analyte(s) of interest from the magnetic beads. The bound-free magnetic beads are then separated and the supernatant containing the peptides of the analyte is captured.

[0218] In embodiments of the sixth aspect of the present invention, step 5) comprises a chromatographic step iv), in particular liquid chromatography such as a HPLC, micro LC, nano LC or rapid LC, of the sample. In embodiments, the chromatographic step is gas or liquid chromatography. Both methods are well known to the skilled person. In embodiments, the liquid chromatography is selected from the group consisting of HPLC, rapid LC, micro- LC, nano LC, flow injection, and trap and elute. In particular embodiments, the chromatographic separation comprises the use of a single chromatic column, or the use of two or more chromatic columns. In particular embodiments, wherein two or more chromatic columns are used, the columns are positioned downstream of each other, i.e. a second column is positioned downstream of a first column, and an optional third column is position downstream of the second column, etc.. In embodiments wherein two or more columns are used, these columns may be identical or may differ from each other depending on the desired function. It is well-known to the skilled person to choose the correct columns and set up.

[0219] In embodiments of the sixth aspect of the present invention, the enrichment step 5) comprises i) contacting the sample with a solid phase having a surface for the binding of the peptide of interest under conditions wherein the peptide of interest binds to the solid phase, preferably wherein the binding is a selective binding of the peptide of interest, ii) washing of the solid phase to which the peptide of interest, if present in the sample, is bound, preferably under conditions which do not elute the peptide of interest, iii) eluting the bound peptides from the solid phase, and / or iv) a chromatographic step, in particular liquid chromatography such as a HPLC, micro LC, nano LC or rapid LC, of the sample.

[0220] In embodiments of the sixth aspect of the present invention, the solid phase comprising an antibody is incubated in step 5) i) for up to about 2 hours, preferably around 0. 1 to around 2 hours.

[0221] In embodiments of the sixth aspect of the present invention, step 6) comprises an MS / MS analysis, preferably a triple quadrupole-MS / MS analysis.

[0222] In embodiments of the sixth aspect of the present invention, the ratio of the internal standard of the analyte to the analyte is determined and the analyte is quantified.

[0223] In embodiments of the sixth aspect of the present invention, the internal standard of the analyte is stable isotope labeled with13C and / or15N.

[0224] In a seventh aspect, the invention relates to a method for determination or quantification of an analyte by mass spectrometry comprising the steps of:

[0225] 1) providing a sample comprising proteins;

[0226] 2) optionally adding an internal standard of the analyte, preferably wherein the internal standard is stable isotope labeled;

[0227] 3) subjecting the sample from step 1) or 2) to a digestion step to provide peptide fragments of the proteins including the analyte if present, wherein the digestion comprises i) adding a digestion buffer comprising an organic solvent, preferable acetronitrile, 2-propanol, methanol or ethanol, more preferably 2-propanol, and ii) adding a endopeptidase, preferably Trypsin, and incubating less than about 60 min;

[0228] 4) inhibiting the endopeptidase, preferably Trypsin, pH independent, wherein an inhibitor of the endopeptidase, preferably Trypsin, is added to the mixture of 3);

[0229] 5) optionally enriching a peptide of interest, wherein the peptide of interest is a peptide of the analyte; and

[0230] 6) subjecting the mixture of 4) or 5) to an analysis by mass-spectrometry (MS), wherein the presence or concentration of the analyte is determined.

[0231] The immunometric quantification of analytes can face challenges. For instance, Tg faces the challenge of Tg autoantibody (aAB) interference, which is present in roughly 20-30% of the patients with differentiated thyroid cancer (DTC). LC-MS / MS based methodologies overcome this issue by protein denaturation and enzymatic digestion. However, there are certain shortcomings of the prior art.

[0232] One of the shortcomings is the high number of used chemicals and thus the need for instruments, e.g. centrifuges, which are incompatible with automation. In the prior art two types of workflows are described: Precipitation based workflow and chemical denaturationbased workflow. The precipitation-based workflow often use ammonium sulfate for precipitation, centrifugation, denaturation and renaturation (Clarke et al 2012, Netzel et al 2015). Chemical denaturation-based workflows use denaturation, reduction and / or alkylation before digesting the proteins (Hoofnagle et al. 2008, Kushnir et al 2013, Schuford et al. 2020, Patents: US 7,807,172 B2). A further shortcoming of the prior art is the very low throughput. Due to the long protein digestion times of about 2.5 to 22 hours the total workflow in the prior art lasts for about 4.5 hours to 22 hours.

[0233] The present inventors have developed a method for determination and / or quantification of an analyte, which overcome the above described shortcomings. The method i.a. comprise a very fast digestion and allow a sensitive peptide immunoenrichment, if needed. The method eliminates the use of chemical denaturation agent, which partially inactivates proteases such as trypsin, prolongs digestion time, requires clean-up steps, and potentially impairs the immune enrichment step. The inventors surprisingly discovered that organic solvents in the buffer could strongly aid in endopeptidase digestion efficiency (see Example 8, FIG. 10 A- D). Further the method comprise a chemical inactivation of the protease to enable the seamless connection with downstream process steps like immunocapture. Thus, the inventive method is a fully integrated workflow that bridges very fast protein digestion with sensitive immune- enrichment of a peptide from complex protein digest for accurate protein quantification.

[0234] In embodiments of the seventh aspect, the final organic solvent concentration is about 15 to 40 %. In embodiments of the seventh aspect, the digestion buffer has a pH of about 7 to about 10. In embodiments of the seventh aspect, the digestion buffer comprises 50 mM Tris- HCL.

[0235] In embodiments of the seventh aspect, the incubation in step 3) ii) is carried out at a temperature range from about 35 °C to about 40°C, preferably at about 37°C. In embodiments of the seventh aspect, the digestion of step 3) is carried out with Trypsin, particularly in a ratio protein in the sample: Trypsin of 1 : 100 to 1:5 (w / w) in a digestion buffer comprising of about 15 to 40% 2-Propanol at a pH of about 8.5, for a time period of about 5 to 30 min at a temperatur of about 37°C, and the digestion is stopped by adding 4-(2-Aminoethyl)- benzylsulfonylfluorid hydrochlorid up to a final concentration of 5- 50 mM.

[0236] In embodiments of the seventh aspect, the sample is whole blood, serum, plasma, bronchioalveolar lavage (BAL), epithelial lining fluid (ELF), urine, sputum or sweat, preferably serum or plasma.

[0237] A blood sample may be a whole blood sample, or a processed blood sample e.g., serum, plasma etc. Methods for obtaining biological fluid samples (e.g., whole blood, serum, plasma, etc) from a subject are well known in the art. For example, methods for obtaining blood samples from a subject are well known and include established techniques used in phlebotomy. The obtained blood samples may be further processed using standard techniques to obtain e.g., a serum sample, or a plasma sample. Advantageously, methods for obtaining biological fluid samples from a subject are typically low-invasive or non-invasive.

[0238] A whole blood sample is defined as a blood sample drawn from the body and from which (substantially) no constituents (such as platelets or plasma) have been removed. In other words, the relative ratio of constituents in a whole blood sample is substantially the same as a blood in the body. In this context, “substantially the same” allows for a very small change in the relative ratio of the constituents of whole blood e.g., a change of up to 5%, up to 4%, up to 3%, up to 2%, up to 1% etc. Whole blood contains both the cell and fluid portions of blood. A whole blood sample may therefore also be defined as a blood sample with (substantially) all of its cellular components in plasma, wherein the cellular components (i.e., at least comprising the requisite white blood cells, red blood cells, platelets of blood) are intact.

[0239] In further embodiments of the present invention, the endopeptidase is a serine endopeptidase.

[0240] In embodiments of the present invention, the analyte is a protein or a peptide. In embodiments of the present invention, the analyte is a protein.

[0241] In a further embodiment of the seventh aspect of the present invention, the analyte is Thyroglobulin or ApoAl. In a specific embodiment, the analyte is Thyroglobulin. In a further embodiment the analyte is ApoAl.

[0242] In embodiments of the seventh aspect of the present invention, Thyroglobulin is homologues to the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2 or ApoAl is homologous to the amino acid sequence as shown in SEQ ID NO: 3. In embodiments of the seventh aspect of the present invention, ApoAl comprises SEQ ID NO: 3. In embodiments Thyroglobulin comprises SEQ ID NO: 1 or SEQ ID NO: 2.

[0243] In a further embodiment of the seventh aspect of the present invention, the sample is not subjected to a denaturation step using denaturating agents, reduction, alkylation and / or precipitation before adding the digestion buffer.

[0244] Methods using denaturating steps using denaturating agents, reduction, alkylation and precipitation are well known in the art. For example, ammonium sulfate is often used for precipitation and urea is often used as denaturating agent. Further dithiothreitol is often used for reduction and iodoacetamide for alkylation in the art. Also methods for precipitation are well known in the art.

[0245] In an embodiment of the seventh aspect of the present invention, step 3) ii) is incubated for less than about 45 min, preferably less than about 30 min, more preferably less than about 15 min. In a further specific embodiment of the first aspect of the present invention step 3) ii) is incubated for at least about 60 sec to less than 45 min, preferably for at least about 90 sec to less than about 30 min, more preferably for at least about 2 min to less than about 20 min, most preferably for at least about 5 min to less than about 20 min. In a further embodiment of the seventh aspect of the present invention, the endopeptidase, preferably Trypsin in step 3) ii) is added at a ratio of protein in the sample: endopeptidase, preferably Trypsin of 100: 1 to 5: 1 (w / w).

[0246] In a further embodiment of the seventh aspect of the present invention the endopeptidase, preferably Trypsin is inhibited by adding an inhibitor. In a specific embodiment of the seventh aspect of the present invention the endopeptidase, preferably Trypsin is inhibited by adding an inhibitor of the endopeptidase, preferably Trypsin.

[0247] In embodiments of the seventh aspect of the present invention, the inhibitor is an inhibitor of endopeptidases. In embodiments of the seventh aspect of the present invention the inhibitor is 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid. In embodiments of the seventh aspect of the present invention, 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid is added at a final concentration of 0.1-100 mM, preferably of 1 mM-40 mM, more preferably of 5-20 mM, most preferably of 5- 15 mM.

[0248] In embodiments of the seventh aspect of the present invention, the enrichment step 5) may include one or more enrichment methods, in particular a first and / or a second enrichment step. Enrichment methods are well-known in the art and include but are not limited to chemical enrichment methods including but not limited to chemical precipitation, and enrichment methods using solid phases including but not limited to solid phase extraction methods, bead workflows, and chromatographic methods (e.g. gas or liquid chromatography). Accordingly, in particular embodiments, enrichment step 5) comprise one or more enrichment methods selected from the group consisting of chemical precipitation, methods using solid phase extraction methods, bead workflows, and chromatographic methods.

[0249] In embodiments of the seventh aspect of the present invention, the enrichment step 5) comprises a bound / free separation step 5) i) comprising contacting the sample with a solid phase having a surface for the binding of the peptide of interest under conditions wherein the peptide of interest binds to the solid phase. The solid phase may be comprised of solid particles or of a non-particular solid phase, e.g. a coated surface within a vessel or a well. In particular embodiments, the solid phase is comprised of magnetic or paramagnetic particles, in particular particles having a magnetic or paramagnetic core. In embodiments, said magnetic or paramagnetic core comprises a metal oxide and / or a metal carbide. In an especially particular embodiment, the core comprises FesC The surface of the solid phase, in particular the magnetic or paramagnetic beads, may be a hydrophobic surface, in particular comprising hydrophobic organic groups such as C3-C18 alkyl groups, more particularly C4 alkyl groups. Further, the hydrophobic surface of the solid phase, in particular the surface of the magnetic or superparamagnetic beads, comprises pores. The pore size may be in the range of from 1 nm to 200 nm, in particular less than about 100 nm, in particular less than about 10 nm. Suitable hydrophobic surfaces may e.g. be found in “The HPLC Expert: Possibilities and Limitations of Modem High Performance Liquid Chromatography” DOI: 10.1002 / 9783527677610.

[0250] In specific embodiments, the binding is a selective binding. Abundant plasma proteins might be quantified without enrichment step. Furthermore these proteins can be quantified with an enrichment step comprising a bound / free separation step without a selective binding.

[0251] In embodiments of the seventh aspect of the present invention, the solid phase comprises an antibody selectively binding to the peptide of interest coupled to the surface. Specific antibodies, for instance, may be obtained using the analyte as antigen by methods well known in the art. Antibodies as referred to herein include both polyclonal and monoclonal antibodies, as well as fragments thereof, such as Fv, Fab, and F(ab)2 fragments that are capable of binding the antigen. Moreover, encompassed are single chain antibodies and nanobodies.

[0252] In embodiments of the seventh aspect of the present invention, the enrichment step 5) further comprises

[0253] 5) ii) washing of the solid phase to which the peptide of interest, if present in the sample, is bound, preferably under conditions which do not elute the peptide of interest; and / or

[0254] 5) iii) eluting the bound peptides from the solid phase under conditions suitable to allow the elution of the peptide of interest.

