(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2016/032319 Al 3 March 2016 (03.03.2016) P O P CT

(51) International Patent Classification: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, G01N 33/68 (2006.01) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (21) International Application Number: HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, PCT/NL2014/050587 KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, (22) International Filing Date: MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, 28 August 2014 (28.08.2014) OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, (25) Filing Language: English TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, (26) Publication Language: English ZW. (71) Applicants: ERASMUS UNIVERSITY MEDICAL (84) Designated States (unless otherwise indicated, for every CENTER ROTTERDAM [NL/NL]; Dr. Molewaterplein kind of regional protection available): ARIPO (BW, GH, 50, NL-3015 GE Rotterdam (NL). PEPSCAN SYSTEMS GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, B.V. [NL/NL]; Zuidersluisweg 2, NL-8243 RC Lelystad TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, (NL). DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, (72) Inventors: LUIDER, Theo Marten; c/o Dr. Molewater LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, plein 50, NL-3015 GE Rotterdam (NL). VAN DIJK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, Evert; c/o Zuidersluisweg 2, NL-8243 RC Lelystad (NL). GW, KM, ML, MR, NE, SN, TD, TG).

(74) Agent: JANSEN, CM.; V.O., Carnegieplein 5, 25 17 KJ Published: Den Haag (NL). — with international search report (Art. 21(3)) (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM,

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© (54) Title: PROTEIN QUANTITATION WITH MASS SPECTROMETRY. (57) Abstract: The present invention is directed to a method to quantitate a target protein in a biological sample by mass spectro - metry using a homopeptide as internal reference. The invention is further directed to kits comprising a homopeptide for quantitation S of a target protein in a biological sample by mass spectrometry. Title: Protein quantitation with Mass spectrometry.

The invention The present invention is directed to methods to determine and quantitate a target protein in a sample, and to determine the quality of the sample. The present invention provides a higher sensitivity and reliability of the quantitation of target proteins, especially in samples with high background and/or low concentrations of a target protein. Background Many markers for diseases are protein markers. The detection of protein markers may be straightforward when proteins are truncated by a nonsense mutation or fused to other proteins, e.g by simple Western blotting of cellular extracts. However, a large number of markers are missense mutations that alter the encoded proteins only subtly or are different in expression levels of proteins. Although it is theoretically possible to detect abnormal proteins directly with antibodies directed against mutant epitopes, doing so has been difficult to accomplish in practice. Because many different mutations can occur in a single cancer-causing protein, it is necessary to develop a specific antibody for each possible mutant epitope, thus increasing the difficulty of such a strategy. Thus, there is a critical need for assays that would permit quantitation of proteins in a generic fashion. Recent advances in Mass Spectrometry (MS) permit sampling of a large fraction of normal and abnormal cellular proteomes in an unbiased and specific fashion (Mann M, Kelleher NL (2008) Precision proteomics: The case for high resolution and high mass accuracy. Proc Natl Acad Sci USA 105:18132-18138). MS already has become the method of choice for quantifying protein levels, and a number of quantitative proteomics strategies for this purpose have been described (Gerber SA, Rush J , Stemman O, Kirschner MW, Gygi SP (2003) Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS (Proc Natl Acad Sci USA 100:6940-6945). Prior studies have shown that it is possible to identify post-translational altered proteins using MS, as well as to identify highly abundant abnormal proteins, such as those responsible for amyloidosis (Nepomuceno AI, Mason CJ, Muddiman DC, Bergen HR, 3rd, Zeldenrust SR (2004)). Detection of genetic variants of transthyretin by hquid-chromatography-dual-electrospray-ionization-fourier-transform-ion- cyclotron-resonance-mass-spectrometry (Clin Chem 50:1535-1543). Although the sensitivity and resolution of MS lies in the attomolar range, the dynamic range of a sample, i.e. the difference between the highest abundant and lowest abundant protein may be problematic. In such cases where the protein of interest is only present in a sample in a very low amount and other proteins are more abundantly present, the detection and more so the quantitation of such a low-abundance protein is challenging.. Especially for complex samples with a large dynamic range such as e.g. human serum or plasma samples there is a need for an improved