[0255] In embodiments, the method comprises a washing step (Wl) after incubation with solid phase, preferably magnetic beads. Depending on the analyte(s) one or more additional washing steps (W2) are performed. One washing step (Wl, W2) comprises a series of steps including magnetic bead separation by a magnetic bead handling unit comprising magnets or electromagnets, aspiration of liquid, addition of a washing buffer, resuspension of the magnetic beads, another magnetic bead separation step and another aspiration of the liquid. Moreover, washing steps may differ in terms of type of solvent (water / organic / salt / pH), apart from volume and number or combination of washing cycles. It is well-known to the skilled person how to choose the respective parameters. The last of the washing steps (Wl, W2) is followed by the addition of an elution reagent followed by resuspension of the magnetic beads and a pre-defined incubation period for releasing the peptides of the analyte(s) of interest from the magnetic beads. The bound-free magnetic beads are then separated and the supernatant containing the peptides of the analyte is captured.

[0256] In embodiments of the seventh aspect of the present invention, step 5) comprises a chromatographic step iv), in particular liquid chromatography such as a HPLC, micro LC, nano LC or rapid LC, of the sample. In embodiments, the chromatographic step is gas or liquid chromatography. Both methods are well known to the skilled person. In embodiments, the liquid chromatography is selected from the group consisting of HPLC, rapid LC, micro- LC, nano LC, flow injection, and trap and elute. In particular embodiments, the chromatographic separation comprises the use of a single chromatic column, or the use of two or more chromatic columns. In particular embodiments, wherein two or more chromatic columns are used, the columns are positioned downstream of each other, i.e. a second column is positioned downstream of a first column, and an optional third column is position downstream of the second column, etc.. In embodiments wherein two or more columns are used, these columns may be identical or may differ from each other depending on the desired function. It is well-known to the skilled person to choose the correct columns and set up.

[0257] In embodiments of the seventh aspect of the present invention, the enrichment step 5) comprises i) contacting the sample with a solid phase having a surface for the binding of the peptide of interest under conditions wherein the peptide of interest binds to the solid phase, preferably wherein the binding is a selective binding of the peptide of interest, ii) washing of the solid phase to which the peptide of interest, if present in the sample, is bound, preferably under conditions which do not elute the peptide of interest, iii) eluting the bound peptides from the solid phase, and / or iv) a chromatographic step, in particular liquid chromatography such as a HPLC, micro LC, nano LC or rapid LC, of the sample. In this embodiment the enrichment step 5) comprises a first enrichment step 5) i) and a second enrichment step 5) iv), wherein the second enrichment step 5) iv) is performed subsequent to the first enrichment step 5) i). In particular embodiments, the first enrichment step 5) i) comprises bead workflow as described in detail above, and the second enrichment step 5) iv) comprises liquid chromatography, in particular selected from the group consisting of HPLC, rapid LC, micro-LC, nano LC, flow injection, and / or trap and elute.

[0258] In embodiments of the seventh aspect of the present invention, the solid phase comprising an antibody is incubated in step 5) i) for up to about 2 hours, preferably around 0. 1 to around 2 hours.

[0259] In embodiments of the seventh aspect of the present invention, step 6) comprises an MS / MS analysis, preferably a triple quadrupole-MS / MS analysis.

[0260] In embodiments of the seventh aspect of the present invention, the ratio of the internal standard of the analyte to the analyte is determined and the analyte is quantified.

[0261] In embodiments of the seventh aspect of the present invention, the internal standard of the analyte is stable isotope labeled with13C and / or15N.

[0262] The eight aspect of the present invention refers to the use of the kit according to the invention in a method according to the invention.

[0263] The ninth aspect of the present invention refers to a diagnostic system for determining an analyte in a sample adapted for performing the method according to the invention. In embodiments of the ninth aspect of the invention the diagnostic system quantifies the analyte in the sample.

[0264] The tenth aspect of the present invention refers to the use of the diagnostic system according to the invention in a method according to the invention.

[0265] The eleventh aspect of the present invention relates to a method for determination or quantification of Thyroglobulin or ApoAl by mass spectrometry comprising the steps of:

[0266] 1) providing a sample comprising proteins;

[0267] 2) optionally adding an internal standard of Thyroglobulin or ApoAl, preferably wherein the internal standard is isotope labeled, preferably wherein the internal standard is stable isotope labeled with13C and / or15N; 3) subjecting the sample from step 1) or 2) to a digestion step to provide peptide fragments of the proteins including Thyroglobulin or ApoAl, if present, wherein the digestion comprises i) adding a digestion buffer comprising an organic solvent, preferable acetronitrile, 2-propanol, methanol or ethanol, more preferably 2-propanol, and ii) adding Trypsin and incubating less than about 60 min;

[0268] 4) inhibiting Trypsin pH independent, wherein an inhibitor of Trypsin is added to the mixture of 3);

[0269] 5) optionally enriching a peptide of interest, wherein the peptide of interest is a peptide of the Thyroglobulin or ApoAl; and

[0270] 6) subjecting the mixture of 4) or 5) to an analysis by mass-spectrometry (MS), wherein the presence or concentration of the Thyroglobulin or ApoAl is determined.

[0271] The immunometric quantification of analytes can face challenges. For instance, Tg faces the challenge of Tg autoantibody (aAB) interference, which is present in roughly 20-30% of the patients with differentiated thyroid cancer (DTC). LC-MS / MS based methodologies overcome this issue by protein denaturation and enzymatic digestion. However, there are certain shortcomings of the prior art.

[0272] One of the shortcomings is the high number of used chemicals and thus the need for instruments, e.g. centrifuges, which are incompatible with automation. In the prior art two types of workflows are described: Precipitation based workflow and chemical denaturationbased workflow. The precipitation-based workflow often use ammonium sulfate for precipitation, centrifugation, denaturation and renaturation (Clarke et al 2012, Netzel et al 2015). Chemical denaturation-based workflows use denaturation, reduction and / or alkylation before digesting the proteins (Hoofnagle et al. 2008, Kushnir et al 2013, Schuford et al. 2020, Patents: US 7,807,172 B2). A further shortcoming of the prior art is the very low throughput. Due to the long protein digestion times of about 2.5 to 22 hours the total workflow in the prior art lasts for about 4.5 hours to 22 hours.

[0273] The present inventors have developed a method for determination and / or quantification of Thyroglobulin or ApoAl, which overcome the above described shortcomings. The method i.a. comprise a very fast digestion and allow a sensitive peptide immunoenrichment, if needed. The method eliminates the use of chemical denaturation agent, which partially inactivates proteases such as trypsin, prolongs digestion time, requires clean-up steps, and potentially impairs the immune enrichment step. The inventors surprisingly discovered that organic solvents in the buffer could strongly aid in endopeptidase digestion efficiency (see Example 8, FIG. 10 A-D).

[0274] Further the method comprise a chemical inactivation of the protease to enable the seamless connection with downstream process steps like immunocapture. Thus, the inventive method is a fully integrated workflow that bridges very fast protein digestion with sensitive immune- enrichment of a peptide from complex protein digest for accurate protein quantification.

[0275] In embodiments of the eleventh aspect of the present invention, Thyroglobulin is homologues to the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2 or ApoAl is homologous to the amino acid sequence as shown in SEQ ID NO: 3. In embodiments of the first aspect of the present invention, ApoAl comprises SEQ ID NO: 3. In embodiments Thyroglobulin comprises SEQ ID NO: 1 or SEQ ID NO: 2.

[0276] In embodiments of the eleventh aspect of the present invention, the final organic solvent concentration is about 15 to 40 %. In embodiments of the eleventh aspect of the present invention, the digestion buffer has a pH of about 7 to about 10.

[0277] In embodiments of the eleventh aspect of the present invention, the digestion buffer comprises 50 mM Tris-HCL.

[0278] In embodiments of the eleventh aspect of the present invention, the incubation in step 3) ii) is carried out at a temperature range from about 35 °C to about 40°C, preferably at about 37°C.

[0279] In embodiments of the eleventh aspect of the present invention the digestion of step 3) is carried out with Trypsin in a ratio protein in the sample: Trypsin of 1: 100 to 1:5 (w / w) in a digestion buffer comprising of about 15 to 40% 2-Propanol at a pH of about 8.5, for a time period of about 5 to 30 min at a temperatur of about 37°C, and the digestion is stopped by adding 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid up to a final concentration of 5- 50 mM.

[0280] In one embodiment of the eleventh aspect of the present invention, the sample is whole blood, serum, plasma, bronchioalveolar lavage (BAL), epithelial lining fluid (ELF), urine, sputum or sweat, preferably serum or plasma. A blood sample may be a whole blood sample, or a processed blood sample e.g., serum, plasma etc. Methods for obtaining biological fluid samples (e.g., whole blood, serum, plasma, etc) from a subject are well known in the art. For example, methods for obtaining blood samples from a subject are well known and include established techniques used in phlebotomy. The obtained blood samples may be further processed using standard techniques to obtain e.g., a serum sample, or a plasma sample. Advantageously, methods for obtaining biological fluid samples from a subject are typically low-invasive or non-invasive.

[0281] A whole blood sample is defined as a blood sample drawn from the body and from which (substantially) no constituents (such as platelets or plasma) have been removed. In other words, the relative ratio of constituents in a whole blood sample is substantially the same as a blood in the body. In this context, “substantially the same” allows for a very small change in the relative ratio of the constituents of whole blood e.g., a change of up to 5%, up to 4%, up to 3%, up to 2%, up to 1% etc. Whole blood contains both the cell and fluid portions of blood. A whole blood sample may therefore also be defined as a blood sample with (substantially) all of its cellular components in plasma, wherein the cellular components (i.e., at least comprising the requisite white blood cells, red blood cells, platelets of blood) are intact.

[0282] In embodiments of the present invention, the analyte is a protein or a peptide. In embodiments of the present invention, the analyte is a protein.

[0283] In a further embodiment of the eleventh aspect of the present invention, the sample is not subjected to a denaturation step using denaturating agents, reduction, alkylation and / or precipitation before adding the digestion buffer.

[0284] Methods using denaturating steps using denaturating agents, reduction, alkylation and precipitation are well known in the art. For example, ammonium sulfate is often used for precipitation and urea is often used as denaturating agent. Further dithiothreitol is often used for reduction and iodoacetamide for alkylation in the art. Also methods for precipitation are well known in the art.

[0285] In an embodiment of the eleventh aspect of the present invention, step 3) ii) is incubated for less than about 45 min, preferably less than about 30 min, more preferably less than about 15 min. In a further specific embodiment of the eleventh aspect of the present invention, step 3) ii) is incubated for at least about 60 sec to less than 45 min, preferably for at least about 90 sec to less than about 30 min, more preferably for at least about 2 min to less than about 20 min, most preferably for at least about 5 min to less than about 20 min.

[0286] In a further embodiment of the eleventh aspect of the present invention, Trypsin in step 3) ii) is added at a ratio (w / w) protein in the sample : Trypsin of 100 : 1 to 5 : 1.

[0287] In a further embodiment of the eleventh aspect of the present invention, the inhibitor is an inhibitor of Trypsin, preferably 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid. In embodiments of the eleventh aspect of the present invention, 4-(2-Aminoethyl)- benzylsulfonylfluorid hydrochlorid is added at a final concentration of 0.1-100 mM, preferably of 1 mM-40 mM, more preferably of 5-20 mM, most preferably of 5-15 mM.

[0288] In embodiments of the eleventh aspect of the present invention, the enrichment step 5) may include one or more enrichment methods, in particular a first and / or a second enrichment step. Enrichment methods are well-known in the art and include but are not limited to chemical enrichment methods including but not limited to chemical precipitation, and enrichment methods using solid phases including but not limited to solid phase extraction methods, bead workflows, and chromatographic methods (e.g. gas or liquid chromatography). Accordingly, in particular embodiments, enrichment step 5) comprise one or more enrichment methods selected from the group consisting of chemical precipitation, methods using solid phase extraction methods, bead workflows, and chromatographic methods.

[0289] In embodiments of the eleventh aspect of the present invention, the enrichment step 5) comprises a bound / free separation step 5) i) comprising contacting the sample with a solid phase having a surface for the binding of the peptide of interest under conditions wherein the peptide of interest binds to the solid phase. The solid phase may be comprised of solid particles or of a non-particular solid phase, e.g. a coated surface within a vessel or a well. In particular embodiments, the solid phase is comprised of magnetic or paramagnetic particles, in particular particles having a magnetic or paramagnetic core. In embodiments, said magnetic or paramagnetic core comprises a metal oxide and / or a metal carbide. In an especially particular embodiment, the core comprises FesC

[0290] The surface of the solid phase, in particular the magnetic or paramagnetic beads, may be a hydrophobic surface, in particular comprising hydrophobic organic groups such as C3-C18 alkyl groups, more particularly C4 alkyl groups. Further, the hydrophobic surface of the solid phase, in particular the surface of the magnetic or superparamagnetic beads, comprises pores. The pore size may be in the range of from 1 nm to 200 nm, in particular less than about 100 nm, in particular less than about 10 nm. Suitable hydrophobic surfaces may e.g. be found in “The HPLC Expert: Possibilities and Limitations of Modem High Performance Liquid Chromatography” DOI: 10.1002 / 9783527677610.

[0291] In specific embodiments, the binding is a selective binding. Abundant plasma proteins might be quantified without enrichment step, furthermore these proteins can be quantified with an enrichment step comprising a bound / free separation step without a selective binding.

[0292] In embodiments of the eleventh aspect of the present invention, the solid phase comprises an antibody selectively binding to the peptide of interest coupled to the surface. Specific antibodies, for instance, may be obtained using the analyte as antigen by methods well known in the art. Antibodies as referred to herein include both polyclonal and monoclonal antibodies, as well as fragments thereof, such as Fv, Fab, and F(ab)2 fragments that are capable of binding the antigen. Moreover, encompassed are single chain antibodies and nanobodies.

[0293] In embodiments of the eleventh aspect of the present invention, the enrichment step 5) further comprises

[0294] 5) ii) washing of the solid phase to which the peptide of interest, if present in the sample, is bound, preferably under conditions which do not elute the peptide of interest; and / or

[0295] 5) iii) eluting the bound peptides from the solid phase under conditions suitable to allow the elution of the peptide of interest.

[0296] In embodiments of the eleventh aspect of the present invention, step 5) comprises a chromatographic step iv), in particular liquid chromatography such as a HPLC, micro LC, nano LC or rapid LC, of the sample. In embodiments, the chromatographic step is gas or liquid chromatography. Both methods are well known to the skilled person. In embodiments, the liquid chromatography is selected from the group consisting of HPLC, rapid LC, micro- LC, nano LC, flow injection, and trap and elute. In particular embodiments, the chromatographic separation comprises the use of a single chromatic column, or the use of two or more chromatic columns. In particular embodiments, wherein two or more chromatic columns are used, the columns are positioned downstream of each other, i.e. a second column is positioned downstream of a first column, and an optional third column is position downstream of the second column, etc.. In embodiments wherein two or more columns are used, these columns may be identical or may differ from each other depending on the desired function. It is well-known to the skilled person to choose the correct columns and set up.

[0297] In embodiments of the eleventh aspect of the present invention, the enrichment step 5) comprises i) contacting the sample with a solid phase having a surface for the binding of the peptide of interest under conditions wherein the peptide of interest binds to the solid phase, preferably wherein the binding is a selective binding of the peptide of interest, ii) washing of the solid phase to which the peptide of interest, if present in the sample, is bound, preferably under conditions which do not elute the peptide of interest, iii) eluting the bound peptides from the solid phase, and / or iv) a chromatographic step, in particular liquid chromatography such as a HPLC, micro LC, nano LC or rapid LC, of the sample.

[0298] In this embodiment the enrichment step 5) comprises a first enrichment step 5) i) and a second enrichment step 5) iv), wherein the second enrichment step 5) iv) is performed subsequent to the first enrichment step 5) i). In particular embodiments, the first enrichment step 5) i) comprises bead workflow as described in detail above, and the second enrichment step 5) iv) comprises liquid chromatography, in particular selected from the group consisting of HPLC, rapid LC, micro-LC, nano LC, flow injection, and / or trap and elute.

[0299] In embodiments of the eleventh aspect of the present invention, the solid phase comprising an antibody is incubated in step 5) i) for up to about 2 hours, preferably around 0. 1 to around 2 hours.

[0300] In embodiments of the eleventh aspect of the present invention, step 6) comprises an MS / MS analysis, preferably a triple quadrupole-MS / MS analysis.

[0301] In embodiments of the eleventh aspect of the present invention, the ratio of the internal standard of Thyroglobulin or ApoAl to Thyroglobulin or ApoAl is determined and the concentration of Thyroglobulin or ApoAl is quantified. In embodiments of the eleventh aspect of the present invention, the ratio of the internal standard of Thyroglobulin to Thyroglobulin is determined and the concentration of Thyroglobulin is quantified. In embodiments of the eleventh aspect of the present invention, the ratio of the internal standard of ApoAl to ApoAl is determined and the concentration of ApoAl is quantified.

[0302] In a twelfth aspect, the invention relates to a method for quantification of Thyroglobulin or ApoAl by mass spectrometry comprising the steps of:

[0303] 1) providing a sample comprising proteins;

[0304] 2) optionally adding an internal standard of Thyroglobulin or ApoAl, preferably wherein the internal standard is isotope labeled, preferably wherein the internal standard is stable isotope labeled with13C and / or15N;

[0305] 3) subjecting the sample from step 1) or 2) to a digestion step to provide peptide fragments of the proteins including Thyroglobulin or ApoAl, if present, wherein the digestion comprises i) adding a digestion buffer comprising the organic solvent 2-propanol, wherein the final 2-propanol concentration is about 15 to 40 %, and ii) adding Trypsin, wherein Trypsin is added at a ratio (w / w) protein in the sample :Trypsin 1: 15-1:40, and incubating less than about 60 min;

[0306] 4) inhibiting Trypsin pH independent, wherein an inhibitor of Trypsin is added to the mixture of 3);

[0307] 5) optionally enriching a peptide of interest, wherein the peptide of interest is a peptide of Thyroglobulin or ApoAl, wherein the enrichment step 5) comprises a bound / free separation step i) comprising contacting the sample with a solid phase having a surface for the binding of the peptide of interest under conditions wherein the peptide of interest binds to the solid phase, wherein the enrichment step 5) further comprises ii) washing of the solid phase to which the peptide of interest, if present in the sample, is bound, preferably under conditions which do not elute the peptide of interest; and / or iii) eluting the bound peptides from the solid phase under conditions suitable to allow the elution of the peptide of interest,, and iv) a chromatographic step such as a HPLC of the sample; and

[0308] 6) subjecting the mixture of 5) or 4) to an analysis by mass-spectrometry (MS), wherein the concentration of Thyroglobulin or ApoAl is determined, wherein the ratio of the internal standard of Thyroglobulin or ApoAl to Thyroglobulin or ApoAl, respectively, is determined and the concentration of Thyroglobulin or ApoAl is quantified; wherein step 6) comprises an MS / MS analysis; and wherein steps 1 to 6 are carried out in about 2.5 to 3.5 hours.

[0309] In embodiments of the twelfth aspect of the present invention, the digestion buffer has a pH of about 7 to about 10.

[0310] In embodiments of the twelfth aspect of the present invention, the digestion buffer comprises 50 mM Tris-HCL.

[0311] In embodiments of the twelfth aspect of the present invention, the incubation in step 3) ii) is carried out at a temperature range from about 35°C to about 40°C, preferably at about 37°C.

[0312] In embodiments of the twelfth aspect of the present invention, the digestion of step 3) is carried out with Trypsin, particularly in a ratio (w / w) protein in the sample :Trypsin of 1 : 15- 1:40 in a digestion buffer comprising of about 15 to 40% 2-Propanol at a pH of about 8.5, for a time period of about 5 to 30 min at a temperatur of about 37°C, and the digestion is stopped by adding 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid up to a final concentration of 5- 50 mM.

[0313] In embodiments of the twelfth aspect of the present invention, the sample is whole blood, serum, plasma, bronchioalveolar lavage (BAL), epithelial lining fluid (ELF), urine, sputum or sweat, preferably serum or plasma.

[0314] In embodiments of the present invention, the analyte is a protein or a peptide. In embodiments of the present invention, the analyte is a protein. In embodiments of the twelfth aspect of the present invention, the sample is not subjected to a denaturation step using denaturating agents, reduction, alkylation and / or precipitation before adding the digestion buffer.

[0315] In embodiments of the twelfth aspect of the present invention, step 3) ii) is incubated for less than about 45 min, preferably less than about 30 min, more preferably less than about 15 min.

[0316] In embodiments of the twelfth aspect of the present invention, step 3) ii) is incubated for at least about 60 sec to less than 45 min, preferably for at least about 90 sec to less than about 30 min, more preferably for at least about 2 min to less than about 20 min, most preferably for at least about 5 min to less than about 20 min.

[0317] In embodiments of the twelfth aspect of the present invention, the inhibitor is an inhibitor of Trypsin, preferably 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid.

[0318] In embodiments of the twelfth aspect of the present invention, 4-(2-Aminoethyl)- benzylsulfonylfluorid hydrochlorid is added at a final concentration of 0.1-100 mM, preferably of 1 mM-40 mM, more preferably of 5-20 mM, most preferably of 5-15 mM.

[0319] In embodiments of the twelfth aspect of the present invention, the solid phase comprising an antibody is incubated in step 5) i) for up to around 2 hours, preferably around 0. 1 to around 2 hours.

[0320] The thirteenth aspect of the present invention refers to the use of the kit according to second aspect of the present invention or embodiments thereof in a method according to the invention.

[0321] The fourteenth aspect of the present invention refers to a diagnostic system for determining an analyte in a sample adapted for performing the method according to the invention. In embodiments of the fourth aspect of the invention the diagnostic system quantifies the analyte in the sample.

[0322] The fifteenth aspect of the present invention refers to the use of the diagnostic system according to the invention in a method according to the invention.

[0323] The present invention in particular also relates to the following items: 1. A method for determination or quantification of an analyte by mass spectrometry comprising the steps of:

[0324] 1) providing a sample comprising proteins;

[0325] 2) optionally adding an internal standard of the analyte, preferably wherein the internal standard is stable isotope labeled;

[0326] 3) subjecting the sample from step 1) or 2) to a digestion step to provide peptide fragments of the proteins including the analyte, if present, wherein the digestion comprises i) adding a digestion buffer, and ii) adding a endopeptidase, preferably Trypsin and incubating less than about 60 min;

[0327] 4) inhibiting the endopeptidase, preferably Trypsin, pH independent, wherein an inhibitor of the endopeptidase, preferably Trypsin, is added to the mixture of 3);

[0328] 5) optionally enriching a peptide of interest, wherein the peptide of interest is a peptide of the analyte; and

[0329] 6) subjecting the mixture of 4) or 5) to an analysis by mass-spectrometry (MS), wherein the presence or concentration of the analyte is determined.

[0330] 2. The method of item 1, wherein the sample is whole blood, serum, plasma, bronchioalveolar lavage (BAL), epithelial lining fluid (ELF), urine, sputum or sweat, preferably serum or plasma.

[0331] 3. The method of item 1 or 2, wherein the analyte is Thyroglobulin or ApoAl.

[0332] 4. The method of any one of items 1 to 3, wherein Thyroglobulin is homologues to the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2 or ApoAl is homologues to the amino acid sequence as shown in SEQ ID NO: 3

[0333] 5. The method of any one of items 1 to 4, wherein the sample is not subjected to a denaturation step using denaturating agents, reduction, alkylation and / or precipitation before adding the digestion buffer. 6. The method of any one of items 1 to 5, wherein step 3) ii) is incubated for less than about 45 min, preferably less than about 30 min, more preferably less than about 15 min.

[0334] 7. The method of any one of items 1 to 6, wherein step 3) ii) is incubated for at least about 60 sec to less than 45 min, preferably for at least about 90 sec to less than about 30 min, more preferably for at least about 2 min to less than about 20 min, most preferably for at least about 5 min to less than about 20 min.

[0335] 8. The method of any one of items 1 to 7, wherein the endopeptidase, preferably Trypsin in step 3) ii) is added at a ratio of protein in the sample: endopeptidase, preferably Trypsin of 100: 1 to 5: 1 (w / w).

[0336] 9. The method of any one of items 1 to 8, wherein the endopeptidase, preferably Trypsin is inhibited by adding an inhibitor of endopeptidases.

[0337] 10. The method of item 9, wherein the inhibitor is an inhibitor of endopeptidases, preferably 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid.

[0338] 11. The method of item 10, wherein 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid is added at a final concentration of 0.1-100 mM, preferably of 1 mM-40 mM, more preferably of 5-20 mM, most preferably of 5-15 mM.

[0339] 12. The method of any one of items 1 to 11, wherein the enrichment step 5) comprises a bound / free separation step 5) i) comprising contacting the sample with a solid phase having a surface for the binding of the peptide of interest under conditions wherein the peptide of interest binds to the solid phase.

[0340] 13. The method of item 12, wherein the solid phase is comprised of particles, preferably of magnetic or paramagnetic particles.

[0341] 14. The method of item 12 or 13, wherein the binding is a selective binding.

[0342] 15. The method of any one of items 12-14, wherein the solid phase comprises an antibody selectively binding to the peptide of interest coupled to the surface.

[0343] 16. The method of any one of items 12 to 15, wherein the enrichment step 5) further comprises 5) ii) washing of the solid phase to which the peptide of interest, if present in the sample, is bound, preferably under conditions which do not elute the peptide of interest; and / or

[0344] 5) iii) eluting the bound peptides from the solid phase under conditions suitable to allow the elution of the peptide of interest.

[0345] 17. The method of any one of items 1 to 16, wherein step 5) comprises a chromatographic step iv), in particular liquid chromatography such as a HPLC, micro LC, nano LC or rapid LC, of the sample.

[0346] 18. The method of any one of items 1 to 17, wherein enrichment step 5) comprises i) contacting the sample with a solid phase having a surface for the binding of the peptide of interest under conditions wherein the peptide of interest binds to the solid phase, preferably wherein the binding is a selective binding of the peptide of interest, ii) washing of the solid phase to which the peptide of interest, if present in the sample, is bound, preferably under conditions which do not elute the peptide of interest, iii) eluting the bound peptides from the solid phase, and / or iv) a chromatographic step, in particular liquid chromatography such as a HPLC, micro LC, nano LC or rapid LC, of the sample.

[0347] 19. The method of any one of items 12 to 18, wherein the solid phase comprising an antibody is incubated in step 5) i) for up to about 2 hours, preferably around 0.1 to around 2 hours.

[0348] 20. The method of any one of items 1 to 19, wherein step 6) comprises an MS / MS analysis, preferably a triple quadrupole-MS / MS analysis.

[0349] 21. The method of any one of items 1 to 20, wherein the ratio of the internal standard of the analyte to the analyte is determined and the analyte is quantified.

[0350] 22. The method of any one of items 1 to 21, wherein the digestion buffer in step 3) i) is a buffer comprising calcium chloride.