quantitative assay. Such quantitative assays were conventionally performed as immunoassays such as ELISA. Selected reaction monitoring (SRM) can be used for targeted quantitative proteomics by mass spectrometry. SRM is a method used in tandem mass spectrometry in which a precursor ion of a particular mass is selected in the first stage of a tandem mass spectrometer and an ionic product of a fragmentation reaction of the precursor ion is selected in the second mass spectrometer stage for detection. Following ionization in an electrospray source, a peptide precursor is first isolated to obtain a substantial ion population of mostly the intended species. This population is then fragmented to yield product ions whose signal abundances are indicative of the abundance of the peptide in the sample. This experiment is primarily performed on triple quadrupole mass spectrometers. By spiking in stable-isotope-labelled (e.g., 2H, 1 C, or 1 N) peptides to a complex matrix as concentration standards, SRM can be used to construct a calibration curve that can provide the absolute quantitation (i.e. number of proteins per volume) of the native peptide, and by extension, its parent protein. Although the use of stable-isotope-labelled (SIL) peptides as an internal reference allows mass spectrometry to provide quantitation of proteins, there remains an issue of sensitivity of MS when applied to complex mixtures such as those created by digestion of whole plasma proteins to peptides, where the background of the sample is very complex and abundantly present. For patient samples yielding detection levels of low ng protein/ml, the variability in background becomes a point of concern. In current methods, to identify proteins of interest, SIL peptides are used. The retention time of SIL peptides are identical to the protein fragments under study, and thus allow for quantitation of the protein/pep tide of interest. However if background peptides have the same mass as the peptide of interest, such a SIL peptide does not allow discrimination between peptide of interest and background peptides. This affects severely the sensitivity and the reproducibility of the method among patient samples and leads to undesirable false positive or false negative results. Methods of dealing with high and variable background in complex samples have been used. EP2286237 discloses a method to resolve the problem of different peptides with the same mass. It uses a cleavable mass label to select only the peptide of interest. WO201 1116028 discloses an improved mass spectrometric assay for peptides using a standard-isotope-labelled version of a peptide to provide a standardized sample. In preferred embodiments, the standard isotope labelled version of a peptide is linked to a carrier molecule for easy quantitation. WO20 12/170549 discloses a method for quantitation of a target analyte by mass spectrometry using two calibrators wherein the two calibrators are distinguishable in mass. The difference in mass may be due to different isotopes or different chemical moieties. The use of homo amino acids is not described. There thus remains a need to quantify a target protein in sample wherein the target protein is present in a low concentration. There also remains a need to determine the amount of a target protein in a sample wherein the background is present in a large amount. There also remains a need for a quantitation method for a target protein wherein the method has a high sensitivity. There also remains a need for a quantitation method for a target protein wherein the method has a high reliability. There also remains a need for a quantitation method for a target protein that is present in a low concentration in a sample wherein the method has a high reliability and/or sensitivity. Summary of the invention In one aspect, the invention is directed to a method for quantitation of a target protein in a sample by mass spectrometric analysis wherein a homopeptide of a signal peptide of said target protein is used as internal reference peptide. In a preferred embodiment of aspects and/or embodiments of the invention, two or more internal reference peptides are used. In a preferred embodiment of aspects and/or embodiments of the invention, at least one of the reference peptides is a homopeptide and at least one of the reference peptides is a stable-isotope-labelled (SIL) peptide. In a preferred embodiment of aspects and/or embodiments of the invention, the homopeptide and SIL peptide have the same amino acid sequence as a signal peptide of said target protein but differ in their respective mass or mass fragmentation pattern from each other and from said signal peptide. In a preferred embodiment of aspects and/or embodiments of the invention, said homopeptide comprises at least one homo amino acid, preferably at least 2, 3, 4, 5, or 6 homo amino acids. In a preferred embodiment of aspects and/or embodiments of the invention, the homo amino acids can be different or identical. The homo amino acid may be an alpha amino acid or beta-amino acid. In a preferred embodiment of aspects and/or embodiments of the invention, said homo amino acid preferably is an a amino acid. In a preferred embodiment of aspects and/or embodiments of the invention, said homo amino acid is selected from the group consisting of homo-arginine, homo-lysine, homo-histidine, homo-aspartic acid, homo- glutamic acid, homo-serine, homo-threonine, homo-asparagine, homo- glutamine, homo-cysteine, homo-glycine , homo-proline, homo-alanine, homo-valine, homo-isoleucine, homo-leucine, homo-methionine, homo- phenylalanine, homo-tryptophan, homo-selenocysteine. In a preferred embodiment of aspects and/or embodiments of the invention, the method comprises the following steps: i) Digesting of a sample suspected to comprise said target protein to provide a signal peptide of said target protein; ii) Addition of one or more homopeptides of said signal peptide in a known quantity; hi) Optionally addition of one or more SIL peptides of said signal peptide in a known quantity; iv) Quantitation of the target protein by mass spectrometric analysis. In a preferred embodiment of aspects and/or embodiments of the invention, the homopeptide and/or the SIL peptide is added before the digesting step. In a preferred embodiment of aspects and/or embodiments of the invention, said target protein is selected from the group consisting of serum proteins, tissue proteins, cancer tissue proteins. In a preferred embodiment of aspects and/or embodiments of the invention, the method further comprises the steps v) Repeat step i) to iv) with a corresponding reference peptide preparation of known quantity spiked into said sample to obtain a target protein standard curve; vi) Quantity of said target protein obtained in step iv) is compared to the protein standard curve obtained in step v); vii) Quantitation of said target protein in said sample is obtained. In a preferred embodiment of aspects and/or embodiments of the invention, the method to quantitate the target protein is SRM (selected reaction monitoring)or MRM (multiple reaction monitoring). In a preferred embodiment of aspects and/or embodiments of the invention, the mass spectrometry comprises a technique selected from the group comprising fourier-transform ion cyclotron resonance (FTICR), TOF, Q-TOF triple quadrupole based methods, MALDI-TOF, LC-MS. LC-MS/MS, LC-ESI-MS/MS, LC-MALDI-MS/MS including FT mass spectrometry based devices, multiple reaction monitoring (MRM), selected reaction monitoring (SRM), consecutive reaction monitoring (CRM), and parallel reaction monitoring (PRM) based methods and combinations thereof. In a preferred embodiment of aspects and/or embodiments of the invention, the sample is a sample selected from the group consisting of blood, serum, plasma, or tissue. In a preferred embodiment of aspects and/or embodiments of the invention, the method is used for diagnosing a disease. In a preferred embodiment of aspects and/or embodiments of the invention, said disease is selected from the group consisting of bacterial infection, viral infection, cancer, neurological diseases such as multiple sclerosis and Alzheimer. In a second aspect the invention is directed to a kit for carrying out the methods according to the first aspect of the invention and/or embodiments thereof comprising i) at least one homopeptide of a signal peptide of a target protein, ii) optionally at least one SIL peptide of said signal peptide, wherein said SIL peptide and said homopeptide have the same amino acid sequence, iii) optionally MS matrix material, and iv) optionally instructions for use. In a preferred embodiment of aspects and/or embodiments of the invention, the homopeptide and optionally the SIL peptide have the sequence of a signal peptide of said target protein but differ in respective mass and/or mass fragmentation pattern from each other of said signal peptide. In a preferred embodiment of aspects and/or embodiments of the invention, the kit comprises at least 2 homopeptides and/or at least 2 SIL peptides. Detailed description Description of figures

Figure 1: variation in quantitation of mass of a signal peptide in different samples and between samples when only using the endogenous signal peptide for quantitation. Figure 2 : variation in quantitation of mass of a signal peptide in different samples and between samples when using the SIL peptide and the endogenous signal peptide for quantitation. Figure 3: variation in quantitation of mass of a signal peptide in different samples and between samples when using the homopeptide the endogenous signal peptide for quantitation. . Definition and abbreviations Homo amino acids are amino acids that differ from the normal amino acid by the insertion of an additional methylene unit, -CH2-. For example homo-serine, also known as iso-threonine, is an amino acid with the chemical formula H2NCH(CH2CH20H)C02H. Compare serine with homo-serine:

Homovariants of natural amino acids are commercially available. Most of the common natural amino acids are alpha amino acids or a amino acids. Beta amino acids or β amino acids have their amino group bound to a different carbon atom than the carboxyl group, usually the carbon atom adjacent to the carbon atom on which the carboxyl group