[0351] 23. The method of item 22, wherein the digestion buffer in step 3) i) is a buffer comprising at least about 250 mM calcium chloride, preferable at least about 300 mM, more preferable at least about 500 mM. 24. The method of any one of items 1 to 23, wherein the digestion buffer in step 3) i) comprises calcium chloride and has a pH of about 7 to about 10, preferably a pH of about 8 to about 10.

[0352] 25. The method of any one of items 1 to 24, wherein the digestion buffer comprises 100 mM Tris-HCl, preferably pH about 9.5, and 250-800 mM calcium chloride.

[0353] 26. The method of any one of items 1 to 25, wherein the digestion buffer in step 3) i) comprises calcium chloride and wherein the incubation in step 3) ii) is carried out at a temperature range from about 50°C to about 75°C, preferably at about 60°C to about 70°C.

[0354] 27. The method of any one of items 1 to 26, wherein the digestion of step 3) is carried out with Trypsin, particularly in a ratio of protein in the sample: Trypsin of 1: 100 to 1:5 (w / w) in a digestion buffer comprising of about 650 mM calcium chloride at a pH of about 9.5, for a time period of about 5 to 15 min at a temperature of about 60°C, and the digestion is stopped by adding 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid up to a final concentration of 5- 50 mM.

[0355] 28. The method of any one of items 1 to 21, wherein the digestion buffer comprises an organic solvent, preferable acetonitrile, 2-propanol, methanol or ethanol, more preferably 2- propanol.

[0356] 29. The method of item 28, wherein the digestion buffer comprises an organic solvent and wherein the final organic solvent concentration is about 15 to 40 %.

[0357] 30. The method of any one of items 1 to 21 or 28 or 29, wherein the digestion buffer comprises an organic solvent and wherein the digestion buffer has a pH of about 7 to about 10

[0358] 31. The method of any one of items 1 to 21 or 28 to 30, wherein the digestion buffer comprises an organic solvent and wherein the incubation in step 3) ii) is carried out at a temperature range from about 35°C to about 40°C, preferably at about 37°C.

[0359] 32. The method of any one of items 1 to 21 or 28 to 31, wherein the digestion buffer comprises an organic solvent and wherein the digestion of step 3) is carried out with Trypsin, particularly in a ratio of protein in the sample: Trypsin of 1: 100 to 1:5 (w / w) in a digestion buffer comprising of about 15 to 40% 2-propanol at a pH of about 8.5, for a time period of about 5 to 15 min at a temperature of about 37°C, and the digestion is stopped by adding 4- (2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid up to a final concentration of 5- 50 mM.

[0360] 33. The method of any one of items 1 to 32, wherein the internal standard of the analyte is stable isotope labeled with13C and / or15N.

[0361] 34. A kit comprising

[0362] (i) a endopeptidase, preferably Trypsin,

[0363] (ii) a digestion buffer, comprising calcium chloride or an organic solvent, preferably 2-propanol,

[0364] (iii) an inhibitor of the endopeptidase, preferably Trypsin,

[0365] (iv) optionally enrichment means for enriching peptides of interest, and

[0366] (v) optionally an elution medium for use in eluting sample components from the enrichment means of (iv).

[0367] 35. The kit of item 34, wherein the inhibitor of (iii) is 4-(2-Aminoethyl)- benzylsulfonylfluorid hydrochlorid.

[0368] 36. The kit of item 34 or 35, wherein the digestion buffer comprising calcium chloride comprises at least about 250 mM calcium chloride, preferable at least about 300 mM, more preferable at least about 500 mM.

[0369] 37. The kit of any one of items 34 to 36, wherein the digestion buffer comprising calcium chloride has a pH of about 7 to about 10, preferably a pH of about 9.5.

[0370] 38. The kit of any one of items 34 to 37, wherein the digestion buffer comprising calcium chloride comprises 100 mM Tris-HCl, pH about 9.5 and 250-800 mM calcium chloride.

[0371] 39. The kit of item 34 or 35, wherein the digestion buffer comprises an organic solvent and wherein the final organic solvent concentration is about 15 to 40 %.

[0372] 40. The kit of any one of items 34, 35 or 39, wherein the digestion buffer comprises an organic solvent and wherein the digestion buffer has a pH of about 7 to about 10, preferably about 8.5. 41. Use of the kit of any one of items 34 to 40 in a method according to any one of items 1 to 33.

[0373] 42. A diagnostic system for determining an analyte in a sample adapted for performing the method according to any one of items 1 to 33.

[0374] 43. Use of the diagnostic system according to item 42 in the method of any one of items 1 to 33.

[0375] 44. A method for determination or quantification of an analyte by mass spectrometry comprising the steps of:

[0376] 1) providing a sample comprising proteins;

[0377] 2) optionally adding an internal standard of the analyte, preferably wherein the internal standard is stable isotope labeled;

[0378] 3) subjecting the sample from step 1) or 2) to a digestion step to provide peptide fragments of the proteins including the analyte if present, wherein the digestion comprises i) adding a digestion buffer, comprising calcium chloride, and ii) adding a endopeptidase, preferably Trypsin, and incubating at a temperature range from about 50° to about 75°C less than about 60 min;

[0379] 4) inhibiting the endopeptidase, preferably Trypsin, pH independent, wherein an inhibitor of the endopeptidase, preferably Trypsin, is added to the mixture of 3);

[0380] 5) optionally enriching a peptide of interest, wherein the peptide of interest is a peptide of the analyte; and

[0381] 6) subjecting the mixture of 4) or 5) to an analysis by mass-spectrometry (MS), wherein the presence or concentration of the analyte is determined.

[0382] 45. The method of item 44, wherein the incubation in step 3) ii) is carried out at a temperature range from about 60°C to about 70°C.

[0383] 46. The method of item 44 or 45, wherein the digestion buffer in step 3) i) is a buffer comprising at least about 250 mM calcium chloride, preferable at least about 400 mM, more preferable at least about 600 mM. 47. The method of any one of items 44 to 46, wherein the digestion buffer in step 3) i) has a pH of about 7 to about 10, preferably a pH of about 8 to about 10.

[0384] 48. The method of any one of items 44 to 47, wherein the digestion buffer comprises 100 mM Tris-HCl, pH about 9.5 and 250-800 mM calcium chloride.

[0385] 49. The method of any one of items 44 to 48, wherein the digestion buffer further comprises glycerol, preferably 1.5-5% glycerol.

[0386] 50. The method of any one of items 44 to 49, wherein the digestion of step 3) is carried out with Trypsin, particularly in a ratio of protein in the sample: Trypsin of 1: 100 to 1:5 (w / w), in a digestion buffer comprising of about 400- 750 mM calcium chloride at a pH of about 9.5, for a time period of about 5 to 15 min at a temperature of about 60°C, and the digestion is stopped by adding 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid up to a final concentration of 5- 50 mM.

[0387] 51. The method of item 50, wherein the digestion buffer comprises 100 mM Tris-HCl, pH about 9.5.

[0388] 52. The method of any one of items 44 to 51, wherein the sample is whole blood, serum, plasma, bronchioalveolar lavage (BAL), epithelial lining fluid (ELF), urine, sputum or sweat, preferably serum or plasma.

[0389] 53. The method of any one of items 44 to 52, wherein the analyte is Thyroglobulin or ApoAl.

[0390] 54. The method of any one of items 44 to 53, wherein Thyroglobulin is homologues to the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2 or ApoAl is homologues to the amino acid sequence as shown in SEQ ID NO: 3

[0391] 55. The method of any one of items 44 to 54, wherein the sample is not subjected to a denaturation step using denaturating agents, reduction step, alkylation step and / or precipitation step before adding the digestion buffer.

[0392] 56. The method of any one of items 44 to 55, wherein step 3) ii) is incubated for less than about 45 min, preferably less than about 30 min, more preferably less than about 15 min.

[0393] 57. The method of any one of items 44 to 56, wherein step 3) ii) is incubated for at least about 60 sec to less than about 45 min, more preferably for at least about 90 sec to less than about 30 min, more preferably for at least about 2 min to less than about 20 min, most preferably for at least about 5 min to less than about 20 min.

[0394] 58. The method of any one of items 44 to 57, wherein the endopeptidase, preferablyTrypsin in step 3) ii) is added at a ratio of protein in the sample: endopeptidase, preferablyTrypsin of 100: 1 to 5: 1 (w / w).

[0395] 59. The method of any one of items 44 to 58, wherein the inhibitor is an inhibitor of endopeptidases, preferably 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid.

[0396] 60. The method of item 59, wherein 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid is added at a final concentration of 0.1-100 mM, preferably of 1 mM-40 mM, more preferably of 5-20 mM, most preferably of 5- 15 mM.

[0397] 61. The method of any one of items 44 to 60, wherein the enrichment step 5) comprises a bound / free separation step 5) i) comprising contacting the sample with a solid phase having a surface for the binding of the peptide of interest under conditions wherein the peptide of interest binds to the solid phase.

[0398] 62. The method of item 61, wherein the solid phase is comprised of particles, preferably of magnetic or paramagnetic particles.

[0399] 63. The method of item 61 or 62, wherein the binding is a selective binding.

[0400] 64. The method of any one of items 61 to 63, wherein the solid phase comprises an antibody selectively binding to the peptide of interest coupled to the surface.

[0401] 65. The method of any one of items 61 to 64, wherein the enrichment step 5) further comprises

[0402] 5) ii) washing of the solid phase to which the peptide of interest, if present in the sample, is bound, preferably under conditions which do not elute the peptide of interest; and / or

[0403] 5) iii) eluting the bound peptides from the solid phase under conditions suitable to allow the elution of the peptide of interest. 66. The method of any one of items 44 to 65, wherein step 5) comprises a chromatographic step iv), in particular liquid chromatography such as a HPLC, micro LC, nano LC or rapid LC, of the sample.

[0404] 67. The method of any one of items 44 to 66, wherein enrichment step 5) comprises i) contacting the sample with a solid phase having a surface for the binding of the peptide of interest under conditions wherein the peptide of interest binds to the solid phase, preferably wherein the binding is a selective binding of the peptide of interest, ii) washing of the solid phase to which the peptide of interest, if present in the sample, is bound, preferably under conditions which do not elute the peptide of interest, iii) eluting the bound peptides from the solid phase, and / or iv) a chromatographic step), in particular liquid chromatography such as a HPLC, micro LC, nano LC or rapid LC, of the sample.

[0405] 68. The method of any one of items 64 to 67, wherein the solid phase comprising an antibody is incubated in step 5) i) for up to about 2 hours, preferably around 0.1 to around 2 hours.

[0406] 69. The method of any one of items 44 to 68, wherein step 6) comprises an MS / MS analysis, preferably a triple quadrupole-MS / MS analysis.

[0407] 70. The method of any one of items 44 to 69, wherein the ratio of the internal standard of the analyte to the analyte is determined and the analyte is quantified.

[0408] 71. The method of any one of items 44 to 70, wherein the internal standard of the analyte is stable isotope labeled with13C and / or15N.

[0409] 72. A method for determination or quantification of an analyte by mass spectrometry comprising the steps of:

[0410] 1) providing a sample comprising proteins;

[0411] 2) optionally adding an internal standard of the analyte, preferably wherein the internal standard is stable isotope labeled;

[0412] 3) subjecting the sample from step 1) or 2) to a digestion step to provide peptide fragments of the proteins including the analyte if present, wherein the digestion comprises i) adding a digestion buffer comprising an organic solvent, preferable acetronitrile, 2-propanol, methanol or ethanol, more preferably 2-propanol, and ii) adding a endopeptidase, preferably Trypsin, and incubating less than about 60 min;

[0413] 4) inhibiting the endopeptidase, preferably Trypsin, pH independent, wherein an inhibitor of the endopeptidase, preferably Trypsin, is added to the mixture of 3);

[0414] 5) optionally enriching a peptide of interest, wherein the peptide of interest is a peptide of the analyte; and

[0415] 6) subjecting the mixture of 4) or 5) to an analysis by mass-spectrometry (MS), wherein the presence or concentration of the analyte is determined.

[0416] 73. The method of item 72, wherein the final organic solvent concentration is about 15 to 40 %.

[0417] 74. The method of item 72 or 73, wherein the digestion buffer has a pH of about 7 to about 10.

[0418] 75. The method of any one of items 72 to 74, wherein the digestion buffer comprises 50 mM Tris-HCL.

[0419] 76. The method of any one of items 72 to 75, wherein the incubation in step 3) ii) is carried out at a temperature range from about 35°C to about 40°C, preferably at about 37°C.

[0420] 77. The method of any one of items 72 to 76, wherein the digestion of step 3) is carried out with Trypsin, particularly in a ratio protein in the sample: Trypsin of 1: 100 to 1:5 (w / w) in a digestion buffer comprising of about 15 to 40% 2-Propanol at a pH of about 8.5, for a time period of about 5 to 30 min at a temperatur of about 37°C, and the digestion is stopped by adding 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid up to a final concentration of 5- 50 mM.

[0421] 78. The method of any one of items 72 to 77, wherein the sample is whole blood, serum, plasma, bronchioalveolar lavage (BAL), epithelial lining fluid (ELF), urine, sputum or sweat, preferably serum or plasma.

[0422] 79. The method of any one of items 72 to 78, wherein the analyte is Thyroglobulin or ApoAl. 80. The method of any one of items 72 to 79, wherein Thyroglobulin is homologues to the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2 or ApoAl is homologues to the amino acid sequence as shown in SEQ ID NO: 3.