occurs. L-a-alanine ^-alanine In a amino acids (molecule at left), both the carboxylic acid group and the amino group are bound to the same carbon center, termed the a carbon (C ). In β amino acids (molecule to the right), the amino group is bound to the carbon atom adjacent to the a-carbon atom, also referred to as the carbon (C ). Most of the 20 standard amino acids comprise a carbon atom. Only glycine lacks a carbon, which means that β-glycine does not exist. The only common naturally occurring β amino acid is 6-alanine. Homopeptides are peptides that contain at least one homo amino acid. The addition of one methylene units increases the length of the carbon backbone (for β homo amino acids) or side chain (for a homo amino acids) of the amino acids and adds an additional mass of 14 Da. The change in length of the backbone and/or side chain of the amino acids creates a different retention time of the peptide as it will be slightly more hydrophobic than their natural counterpart. The increase of mass of at least 14 Da, creates systematically a separate peak in the mass spectrum making it easier to identify. In the present invention, a homopeptide is used as an internal reference in mass spectrometry. The homopeptide has the amino acid sequence of a signal peptide of the target protein. It should be understood that the homopeptide has the same primary sequence as the signal peptide, the amino acids in the same order, however one or more of the amino acids in the homopeptide is the homo-variant of the amino acid in the signal peptide. For example a signal peptide has the amino acid sequence LGPLVEQGR, the homopeptide variant thereof has the same sequence LGPLVEQGR however one of the amino acids is a homo amino acid, for example the arginine: LGPLVEQGR , wherein the R denotes homo- arginine. Stable-isotope-labelled peptide (SIL peptide) is a peptide that is labelled with stable heavy isotopes such as deuterium, carbon- 13, nitrogen- 15, and oxygen- 18. SIL peptides are added as an internal reference to a sample containing or suspected to contain a target protein. Because of the ionization variability for different peptides, the best internal reference for peptide and protein quantitation is a SIL peptide having the same amino acid sequence as a signal peptide but then labelled with stable isotopes. This way, relative protein quantitation is achieved where the SIL peptide serves as a internal reference. The internal reference and signal peptide differ only by the incorporation of the heavy stable isotopes, and the native isotopes, respectively. These two types of peptides with different mass have the same chemical properties and behave with essentially identical chromatographical characteristics under any isolation or separation step. Thus the ratio between intensities of the SIL/Signal peptide pair provides an accurate relative peptide and therefore protein abundance measure. There are several protocols for the use of stable isotopes in proteomics. Beta peptide or β-peptide is a peptide that comprises beta amino acids. Two main types of β-peptides or beta peptides exist: those with the amino acid residue side chain (R) next to the amine are called 6 -peptides and those with the amino acid residue side chain positioned next to the carbonyl group are called 62-peptides.

Because in β amino acids the amine is bound to the carbon in stead of the a carbon, the peptide backbone of 6-peptides is longer than those of peptides that consist of a amino acids. The difference in peptide backbone, results in that 6-peptides form different secondary structures. The methylenes at the a and positions in a amino acid favor a gauche conformation about the bond between the a-carbon and 6-carbon. This also affects the thermodynamic stability of the structure. 6-peptides are stable against proteolytic degradation. Target protein or target peptide means the protein or peptide of interest. The target protein or the target peptide is the protein or peptide that is quantified. The term 'signal peptide(s)' means one or more different peptide(s) selected as a monitor peptide or fragment of the target protein in a sample. A signal peptide is a peptide fragment from a target protein whereby the sequence of the peptide fragment is unique and/or specific for said target protein. In certain cases, wherein the target protein is small, the signal peptide is the whole target protein. Background proteins or background peptides are proteins and peptides that are present in a sample but are not the target protein or the peptides of interest from the target protein. 'Internal reference peptides', internal reference' or 'reference peptide' may be any altered version of the respective signal peptide that is recognized as equivalent or similar to the signal peptide by an appropriate binding agent or chemically equivalent or similar by biophysical properties and differs from it in a manner that may be distinguished by a mass spectrometer, either through direct measurement of molecular mass or through mass measurement of fragments (e. g. through MS/MS analysis), or by another equivalent means. Multiple reaction monitoring (MRM), selected reaction monitoring (SRM) , consecutive reaction monitoring (CRM), and parallel reaction monitoring (PRM), are a mode of analysis of a mass spectrometer. Single/selected reaction monitoring (SRM) analysis utilizes a mass spectrometer (e.g. a triple quadrupole type of instrument) to select and analyze a specific analyte (such as a peptide or a small molecule). Selected reaction monitoring (SRM) is a method used in tandem mass spectrometry in which an ion of a particular mass is selected in the first stage of a tandem mass spectrometer and an ion product of a fragmentation reaction of the precursor ion is selected in the second mass spectrometer stage for detection. Following ionization in an electrospray source, a peptide precursor is first isolated to obtain a substantial ion population of mostly the intended species. This population is then fragmented to yield product ions whose signal abundances are indicative of the abundance of the peptide in the sample. These peptide monitoring experiments are primarily performed on triple quadrupole mass spectrometers, where mass-resolving

Q i isolates the precursor, q 2 acts as a collision cell, and mass-resolving 3 is cycled through the product ions which are detected upon exiting the last quadrupole by an electron multiplier. A precursor/product pair is often referred to as a transition. Much work goes into ensuring that transitions are selected that have maximum specificity. By spiking in heavy -labelled (e.g., D, 1 C, or 1 N) peptides to a complex matrix as concentration standards, SRM can be used to construct a calibration curve that can provide the absolute quantitation of the native, peptide, and by extension, its parent protein. Therefore, SRM is a highly specific detection/monitoring method with low background interference. When multiple parent ions are monitored in a single MS run, this type of analysis is known as multiple reaction monitoring (MRM). Using MRM analysis, multiple proteins and multiple regions (signal peptides) of a protein can be monitored in single MS run. Consecutive reaction monitoring (CRM) is the serial application of three or more stages of selected reaction monitoring. Parallel reaction monitoring (PRM) is the application of selected reaction monitoring with parallel detection of all transitions in a single analysis using a high resolution mass spectrometer.

Detailed description In a first aspect the invention is directed to quantitation of a target protein by mass spectrometry using a homopeptide as an internal reference. The homopeptide has the same sequence as a signal peptide of said target protein. It was found that the use of a homopeptide as internal reference peptide provides a better accuracy of the quantitation of said target protein than the use of a SIL peptide which is normally used in quantitation of proteins by mass spectrometry. It was also found that the sensitivity and reliability of the quantitation of protein by mass spectrometry was improved with the method of the invention. The improvement of sensitivity of quantitation is important for samples having a low concentration of target protein. The improvement of reliability of quantitation is important for samples wherein the background is high and/or where the risk of overlap of background and protein target is high. Especially in biological samples, such as patient samples with a lot of protein such as blood and serum samples and tissue samples, the background may become a problem and may interfere with the quantitation of the target protein. It is known that background, i.e. proteins that are not the target protein, may vary a lot between patients, and for example samples from smokers have been known to have a very varied background. Also if patients are ill, several proteins may be unregulated, e.g. proteins involved in inflammation and have a high concentration in a sample from the patient. The high amount of background proteins may hamper the quantitation of a target protein. The present method provides a reliable method for quantitation of a target protein, also in samples wherein the target protein is present in a low concentration and/or wherein the background is high. The method of the present invention also has an improved sensitivity enabling the quantitation of low concentrations of target peptide in a sample. It was also found that the combination of the use of a homopeptide and SIL peptide as an internal reference provides even better results in terms of sensitivity, accuracy and/or reliability. Suitably the sequence of the homopeptide is selected using one or more of the following criteria: a) The homopeptide has a sequence of a signal peptide that results from cleavage of the target protein with a desired proteolytic enzyme, or in cases of small target protein, such as neural proteins, it has the amino acid sequence of the whole target protein. b) The homopeptide preferably