[0423] 81. The method of any one of items 72 to 80, wherein the sample is not subjected to a denaturation step using denaturating agents, reduction, alkylation and / or precipitation before adding the digestion buffer.

[0424] 82. The method of any one of items 72 to 81, wherein step 3) ii) is incubated for less than about 45 min, preferably less than about 30 min, more preferably less than about 15 min.

[0425] 83. The method of any one of items 72 to 82, wherein step 3) ii) is incubated for at least about 60 sec to less than 45 min, preferably for at least about 90 sec to less than about 30 min, more preferably for at least about 2 min to less than about 20 min, most preferably for at least about 5 min to less than about 20 min.

[0426] 84. The method of any one of items 72 to 83, wherein Trypsin in step 3) ii) is added at a ratio (w / w) protein in the sample: endopeptidase, preferably Trypsin, of 100: 1 to 5: 1.

[0427] 85. The method of any one of items 72 to 84, wherein the inhibitor is an inhibitor of endopeptidases, preferably 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid.

[0428] 86. The method of item 85, wherein 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid is added at a final concentration of 0.1-100 mM, preferably of 1 mM-40 mM, more preferably of 5-20 mM, most preferably of 5- 15 mM.

[0429] 87. The method of any one of items 72 to 86, wherein the enrichment step 5) comprises a bound / free separation step 5) i) comprising contacting the sample with a solid phase having a surface for the binding of the peptide of interest under conditions wherein the peptide of interest binds to the solid phase.

[0430] 88. The method of item 87, wherein the solid phase is comprised of particles, preferably of magnetic or paramagnetic particles.

[0431] 89. The method of item 87 or 88, wherein the binding is a selective binding.

[0432] 90. The method of any one of items 87 to 89, wherein the solid phase comprises an antibody selectively binding to the peptide of interest coupled to the surface. 91. The method of any one of items 87 to 90 wherein the enrichment step 5) further comprises

[0433] 5) ii) washing of the solid phase to which the peptide of interest, if present in the sample, is bound, preferably under conditions which do not elute the peptide of interest; and / or

[0434] 5) iii) eluting the bound peptides from the solid phase under conditions suitable to allow the elution of the peptide of interest.

[0435] 92. The method of any one of items 72 to 91, wherein step 5) comprises a chromatographic step iv), in particular liquid chromatography such as a HPLC, micro LC, nano LC or rapid LC, of the sample.

[0436] 93. The method of any one of items 72 to 92, wherein enrichment step 5) comprises i) contacting the sample with a solid phase having a surface for the binding of the peptide of interest under conditions wherein the peptide of interest binds to the solid phase, preferably wherein the binding is a selective binding of the peptide of interest, ii) washing of the solid phase to which the peptide of interest, if present in the sample, is bound, preferably under conditions which do not elute the peptide of interest, iii) eluting the bound peptides from the solid phase, and / or iv) a chromatographic step, in particular liquid chromatography such as a HPLC, micro LC, nano LC or rapid LC, of the sample.

[0437] 94. The method of any one of items 90 to 93, wherein the solid phase comprising an antibody is incubated in step 5) i) for up to about 2 hours, preferably around 0.1 to around 2 hours.

[0438] 95. The method of any one of items 72 to 94, wherein step 6) comprises an MS / MS analysis, preferably a triple quadrupole-MS / MS analysis.

[0439] 96. The method of any one of items 72 to 95, wherein the ratio of the internal standard of the analyte to the analyte is determined and the analyte is quantified.

[0440] 97. The method of any one of items 72 to 96, wherein the internal standard of the analyte is stable isotope labeled with13C and / or15N. 98. Use of the kit of any one of items 34 to 40 in a method according to any one of items 44 to 97.

[0441] 99. A diagnostic system for determining an analyte in a sample adapted for performing the method according to any one of items 44 to 97.

[0442] 100. Use of the diagnostic system according to item 99 in the method of any one of items 44 to 97.

[0443] 101. A method for determination or quantification of Thyroglobulin or ApoAl by mass spectrometry comprising the steps of:

[0444] 1) providing a sample comprising proteins;

[0445] 2) optionally adding an internal standard of Thyroglobulin or ApoAl, preferably wherein the internal standard is isotope labeled, preferably wherein the internal standard is stable isotope labeled with13C and / or15N;

[0446] 3) subjecting the sample from step 1) or 2) to a digestion step to provide peptide fragments of the proteins including Thyroglobulin or ApoAl, if present, wherein the digestion comprises i) adding a digestion buffer comprising an organic solvent, preferable acetronitrile, 2-propanol, methanol or ethanol, more preferably 2-propanol, and ii) adding Trypsin and incubating less than about 60 min;

[0447] 4) inhibiting Trypsin pH independent, wherein an inhibitor of Trypsin is added to the mixture of 3);

[0448] 5) optionally enriching a peptide of interest, wherein the peptide of interest is a peptide of the Thyroglobulin or ApoAl; and

[0449] 6) subjecting the mixture of 4) or 5) to an analysis by mass-spectrometry (MS), wherein the presence or concentration of the Thyroglobulin or ApoAl is determined.

[0450] 102. The method of item 101, wherein the final organic solvent concentration is about 15 to 40 %. 103. The method of item 101 or 102, wherein the digestion buffer has a pH of about 7 to about 10.

[0451] 104. The method of any one of items 101 to 102, wherein the digestion buffer comprises 50 mM Tris-HCL.

[0452] 105. The method of any one of items 101 to 104, wherein the incubation in step 3) ii) is carried out at a temperature range from about 35°C to about 40°C, preferably at about 37°C.

[0453] 106. The method of any one of items 101 to 105, wherein the digestion of step 3) is carried out with Trypsin in a ratio protein in the sample: Trypsin of 1 : 100 to 1:5 (w / w) in a digestion buffer comprising of about 15 to 40% 2-Propanol at a pH of about 8.5, for a time period of about 5 to 30 min at a temperatur of about 37°C, and the digestion is stopped by adding 4- (2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid up to a final concentration of 5- 50 mM.

[0454] 107. The method of any one of items 101 to 106, wherein the sample is whole blood, serum, plasma, bronchioalveolar lavage (BAL), epithelial lining fluid (ELF), urine, sputum or sweat, preferably serum or plasma.

[0455] 108. The method of any one of items 101 to 107, wherein the sample is not subjected to a denaturation step using denaturating agents, reduction, alkylation and / or precipitation before adding the digestion buffer.

[0456] 109. The method of any one of items 101 to 108, wherein step 3) ii) is incubated for less than about 45 min, preferably less than about 30 min, more preferably less than about 15 min.

[0457] 110. The method of any one of items 101 to 109, wherein step 3) ii) is incubated for at least about 60 sec to less than 45 min, preferably for at least about 90 sec to less than about 30 min, more preferably for at least about 2 min to less than about 20 min, most preferably for at least about 5 min to less than about 20 min.

[0458] 111. The method of any one of items 101 to 110, wherein Trypsin in step 3) ii) is added at a ratio (w / w) protein in the sample:Trypsin of 100: 1 to 5: 1.

[0459] 112. The method of any one of items 101 to 112, wherein the inhibitor is an inhibitor of Trypsin, preferably 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid. 113. The method of item 112, wherein 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid is added at a final concentration of 0.1-100 mM, preferably of 1 mM-40 mM, more preferably of 5-20 mM, most preferably of 5-15 mM.

[0460] 114. The method of any one of items 101 to 113, wherein the enrichment step 5) comprises a bound / free separation step 5) i) comprising contacting the sample with a solid phase having a surface for the binding of the peptide of interest under conditions wherein the peptide of interest binds to the solid phase.

[0461] 115. The method of item 114, wherein the solid phase is comprised of particles, preferably of magnetic or paramagnetic particles.

[0462] 116: The method of item 114 or 115, wherein the binding is a selective binding.

[0463] 117. The method of any one of items 114 to 116, wherein the solid phase comprises an antibody selectively binding to the peptide of interest coupled to the surface.

[0464] 118. The method of any one of items 114 to 117, wherein the enrichment step 5) further comprises

[0465] 5) ii) washing of the solid phase to which the peptide of interest, if present in the sample, is bound, preferably under conditions which do not elute the peptide of interest; and / or

[0466] 5) iii) eluting the bound peptides from the solid phase under conditions suitable to allow the elution of the peptide of interest.

[0467] 119. The method of any one of items 101 to 118, wherein step 5) comprises a chromatographic step iv), in particular liquid chromatography such as a HPLC, micro LC, nano LC or rapid LC, of the sample.

[0468] 120. The method of any one of items 101 to 119, wherein enrichment step 5) comprises i) contacting the sample with a solid phase having a surface for the binding of the peptide of interest under conditions wherein the peptide of interest binds to the solid phase, preferably wherein the binding is a selective binding of the peptide of interest, ii) washing of the solid phase to which the peptide of interest, if present in the sample, is bound, preferably under conditions which do not elute the peptide of interest, iii) eluting the bound peptides from the solid phase, and / or iv) a chromatographic step, in particular liquid chromatography such as a HPLC, micro LC, nano LC or rapid LC, of the sample.

[0469] 121. The method of any one of items 117 to 120, wherein the solid phase comprising an antibody is incubated in step 5) i) for up to about 2 hours, preferably around 0. 1 to around 2 hours.

[0470] 122. The method of any one of items 101 to 121, wherein step 6) comprises an MS / MS analysis, preferably a triple quadrupole-MS / MS analysis.

[0471] 123. The method of any one of items 101 to 122, wherein the ratio of the internal standard of Thyroglobulin or ApoAl to Thyroglobulin or ApoAl is determined and the concentration of Thyroglobulin or ApoAl is quantified.

[0472] 124. A method for quantification of Thyroglobulin or ApoAl by mass spectrometry comprising the steps of:

[0473] 1) providing a sample comprising proteins;

[0474] 2) optionally adding an internal standard of Thyroglobulin or ApoAl, preferably wherein the internal standard is isotope labeled, preferably wherein the internal standard is stable isotope labeled with13C and / or15N;

[0475] 3) subjecting the sample from step 1) or 2) to a digestion step to provide peptide fragments of the proteins including Thyroglobulin or ApoAl, if present, wherein the digestion comprises i) adding a digestion buffer comprising the organic solvent 2-propanol, wherein the final 2-propanol concentration is about 15 to 40 %, and ii) adding Trypsin, wherein Trypsin is added at a ratio (w / w) protein in the sample :Trypsin 1: 15-1:40, and incubating less than about 60 min;

[0476] 4) inhibiting Trypsin pH independent, wherein an inhibitor of Trypsin is added to the mixture of 3); 5) optionally enriching a peptide of interest, wherein the peptide of interest is a peptide of Thyroglobulin or ApoAl, wherein the enrichment step 5) comprises a bound / free separation step i) comprising contacting the sample with a solid phase having a surface for the binding of the peptide of interest under conditions wherein the peptide of interest binds to the solid phase, wherein the enrichment step 5) further comprises ii) washing of the solid phase to which the peptide of interest, if present in the sample, is bound, preferably under conditions which do not elute the peptide of interest; and / or iii) eluting the bound peptides from the solid phase under conditions suitable to allow the elution of the peptide of interest,, and iv) a chromatographic step such as a HPLC of the sample; and

[0477] 6) subjecting the mixture of 5) or 4) to an analysis by mass-spectrometry (MS), wherein the concentration of Thyroglobulin or ApoAl is determined, wherein the ratio of the internal standard of Thyroglobulin or ApoAl to Thyroglobulin or ApoAl, respectively, is determined and the concentration of Thyroglobulin or ApoAl is quantified; wherein step 6) comprises an MS / MS analysis; and wherein steps 1 to 6 are carried out in about 2.5 to 3.5 hours.

[0478] 125. The method of item 124, wherein the digestion buffer has a pH of about 7 to about 10.

[0479] 126. The method of item 124 to 125, wherein the digestion buffer comprises 50 mM Tris- HCL.

[0480] 127. The method of any one of items 124 to 126, wherein the incubation in step 3) ii) is carried out at a temperature range from about 35°C to about 40°C, preferably at about 37°C.

[0481] 128. The method of any one of items 124 to 127, wherein the digestion of step 3) is carried out with Trypsin, particularly in a ratio (w / w) protein in the sample: Trypsin of 1: 15-1:40 in a digestion buffer comprising of about 15 to 40% 2-Propanol at a pH of about 8.5, for a time period of about 5 to 30 min at a temperature of about 37°C, and the digestion is stopped by adding 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid up to a final concentration of 5- 50 mM.

[0482] 129. The method of any one of items 124 to 128, wherein the sample is whole blood, serum, plasma, bronchioalveolar lavage (BAL), epithelial lining fluid (ELF), urine, sputum or sweat, preferably serum or plasma.

[0483] 130. The method of any one of items 124 to 129, wherein the sample is not subjected to a denaturation step using denaturating agents, reduction, alkylation and / or precipitation before adding the digestion buffer.

[0484] 131. The method of any one of items 124 to 130, wherein step 3) ii) is incubated for less than about 45 min, preferably less than about 30 min, more preferably less than about 15 min.

[0485] 132. The method of any one of items 124 to 131, wherein step 3) ii) is incubated for at least about 60 sec to less than 45 min, preferably for at least about 90 sec to less than about 30 min, more preferably for at least about 2 min to less than about 20 min, most preferably for at least about 5 min to less than about 20 min.

[0486] 133. The method of any one of items 124 to 132, wherein the inhibitor is an inhibitor of Trypsin, preferably 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid.

[0487] 134. The method of item 133, wherein 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid is added at a final concentration of 0.1-100 mM, preferably of 1 mM-40 mM, more preferably of 5-20 mM, most preferably of 5-15 mM.

[0488] 135. The method of any one of items 124 to 134, wherein the solid phase comprising an antibody is incubated in step 5) i) for up to around 2 hours, preferably around 0. 1 to around 2 hours.

[0489] 136. Use of the kit of any one of items 34 to 40 in a method according to any one of items 101 to 135.

[0490] 137. A diagnostic system for determining Thyroglobulin or ApoAl in a sample adapted for performing the method according to any one of items 101 to 135.

[0491] 138. Use of the diagnostic system according to item 137 in the method of any one of items 101 to 135. Examples

[0492] The following examples are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit.

[0493] A method for ultra-fast proteolytic digestion for determining concentrations of proteins of interest from complex sample matrix

[0494] Trypsin is most commonly used endopeptidases to generate peptides for LC-MS / MS analysis of proteins. Conventionally, in order for trypsin to function effectively, proteins need to be unfolded with harsh denaturation agents. However, these chemicals have negative impacts on throughput and automation capability of the whole workflow: i) they impair the activity of the trypsin, thereby longer digestion time is needed, ii) can inhibit follow-up biological steps e.g. antibody enrichment, iii) can interfere with LC-MS analysis.

[0495] Therefore, further downstream procedures and instrumentation is required to remove them from digestion mix (e.g. clean-up steps, vacuum centrifugation).

[0496] Figure 1 shows a graphical illustration of conventional peptide-centric, immunoenrichment based absolute quantification of protein from complex matrixes by LC-MS / MS.

[0497] Whereas Figure 2 shows a graphical illustration of the developed peptide-centric, immunoenrichment based absolute quantification of protein from complex matrixes by LC- MS / MS.

[0498] Therefore, a method and kit components for high sensitive protein quantification with a medically relevant, low abundant protein biomarker, thyroglobulin from a complex matrix was devised.

[0499] Method and kits component provides solutions for

[0500] 1) Increasing throughput 2) Automation capabilities

[0501] The major bottleneck for the throughput is the long digestion times. To overcome this issue two alternative approaches were devised.

[0502] A) Heat based digestion:

[0503] Example-1: Proteins need to be denatured for efficient and fast digestion

[0504] Conventional trypsin digestion require unfolding of proteins with denaturation agents, such as guanidine hydrochloride, urea, sodium dodecyl sulfate (SDS), sodium deoxycholate (DOC)) for efficient proteolytic digestion. However, these chemicals have three main negative impact on the whole system i) they reduce the activity of the trypsin, ii) they inhibit subsequent biological steps (e.g. antibody enrichment) and iii) if not removed, interfere with LC-MS analysis.

[0505] Some of them can be removed clean-up procedure ( e.g. SPE), however this brings the requirement of additional handling steps and instrumentation (e.g. vacuum centrifugation).

[0506] Description:

[0507] In order to test whether trypsin can digest complex protein mixture (e.g. serum) without use of denaturing agent, 2-pL serum was mixed with 26 pL of conventional trypsin digestion buffer (50 mM NH4HCO3 buffer, pH 7.8). The reaction mix was incubated for 10 min at 37°C for temperature equilibration on the thermoshaker. Then 4 pL (1 pg / pL) Trypsin (Sigma) was added to reaction mix, mixed well, and incubated at 37°C for 1, 3, 5, 10, 30 min, 1 hour (hr), 2 hours (hr), 3 hr, 5.5 hr and 18 hr with shaking at 900 rpm on thermoshaker. Trypsin digestion was quenched by adding formic acid solution in H2O with a final concentration of 2 % (v / v). 25 % digestion solution was run with NuPAGE (Invitrogen) 4- 12 % Bis-Tris gels with MOPS buffer according to manufacturer’s instructions (See NuPAGE® Technical Guide, manual part no. IM-1001) and then stained with Imperial Protein Stain (Thermo Scientific) according to manufacturer’s instructions (see Fig. 3).

[0508] Fig. 3 shows SDS-PAGE image of trypsin digestion of serum proteins with conventional buffer system without denaturation in a time dependent manner. “Input lane” is the negative control where instead of 4 pL ( 1 pg / pL) Trypsin, 4 pL H2O is added to the reaction mix and it was processed as described above with an incubation time of 3 hr at 37°C. Multiple high molecular weight discrete protein bands show how undigested proteins run on the SDS- PAGE gel. As expected, human serum albumin protein, which is the highest abundant protein in serum, is the thickest band at around 55 kDa level.

[0509] Fig. 3 “2 % FA lane” is the second negative control in which, digestion mixture contained 2 % formic acid (v / v) in addition to trypsin. This brings the pH of the reaction mix < pH 3, where trypsin is inactive. Reaction mix was incubated at 37°C for 3 hr with shaking at 900 rpm on the thermoshaker. This indicates that even after short digestion times of 1-10 min, trypsin is completely inhibited in the presence of 2 % formic acid (v / v) and time -dependent digestion can be faithfully interpreted.

[0510] Even though some of the high MW protein bands (> 62 kDa) disappeared over a time, complete digestion of serum proteins was not achieved. This indicates that the denaturation step is essential for an efficient trypsin digestion complex matrix like serum.

[0511] In order to find a denaturing condition which does not reduce the trypsin activity and at the same time to increase the kinetics of the trypsinization digestion a heat-assisted denaturation was tested.

[0512] Example 2: Trypsin is not functional at elevated temperature with conventional buffer

[0513] In order to test whether trypsin would digest complex protein mixture with conventional buffer at elevated temperatures, 2 pL serum was mixed with 26 pL of 50 mM NH4HCO3 buffer, pH 7.8. The reaction mix was initially incubated for 10 min at following temperatures for equilibration: 37°C, 45°C, 55°C, 60°C, 65°C, 70°C, 75°C and 80°C. Then 4 pL (1 pg / pL) Trypsin (Sigma) added to reaction mix, mixed well, and incubated at respective temperature for 3 min with shaking at 900 rpm. After digestion, samples were equilibrated at room temperature for 5 min and then trypsin digestion was quenched by adding formic acid solution with final concentration of 2 % (v / v). 25 % digestion solution was run on SDS- PAGE as previously described in Example-1 (see Fig. 4).

[0514] Fig. 4 shows SDS-PAGE image of trypsin digestion of serum proteins with conventional buffer system without denaturation in a temperature dependent manner. “Input lane” is a control lane in which trypsin addition is omitted; instead 4 pL H2O was added and it was processed as described above with an incubation time of 10 min at 70°C. As expected the serum albumin band, which is the highest abundant protein in serum was seen as the thickest band at around 55 kDa. With increasing digestion temperature until 80 °C, there is no visible difference in the protein band pattern compared to 37 °C and thickness of the serum albumin band. This showed that activity of trypsin has not been increased at elevated temperatures with conventional buffer system.

[0515] Example 3: Trypsin is functional at elevated temperature with supplementation of CaCh in the digestion buffer.

[0516] In order to test whether trypsin would digest complex protein mixture with conventional buffer at elevated temperatures with CaCT supplementation in digestion buffer, 2 pL serum was mixed with 26 pL of conventional digestion buffer, pH 7.8, with increasing CaCT concentration (O-lOOOmM). Reaction mix was pre-heated for 10 min at 60°C. Then 4 pL (1 pg / pL) Trypsin (Sigma) was added to the reaction mix, mixed well, and incubated at a respective temperature for 3 min with shaking at 900 rpm. After 3 min, samples were removed from the thermoshaker, equilibrated at room temperature for 5 min and then trypsin digestion was quenched by adding formic acid solution with final concentration of 2 % (v / v). 25 % digestion solution was run on SDS-PAGE as previously described in the Example- 1 (see Fig. 5).

[0517] Fig. 5 shows SDS-PAGE image for CaCT concentration screening of trypsin digestion with heat-assisted buffer system. ’’Input lane” is a control lane in which trypsin addition is omitted and instead 4 pL H2O was added. It was processed as described above with an incubation time of 10 min at 60°C. As expected serum albumin band, the highest abundant protein in serum was seen the thickest band at around 55 kDa.

[0518] The effect of the calcium chloride on trypsin stability started to be seen at concentration above > 250 mM. The human serum albumin band disappeared at CaC concentration of 500 mM. This result shows that trypsin is still functional at 60°C.

[0519] Example-4: Optimal temperature for trypsin activity is 60-70°C

[0520] To determine optimal temperature for trypsin using heat-assisted digestion buffer, 2 pL serum was mixed with 26 pL of heat-assisted digestion buffer. The reaction mix was incubated for 10 min at following temperatures for equilibration: 37°C, 45°C, 55°C, 60°C, 65°C, 70°C, 75°C and 80°C. Then 4 pL (1 pg / pL) Trypsin (Sigma) was added to reaction mix, mixed well, and incubated at respective temperature for 3 min with shaking at 900 rpm. Samples were removed from the thermoshaker, incubated at room temperature for 5 min and then trypsin digestion was quenched by adding formic acid solution with final concentration of 2 % (v / v). 25 % digestion reaction was run with on SDS-PAGE as previously described (see Fig. 6).

[0521] Fig. 6 “Input lane” is a control lane in which trypsin addition is omitted and instead 4 pL H2O was added and it was processed as described above with an incubation time of 3 min at 70°C.

[0522] Unlike trypsin digestion with conventional buffer (see Fig. 3), > 45°C majority protein bands disappeared, which shows that with heat-assisted digestion buffer trypsin is still active at elevated temperatures (see Fig. 6). For example for albumin already 45°C were sufficient to digest it > 95%.

[0523] Example-5: Quenching of trypsin without changing pH of the solution

[0524] The conventional way to quench trypsin activity is lowering the pH of the solution down to < pH 3. However, lowering pH would be incompatible for subsequent immunoprecipitation of target peptide. On the other hand, presence of minute amounts of active trypsin would pose the issue of antibody digestion, which would result in lower sensitivity. Therefore, alternative irreversible inactivation of trypsin without changing the pH of the solution was sought.

[0525] To test whether protease inhibitors quench trypsin activity at high temperatures, serum trypsin digestion was performed in the presence of these inhibitors. 2 pL serum was mixed with 26 pL of heat-assisted digestion buffer. Digestion mix was supplemented with either Leupeptin (Sigma, final concentration of 20-162 pM) solution or PefaBloc SC (Roche, final concentration of 4-50 mM). The reaction mix was incubated for 10 min at 70 °C for equilibration. Then 4 pL (1 pg / pL) Trypsin (Sigma) was added to the reaction mix and digestion was performed for 10 min. Trypsin digestion was quenched by adding formic acid solution to a final concentration of 2 % (v / v). 25 % digestion reaction was run on SDS-PAGE as previously described in Example 1 (see Fig. 7).

[0526] Fig. 7 “Input lane” is a control lane in which trypsin addition is omitted and instead 4 pL H2O was added and it was processed as described above.

[0527] While Leupeptin did not quench the trypsinization at tested concentrations, Pefabloc SC at concentrations > 5 mM was sufficient to inhibit trypsin even at high temperatures (see Fig. 7). Example- 6: Optimal pH for both trypsin and immunoprecipitation

[0528] To find out the optimal pH value for trypsin digestion and peptide immunoprecipitation, 1 pg Thyroglobulin (Tg)- peptide antibody-# 1 (Abeam Cat.# ab264461) and antibody -#2 (Abeam Cat.# ab264462) was immobilized onto magnetic beads and incubated with synthetic target peptides.

[0529] Fig. 8 shows a histogram plot showing Tg peptide recovery efficiency from immunoprecipitation at different pH values.

[0530] To find optimum pH for the Tg peptide immunoprecipitation, 1 pg of biotinylated Tg- Peptide Antibodies (Abeam, Cat.# ab264461, against “VIFDANAPVAVR” (SEQ ID NO:

[0531] 4) peptide and Abeam, Cat.# ab264462, against “FSPDDSAGASALLR” (SEQ ID NO: 5) peptide ) were separately immobilized onto 75 pg of Streptavidin coated magnetic Roche Elecsys SA Coat-Bead (Cat. #11 865 943 001). Beads were washed 3 times with 75 mM Tris-HCl, pH 7.2, 150 mM CaCF. After last washing step, Ab-immobilized magnetic beads were diluted with 175 pL of 50 mM Tris-HCl, pH 6.5 or 7.2 or 8.5 or 9.5, 500 mM CaCT. 3 % glycerol in H2O and 6.25 finol of following synthetic Tg peptides: Peptide- 1 “VIFDANAPVAVR” (SEQ ID NO: 4), Peptide-2 “FSPDDSAGASALLR” (SEQ ID NO:

[0532] 5). These peptides were spiked into respective bead suspension. Ab-immobilized beads and synthetic peptides were incubated for 30 min at room temperature with shaking at 600 rpm. Beads were washed 2 times with 75 mM Tris-HCl, pH 6.5 or 7.2 or 8.5 or 9.5, 150 mM CaC12 in H2O. Bound peptides were eluted with 50 pL of 2 % formic acid , 5 % acetonitrile in H2O. 40 % of the elution solution was injected into LC-MS. In order to calculate the elution efficiency, 2.5 pL of 1 nM was injected to LC-MS / MS.

[0533] 1 nM of “VIFDANAPVAVR” (SEQ ID NO: 4) and 1 nM of “FSPDDSAGASALLR” (SEQ ID NO: 5) was injected to LC-MS / MS as an input peptide material.