should be soluble in conventional solvents used in enzymatic digestion and affinity chromatography, but should be hydrophobic enough to be retained on a C- 18, or other reverse phase column or equivalent column for desalting. c) The homopeptide should preferably ionize well by either electrospray (ESI) or another type of ionization. d) The homopeptide should preferably have good transition in SRMMRM, PRM or CRM. e) The homopeptide preferably does not contain chemically reactive residues or chemically unstable sequences. Preferably the homopeptide does not contain Tryptophan, Methionine, or Cysteine, or N- term Glutamine, or N-term Aspartic acid. Preferably the homopeptide does not contain Methionine or Cysteine. Preferably the homopeptide does not contain a N-term Glutamine, or a N-term Aspartic acid. f) The homopeptide is preferably chemically stabile. g) The homopeptide preferably does not aggregate and/or does preferably not adhere to one or more undesired surfaces during the experiment. In another preferred embodiment of aspects and/or embodiments of the present invention, the homopeptide comprises at least one homo amino acid, preferably at least 2, 3, 4, 5, or 6 homo amino acids, also all amino acids in the homopeptide may be homo amino acids. Any kind of homo amino acids may be used. In principle any kind of amino acid may be provided with an additional methylene. For example, the homo amino acid is any one selected from the group consisting of homo-arginine, homo-lysine, homo-histidine, homo-aspartic acid, homo-glutamic acid, homo-serine, homo-threonine, homo-asp argine, homo-glutamine, homo-cysteine, homo- glycine , homo-proline, homo-alanine, homo-valine, homo-isoleucine, homo- leucine, homo-methionine, homo-phenylalanine, homo-tryptophan, homo- selenocysteine. In a preferred embodiment the homo amino acid is any one selected from the group consisting of homo-arginine, homo-lysine, homo- histidine, homo-serine, homo-proline, homo-alanine, homo-valine, homo- isoleucine, homo-leucine, homo-phenylalanine, homo-tryptophan. In a more preferred embodiment, the homo amino acids are selected from the group consisting of homo-arginine and homo-lysine. Homo amino acids may be alpha amino acids or beta amino acids, preferably alpha amino acids. In a preferred embodiment of aspects and/or embodiments of the present invention, the homopeptide comprises at least two homo amino acids; these homo amino acids may be the same amino acids or may be different. For example in a homopeptide two homo-lysine residues may be present, or one homo-lysine or one homo-arginine. It is also envisioned in the present invention and/or embodiments thereof that two or more different homo-variants of one amino acid may be used, for example one being a alpha amino acid and the other being a beta amino acid, or wherein the additional methylene is incorporated into different sides of the amino acid. In a preferred embodiment of aspects and/or embodiments of the present invention, two or more internal reference peptides are used. Preferably the internal reference peptides have the same sequence as the signal peptide. Using the known sequence of the target protein, one may select one or more peptide segments within it as 'signal peptides'. A good signal peptide may be defined by a set of criteria designed to select peptides that may preferably be chemically synthesized with high yield, that may be detected quantitatively in an appropriate mass spectrometer, and preferably that elicit antibodies when used as antigens. Any peptide resulting from cleavage with a desired enzyme is a possible choice. In one embodiment one or more of the criteria below may be used for selection of the signal peptide: a) The peptide has a sequence that results from cleavage of the target protein with a desired proteolytic enzyme (e. g. trypsin). All the candidate signal peptides obtainable by enzymatic digestion may be easily computed from the protein sequence by application of generally available software. In cases where the target protein is small, the signal peptide has the sequence of the whole target protein. b) The peptide preferably should be soluble in conventional solvents used in enzymatic digestion and affinity chromatography, but should be hydrophobic enough to be retained on a C-18 column, or other reverse phase column or other equivalent column for desalting. c) The peptide should preferably ionize well by either electrospray (ESI) or another type of ionization. This characteristic may be estimated by software programs or determined experimentally by MS analysis of a digest of the protein in question to see which peptides are detected at highest relative abundance. Another criterion is good transition in MRM, SRM, PRM or CRM. d) The signal peptide preferably does not contain chemically reactive residues or chemically unstable sequences. Preferably the signal peptide does not contain (Tryptophan, Methionine, or Cysteine, or N-term Glutamine, or N-term Aspartic acid. Preferably the signal peptide does not contain Methionine or Cysteine. Preferably the signal