[0534] LC-MS / MS Parameters:

[0535] Detection of the peptides were carried out by Thermo Scientific TSQ Quantiva Triple-Stage Quadrupole Mass Spectrometer coupled to Thermo Scientific Vanquish UPLC. Peptides were loaded on the Cl 8 reverse phase column (Waters, Acquity® UPLC BEH C18 Column 130A, 1.7 pm, 2.1 mm X 150 mm) with 2 % acetonitrile, 0.1 % formic acid in H2O and separated with gradient of 21%-25 % acetonitrile, 0.1 % formic acid in H2O over a 3 min with a with flow rate 0.300 mL / min. Monitored mass transition for the VIFDANAPVAVR (SEQ ID NO: 4) peptide is m / z 636.4 / 1059.6 and for FSPDDSAGASALLR (SEQ ID NO: 5) peptide is m / z 703.9 / 586.8.

[0536] Example- 7: Method comparison between Elecsys TG II Assay and LC-MS / MS method based on heat-assisted digestion and Tg peptide IP

[0537] 16 human serum samples with negligible anti-Tg autoantibody amount (< 5 lU / mL, analyzed by Roche Anti-Tg Assay according to manufacturing instructions) were analyzed by Elecsys TG II assay (Roche, according to manufacturer instructions) and LC-MS / MS based heat- assisted digestion and Tg peptide IP workflow. Results were plotted in Figure 9.

[0538] LC-MS / MS based heat-assisted digestion and Tg peptide Pep IP workflow was as follows: Streptavidin coated magnetic Roche Elecsys SA Coat-Beads (Cat. #11 865 943 001, 0.48 mg / mL) were washed 3 times with 50 mM Tris-HCl, pH 7.2 and brought to concentration of 2.5 mg / ml with 50 mM Tris-HCl, pH 7.2. 3 pg of Tg peptide antibody (Abeam, ab264462, 0.5 mg / ml) was immobilized onto 75 pg of magnetic beads and stored at 4°C till usage. 125 pL of serum samples were mixed with 25 pL of heat-assisted digestion buffer (600 mM Tris- HCl, pH 9.5, 16 % glycerol, 4 M CaCL), then 5 pL stable isotope labeled full length Tg (Arg and Lys were stable isotope labeled, 0.24 pg / mL) was added. 35 pL of trypsin (10 pg / pL) solution was added to reaction mix and reaction mix incubated at 60°C for 10 min with shaking at 900 rpm on the thermoshaker. Trypsin was irreversibly inhibited by adding 5 pL Pefabloc (400 mM). Then 75 pg of Tg peptide antibody immobilized magnetic beads were added to trypsin quenched digestion mix and incubated for 2 hr at room temperature on a rotator. Beads were washed 3 times with 50 mM Tris, pH 8.5, 50 mM CaCh and the peptide of interest was eluted with 0.25 % formic acid, 5 % acetonitrile solution and proceeded with LC-MS / MS analysis.

[0539] LC-MS / MS Parameters: Detection of the peptides were carried out by Thermo Scientific TSQ Quantiva Triple-Stage Quadrupole Mass Spectrometer coupled to Thermo Scientific Vanquish UPLC. Peptides were loaded on the C18 reverse phase column (Waters, Acquity® UPLC BEH C18 Column 130A, 1.7 pm, 2.1 mm X 150 mm) with 2 % acetonitrile, 0.1 % formic acid in H2O and separated with gradient of 21 %-25 % acetonitrile, 0.1 % formic acid in H2O over a 3 min with a with flow rate 0.300 mL / min. Mass transitions of the endogenous FSPDDSAGASALLR (SEQ ID NO: 5) Tg peptide was m / z 703.9 / 586.8 Da and mass transitions of the stable isotope labeled peptide (SEQ ID NO: 5; stable isotope labeled Arg, as described above) was m / z 708.9 / 591.8 Da. The LC-MS / MS method results showed very good linearity and correlation with the Roche Elecsys TG II assay (see Fig. 9), which demonstrates that complete and faithful digestion of Tg digestion was achieved.

[0540] B) Organic Solvent based digestion

[0541] Alternatively, a devise method was set out, which does not require high temperatures but instead can operate at 37°C.

[0542] For this purpose, mild detergent and organic solvents-based buffer systems were screened, in which proteins are denatured but trypsin is still functional.

[0543] Example-8: Organic solvents can strongly aid in trypsin digestion efficiency

[0544] To investigate if trypsin can digest complex protein matrix using organic solvent based buffer systems, 4 pL of serum was mixed with 4.8 pL of 750 mM Tris buffer pH 8.5 and organic solvent in H2O to set final organic solvent concentration to 0 %, 10 %, 20 %, 30 %, 40 %, 50 % and 60 %. Tested organic solvents were acetonitrile, 2-propanol, methanol and ethanol. The reaction mix was incubated for 10 min at 37°C for equilibration. Then 4 pL (1 pg / pL) Trypsin (Sigma) was added to the reaction mix, mixed well, and incubated at 37°C for 9 min with shaking at 900 rpm. Trypsin digestion was quenched by adding formic acid solution at a final concentration of 2 % (v / v). 10 % of the digestion reaction mix was run on SDS-PAGE as previously described (see Fig 10 A-D). Fig. 10 A shows acetonitrile, Fig. 10 B shows 2-propanol, Fig. 11 C shows methanol and Fig. 10 D shows ethanol as organic solvent.

[0545] Fig 10 A-D “Input” is the control sample in which organic solvent and trypsin addition is omitted and instead the same volume of H2O was added. Input sample was processed as described above.

[0546] Fig 10 A-D “Negative Control-1” is the sample, in which the organic solvent concentration is 30 %, however trypsin addition is omitted and instead a solution of 4 pL H2O was added and processed as described above.

[0547] Fig 10 A-D “Negative Control-2” is the sample, in which Trypsin was added however, organic solvent addition is omitted. Instead of organic solvent H2O was added and processed as described above. With increasing organic solvent concentration high molecular weight protein bands and discrete albumin disappeared. Optimal organic solvent concentration for trypsin varies between 20-40 % depending on the individual organic solvent used for digestion.

[0548] Example-9: Thyroglobulin quantification by organic solvent assisted buffer digestion

[0549] 16 human serum samples with negligible anti-Tg autoantibody amount (< 5 lU / mL, analyzed by Roche Anti-Tg Assay according to manufacturing instructions) were analyzed by Elecsys TG II assay (Roche, according to manufacturer instructions) and by the organic solvent- assisted LC-MS / MS Tg Pep IP workflow for method comparison purposes. Results were plotted in Figure 11.

[0550] Organic solvent assisted LC-MS / MS Tg peptide IP workflow was as follows: 70 pL of serum sample was mixed with 10 pL of full length stable isotope labeled-Tg (Arg 10, Lys8 labeling, 0.23 pg / mL). Then 60 pL of 90 % 2-Propanol, 200 mM Tris-HCl, pH 8.5 were supplemented. 15 pL Trypsin (Sigma, 22.5 pg / pL) was added and samples were incubated for 9 min at 37°C with shaking 900 rpm on thermomixer. Trypsin was quenched by addition of 3 pL PefaBloc SC (400 mM). Then 3 pg of Tg peptide antibody (Abeam, ab264462“against “FSPDDSAGASALLR” (SEQ ID NO: 5)) immobilized onto 75 pg of magnetic beads were added to the digestion mix and incubated for 2 hr at room temperature. Beads were washed 3 times with 50 mM Tris, pH 8.5, 50 mM CaCh and the peptide of interest was eluted with 0.25% FA, 5 % ACN solution. LC-MS / MS Parameters: Detection of the peptides were carried out by Thermo Scientific TSQ Quantiva Triple-Stage Quadrupole Mass Spectrometer coupled to Thermo Scientific Vanquish UPLC. Peptides were loaded on the Cl 8 reverse phase column (Waters, Acquity® UPLC BEH C18 Column 130A, 1.7 pm, 2.1 mm X 150 mm) with 2 % acetonitrile, 0.1 % formic acid in H2O and separated with gradient of 21%-25 % acetonitrile, 0.1 % formic acid in H2O over a 3 min with a with flow rate 0.300 mL / min. Mass transitions of the endogenous FSPDDSAGASALLR (SEQ ID NO: 5) Tg peptide was m / z 703.9 / 586.8 Da and mass transitions of the stable isotope labeled peptide (SEQ ID 5, stable isotope labeled Arg, as described above) was m / z 708.9 / 591.8 Da. A set of external calibrators with a defined amount of endogenous Tg was processed as described above to generate an external calibration curve, which is used to calculate endogenous Tg concentration in tested human serum samples. -n -

[0551] The LC-MS / MS method results showed very good linearity and correlation with the Roche Elecsys TG II assay (see Fig. 9), which demonstrates that complete Tg digestion was achieved.

[0552] C) LC-MS / MS Tg Peptide IP method: further experiments

[0553] Example 10: LC-MS / MS Tg Peptide IP is “resilient” against autoantibodies: excess amount of Tg-antibodies spiked into serum to address the endogenous anti-Tg autoantibodies issue.

[0554] To test whether the developed LC-MS / MS Tg Peptide IP method is “resilient” against anti- Tg autoantibodies, a serum with Tg concentration of 5 ng / mL and with negligible amount of anti-Tg autoantibodies was selected (2.3 lU / mL) .To mimic patient derived anti-Tg autoantibodies, two different Elecsys TG II assay capture antibodies (non-biotinylated) was spiked into the serum at a ratio of Elecsys Tg capture Ab:Tg from about 5000 to about 13000. Samples were divided into two sets and one set was analyzed by Elecsys TG II assay (Roche, according to manufacturer instructions) and the other set was analyzed by the heat- assisted LC-MS / MSTg peptide IP method as described above in Example 7. The immunometric approach failed to detect endogenous Tg in all antibody spiked samples. On the other hand, LC-MS / MS method quantified endogenous Tg amount accurately for all 6 samples with a very good CV less than 5 %. Additionally, these results showed that the LC- MS / MS approach provides additional medical value over immunometric quantification of endogenous Tg particularly for patients who produce autoantibodies against Tg.

[0555] Figure 12 shows quantification of endogenous Tg in serum by Elecsys TG II Assay and LC- MS / MS Tg peptide IP upon spiking of non-biotinylated Elecsys TG II capture antibodies to mimic anti-Tg autoantibodies

[0556] Example 11: LC-MS / MS Tg Peptide IP method is “resilient” against autoantibodies: spiking increasing amounts of SIL-Tg into serum with high endogenous Anti-Tg autoantibody amount

[0557] To test whether the endogenous anti-Tg autoantibody would interfere for accurate detection of Tg by the inventive method, a serum with very high anti-Tg autoantibody amount (1998 lU / mL) was selected and an increasing amount of stable isotope labeled Tg was spiked into the serum. Samples were processed as described in Example-7. A very good linear correlation between peak area of SIL-Tg peptide vs total spiked-in Tg amount was measured, which shows that high amounts of endogenous anti-Tg autoantibodies do not interfere with LC-MS / MS Tg Peptide IP method.

[0558] Figure 13 A) shows peak area of stable isotope labeled Tg peptide (SEQ ID NO: 5, stable isotope labeled as described above) vs total amount of spiked in stable isotope labeled Tg. B) zoom into the low Tg spiked region (red square in Figure 13 A).

[0559] Example 12: Comparison of organic solvent assisted LC-MS / MS Tg Peptide IP method with Elecsys Tg II assay for samples containing negligible anti-Tg autoantibodies (< 5 lU / mL) and high anti-Tg autoantibody containing samples serums (30-1998 lU / mL)

[0560] As a method comparison, two sets of serum samples were analyzed. First set of samples had (n=16) negligible anti-Tg autoantibody amount (< 5 lU / mL) and second set had high anti- Tg autoantibody amount (n =9, 30-1998 lU / mL). Samples were processed as described in Example-9. Samples having negligible amount of anti-Tg autoantibodies showed very good correlation between immunometric (Elecsys TG II assay) and LC-MS / MS (see Figure 9). This shows that LC-MS / MS methodology faithfully and accurately quantifies endogenous Tg. For samples having high amount of anti-Tg autoantibody (30-1998 lU / mL), in which immumetric measurements have limitations for accurate quantification, LC-MS / MS method measured higher endogenous Tg concentrations (Fig. 14). This highlights the added value of the LC-MS / MS method.

[0561] Figure 9 shows correlation with organic solvent assisted LC-MS / MS Tg Peptide IP method vs Elecsys Tg II assay with serum negligible anti-Tg autoantibodies

[0562] Figure 14 shows Tg organic solvent assisted LC-MS / MS Tg Peptide IP method vs Elecsys Tg II assay with serum high anti-Tg autoantibodies (n=9)

[0563] Example 13: LC-MS / MS ApoAl method without specific analyte enrichment

[0564] Figure 15 shows SDS-PAGE image of organic solvent elution profile of peptides from carboxylate-modified magnetic beads.

[0565] Bead preparation: Sera-Mag carboxylate-modified (GE Life Sciences, Cat#: 45152105050250, 50 mg / mL) and Sera-Mag carboxylate-modified (GE Life Sciences Cat#, 65152105050250, 50 mg / mL) were mixed 1: 1 ratio and washed three times with 2 % acetonitrile in H2O and then suspended in 90 % acetonitrile with a bead concentration of 50 mg / mL. 2.5 pL of serum was mixed well with 10 pL 80 % 2-isopropanol, 50 mM Tris HC1 pH 8.0. Then 10 pL of Trypsin (Sigma, 1 pg / pL) added to reaction mixture and incubated at 37 C for 9 min. Trypsin was quenched by adding 2 pL PefaBloc SC (400 mM). Upon quenching of the trypsin, 15 pL of magnetic bead suspension (50 mg / mL) was added to digestion mix and immediately 130 pL 100 % acetonitrile added to bring the organic solvent content to 90 %. Reaction mix was incubated at room temperature for 10 min on a rotator. Beads were washed 3 times with 95 % acetonitrile and peptides were eluted from beads with 2 %-10%-25%-40% acetonitrile solution in H2O. To see what remained on the beads, after first elution, beads were incubated with IX NuPAGE LDS sample buffer (Invitrogen , Cat.# NP0007) to elute remaining strongly bound peptides. 5 % of the elution volume was run on SDS-PAGE as previously described in Example-1. “Serum Input” lane shows the input material for organic solvent assisted digestion. “Digestion” lane shows input material for carboxylate-modified magnetic beads purification. The remaining lanes shows peptide elution profile with increasing organic solvent in the elution buffer and second elution with 1 X LDS buffer (Invitrogen , Cat.# NP0007) . With increasing organic solvent content in the elution buffer, less complex peptide solution was eluted from carboxylate-modified magnetic beads.

[0566] Figure 16 shows CV estimation of the ApoAl quantification with spiking of full length stable isotope labeled ApoAl into serum.

[0567] In order to estimate the CV of the LC-MS / MS ApoAl method without specific analyte enrichment full length stable isotope labeled ApoAl (Promise Proteomics, Cat.#: AP176846) was spiked into 6 serum samples with concentration of 25 pg / mL and 50 pg / mL. Samples were processed as described above in the Example- 13 and bound peptides were eluted with 25 % acetonitrile solution. LC-MS / MS Parameters: Detection of the peptides were carried out by Thermo Scientific TSQ Quantiva Triple-Stage Quadrupole Mass Spectrometer coupled to Thermo Scientific Vanquish UPLC. Peptides were loaded on the C18 reverse phase column (Waters, Acquity® UPLC BEH C18 Column 130A, 1.7 pm, 2.1 mm X 150 mm) with 2 % acetonitrile, 0.1 % formic acid in H2O and separated with gradient of 20%-40 % acetonitrile, 0.1 % formic acid in H2O over a 3 min with a with flow rate 0.300 mL / min. Mass transitions of the endogenous ApoAl peptide LLDNWDSVTSTFSK (SEQ ID NO: 6) Tg peptide was m / z 807.0 / 670.4 Da, 807.0 / 971.5 Da and mass transitions of the stable isotope labeled ( N-15) peptide (SEQ ID NO: 6, K stable isotope labeled) was m / z 815.5 / 677.4 Da and 815.5 / 981.5 Da. Data processing was carried out by Chromeleon 7. For both different full length stable isotope labeled ApoAl spiking experiment, endogenous ApoAl was quantified with estimated CV of less than 20 %. Example 14: Effect of the IP time on CV estimation of organic solvent assisted LC- MS / MS Tg peptide IP method.

[0568] In order to show the effect of the immunoprecipitation time on Tg quantification accuracy, full length stable isotope labeled Tg (0.24 pg / mL) was spike into 4 serum samples and were analyzed as described above in Example 8 to estimate and compare the CV. Prolonged immunoprecipitation time does not alter the ratio of endogenous Tg peptide to spiked in internal standard Tg peptide. However, quantification accuracy was improved with prolonged immunoprecipitation (IP). This indicates that shorter IP time can be performed without sacrificing the quantification accuracy. Figure 17 shows the effect of the immunoprecipitation time on CV estimation for organic solvent assisted LC-MS / MS based Tg peptide IP method.

[0569] Table: 1: Sequences disclosed in the present invention WESQLPQPRACQRPQLWQTIQTQGHFQLQLPPGKMCSADYA DLLQTFQVFILDELTARGFCQIQVKTFGTLVSIPVCNNSSVQV GCLTRERLGVNVTWKSRLEDIPVASLPDLHDIERALVGKDLL GRFTDLIQSGSFQLHLDSKTFPAETIRFLQGDHFGTSPRTWFG CSEGFYQVLTSEASQDGLGCVKCPEGSYSQDEECIPCPVGFYQ EQAGSLACVPCPVGRTTISAGAFSQTHCVTDCQRNEAGLQCD QNGQYRASQKDRGSGKAFCVDGEGRRLPWWETEAPLEDSQ CLMMQKFEKVPESKVIFDANAPVAVRSKVPDSEFPVMQCLT DCTEDEACSFFTVSTTEPEISCDFYAWTSDNVACMTSDQKRD ALGNSKATSFGSLRCQVKVRSHGQDSPAVYLKKGQGSTTTL QKRFEPTGFQNMLSGLYNPIVFSASGANLTDAHLFCLLACDR DLCCDGFVLTQVQGGAIICGLLSSPSVLLCNVKDWMDPSEA WANATCPGVTYDQESHQVILRLGDQEFIKSLTPLEGTQDTFT NFQQVYLWKDSDMGSRPESMGCRKDTVPRPASPTEAGLTTE LFSPVDLNQVIVNGNQSLSSQKHWLFKHLFSAQQANLWCLSR CVQEHSFCQLAEITESASLYFTCTLYPEAQVCDDIMESNAQGC RLILPQMPKALFRKKVILEDKVKNFYTRLPFQKLMGISIRNKV PMSEKSISNGFFECERRCDADPCCTGFGFLNVSQLKGGEVTCL TLNSLGIQMCSEENGGAWRILDCGSPDIEVHTYPFGWYQKPI AQNNAPSFCPLVVLPSLTEKVSLDSWQSLALSSVVVDPSIRHF DVAHVSTAATSNFSAVRDLCLSECSQHEACLITTLQTQPGAV RCMFYADTQSCTHSLQGQNCRLLLREEATHIYRKPGISLLSYE ASVPSVPISTHGRLLGRSQAIQVGTSWKQVDQFLGVPYAAPP LAERRFQAPEPLNWTGSWDASKPRASCWQPGTRTSTSPGVSE DCLYLNVFIPQNVAPNASVLVFFHNTMDREESEGWPAIDGSF LAAVGNLIVVTASYRVGVFGFLSSGSGEVSGNWGLLDQVAA LTWVQTHIRGFGGDPRRVSLAADRGGADVASIHLLTARATNS QLFRRAVLMGGSALSPAAVISHERAQQQAIALAKEVSCPMSS SQEVVSCLRQKPANVLNDAQTKLLAVSGPFHYWGPVIDGHF LREPPARALKRSLWVEVDLLIGSSQDDGLINRAKAVKQFEES

[0570] RGRTSSKTAFYQALQNSLGGEDSDARVEAAATWYYSLEHST DDYASFSRALENATRDYFIICPIIDMASAWAKRARGNVFMYH APENYGHGSLELLADVQFALGLPFYPAYEGQFSLEEKSLSLKI MQYFSHFIRSGNPNYPYEFSRKVPTFATPWPDFVPRAGGENY DLLQTFQVFILDELTARGFCQIQVKTFGTLVSIPVCNNSSVQV GCLTRERLGVNVTWKSRLEDIPVASLPDLHDIERALVGKDLL GRFTDLIQSGSFQLHLDSKTFPAETIRFLQGDHFGTSPRTWFG CSEGFYQVLTSEASQDGLGCVKCPEGSYSQDEECIPCPVGFYQ EQAGSLACVPCPVGRTTISAGAFSQTHLMQKFEKVPESKVIFD ANAPVAVRSKVPDSEFPVMQCLTDCTEDEACSFFTVSTTEPEI SCDFYAWTSDNVACMTSDQKRDALGNSKATSFGSLRCQVKV RSHGQDSPAVYLKKGQGSTTTLQKRFEPTGFQNMLSGLYNPI VFSASGANLTDAHLFCLLACDRDLCCDGFVLTQVQGGAIICG LLSSPSVLLCNVKDWMDPSEAWANATCPGVTYDQESHQVIL RLGDQEFIKSLTPLEGTQDTFTNFQQVYLWKDSDMGSRPESM GCRKDTVPRPASPTEAGLTTELFSPVDLNQVIVNGNQSLSSQK HWLFKHLFSAQQANLWCLSRCVQEHSFCQLAEITESASLYFT CTLYPEAQVCDDIMESNAQGCRLILPQMPKALFRKKVILEDK VKNFYTRLPFQKLMGISIRNKVPMSEKSISNGFFECERRCDAD PCCTGFGFLNVSQLKGGEVTCLTLNSLGIQMCSEENGGAWRI

[0571] LDCGSPDIEVHTYPFGWYQKPIAQNNAPSFCPLVVLPSLTEKV SLDSWQSLALSSVVVDPSIRHFDVAHVSTAATSNFSAVRDLC LSECSQHEACLITTLQTQPGAVRCMFYADTQSCTHSLQGQNC RLLLREEATHIYRKPGISLLSYEASVPSVPISTHGRLLGRSQAIQ VGTSWKQVDQFLGVPYAAPPLAERRFQAPEPLNWTGSWDAS KPRASCWQPGTRTSTSPGVSEDCLYLNVFIPQNVAPNASVLV FFHNTMDREESEGWPAIDGSFLAAVGNLIVVTASYRVGVFGF LSSGSGEVSGNWGLLDQVAALTWVQTHIRGFGGDPRRVSLA ADRGGADVASIHLLTARATNSQLFRRAVLMGGSALSPAAVIS HERAQQQAIALAKEVSCPMSSSQEVVSCLRQKPANVLNDAQ TKLLAVSGPFHYWGPVIDGHFLREPPARALKRSLWVEVDLLI GSSQDDGLINRAKAVKQFEESRGRTSSKTAFYQALQNSLGGE DSDARVEAAATWYYSLEHSTDDYASFSRALENATRDYFIICPI IDMASAWAKRARGNVFMYHAPENYGHGSLELLADVQFALG LPFYPAYEGQFSLEEKSLSLKIMQYFSHFIRSGNPNYPYEFSRK VPTFATPWPDFVPRAGGENYKEFSELLPNRQGLKKADCSFWS

[0572] KYISSLKTSADGAKGGQSAESEEEELTAGSGLREDLLSLQEPG SKTYSK

Claims

Patent Claims1. A method for determination or quantification of an analyte by mass spectrometry comprising the steps of:1) providing a sample comprising proteins;2) optionally adding an internal standard of the analyte, preferably wherein the internal standard is stable isotope labeled;3) subjecting the sample from step 1) or 2) to a digestion step to provide peptide fragments of the proteins including the analyte, if present, wherein the digestion comprises i) adding a digestion buffer, and ii) adding a endopeptidase, preferably Trypsin and incubating less than about 60 min;4) inhibiting the endopeptidase, preferably Trypsin, pH independent, wherein an inhibitor of the endopeptidase, preferably Trypsin, is added to the mixture of 3);5) optionally enriching a peptide of interest, wherein the peptide of interest is a peptide of the analyte; and6) subjecting the mixture of 4) or 5) to an analysis by mass-spectrometry (MS), wherein the presence or concentration of the analyte is determined.

2. The method of claim 1, wherein the analyte is Thyroglobulin or ApoAl .

3. The method of claim 2, wherein Thyroglobulin is homologues to the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2 or ApoAl is homologues to the amino acid sequence as shown in SEQ ID NO: 3.

4. The method of any one of claims 1 to 3, wherein the sample is not subjected to a denaturation step using denaturating agents, reduction, alkylation and / or precipitation before adding the digestion buffer.

5. The method of any one of claims 1 to 4, wherein the inhibitor is an inhibitor of endopeptidases, preferably 4-(2-Aminoethyl)-benzylsulfonylfluorid hydrochlorid.

6. The method of any one of claims 1 to 6. wherein the digestion buffer in step 3) i) is a buffer comprising calcium chloride.

7. The method of claim 6, wherein the digestion buffer in step 3) i) is a buffer comprising at least about 250 mM calcium chloride, preferable at least about 300 mM, more preferable at least about 500 mM.

8. The method of any one of claims 1 to 7, wherein the digestion buffer in step 3) i) comprises calcium chloride and wherein the incubation in step 3) ii) is carried out at a temperature range from about 50°C to about 75°C, preferably at about 60°C to about 70°C.

9. The method of any one of claims 1 to 5, wherein the digestion buffer comprises an organic solvent, preferable acetonitrile, 2-propanol, methanol or ethanol, more preferably 2-propanol.

10. The method of claim 9, wherein the digestion buffer comprises an organic solvent and wherein the final organic solvent concentration is about 15 to 40 %.

11. The method of any one of claims 1 to 5 or 9 or 10, wherein the digestion buffer comprises an organic solvent and wherein the incubation in step 3) ii) is carried out at a temperature range from about 35°C to about 40°C, preferably at about 37°C.

12. A kit comprising(i) a endopeptidase, preferably Trypsin,(ii) a digestion buffer, comprising calcium chloride or an organic solvent, preferably 2-propanol,(iii) an inhibitor of the endopeptidase, preferably Trypsin,(iv) optionally enrichment means for enriching peptides of interest, and(v) optionally an elution medium for use in eluting sample components from the enrichment means of (iv).

13. Use of the kit of claim 12 in a method according to any one of claims 1 to 11.

14. A diagnostic system for determining an analyte in a sample adapted for performing the method according to any one of claims 1 to 11.

15. Use of the diagnostic system according to claim 14 in the method of any one of claims I to 11.

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