peptide does not contain a N-term Glutamine, or a N-term Aspartic acid. e) The peptide is preferably chemically stabile. f) The peptide preferably does not aggregate and/or does preferably not adhere to one or more undesired surfaces during the experiment. All possible peptides derived from the target protein may easily be evaluated according to these criteria and one or more signal peptides may be selected that best balance the requirements of the method. In a preferred embodiment of the invention a stable-isotope- labelled (SIL) peptide is used as an internal reference in addition to the homopeptide. It was found that using both a homopeptide and a SIL peptide as an internal reference the sensitivity and reliability of the quantitation of a target peptide is improved. Especially in sample having a lot of background and/or wherein the target protein is present in only a low concentration, the combined use of a homopeptide and a SIL peptide as internal reference provides advantages. In a preferred embodiment of the invention and/or embodiments thereof the SIL peptide is a stable isotope variant of one of the signal peptides. In a preferred embodiment of the invention and/or embodiments thereof the SIL peptide has the same amino acid sequence as the homopeptide. It should be understood that having the same amino acid sequence means that the SIL peptide and the homopeptide have the amino acids in the same order, but differ in that the SIL peptides contains isotope labelling and the homopeptide comprises homo amino acid variants of amino acids of the SIL peptide. Suitable isotopes for use in a SIL peptide include 2H, "B, C, N, ^O, 0 , S, S, S, 4Se, Se, Se, Se, and 2Se. The SIL peptide may comprise one or more stable isotopes, and may comprise at least 2, 3, 4, 5,6 ,7, 8, 9, or 10 stable isotopes, or is even uniformly stable isotope labelled. For example all nitrogen in a SIL peptide are 1 N isotopes In a preferred embodiment, at least one homopeptide and at least one SIL peptide are used as internal references. Preferably at least one of the reference peptides is a homopeptide and at least one of the reference peptides is a SIL peptide. In a preferred embodiment of aspects and/or embodiments of the present invention, the homopeptide and optionally the SIL peptide have the sequence of a signal peptide. In a preferred embodiment of aspects and/or embodiments of the present invention, the homopeptide and SIL peptide have the same amino acid sequence as a signal peptide of said target protein but differ in their mass and/or mass fragmentation pattern from each other and from said signal peptide. The homopeptide and the SIL peptide thus preferably have the same sequence, but the corresponding SIL and homopeptides differ then in mass or mass fragmentation pattern from each other but also from the corresponding signal peptide so that one may be able to identify them. The homopeptide and SIL peptide have the same amino acid sequence as the signal peptide, however the mass of the homopeptide is different from the mass of the SIL peptide and the mass of the signal peptide and the mass of the SIL peptide is different from the mass of the signal peptide. Due to the differences in mass or mass fragmentation pattern the homopeptide, SIL peptide and signal peptide will each have a different peak in the mass spectrum and may therefore be separately identified. The homopeptide has a more hydrophobic character than the corresponding signal peptide due to its additional methylene group. The increased hydrophobicity will lead to a different retention time on a column than the corresponding signal peptide and corresponding SIL peptide. In a preferred embodiment of the invention and/or embodiments thereof at least two of the reference peptides are homopeptides . In a preferred embodiment of the invention at least 2, 3, 4, 5,6 ,7, 8, 9, or 10 homopeptides are used. The at least two homopeptides may have the same or may have different amino acid sequence. For example two or more different signal peptides may be used for quantitation, and thus two or more homopeptides with different amino acid sequences may be used, each having the amino acid sequence of each selected individual signal peptide. In a preferred embodiment of the invention and/or embodiments thereof the at least two homopeptides have the same amino acid sequence but the homo amino acid is different between them, and/or they may have different numbers of homo amino acids. In a preferred embodiment of the invention and/or embodiments thereof at least two SIL peptides are used in addition to the homopeptide. In a preferred embodiment of the invention and/or embodiments thereof at least 2, 3, 4, 5,6 ,7, 8, 9, or 10 SIL peptides are used. The at least two SIL peptides may have the same or may have different amino acid sequence. For example two or more different signal peptides may be used for quantitation, and thus two or more SIL peptides with different amino acid sequences may be used, each having the amino acid sequence of each selected individual signal peptide. In a preferred embodiment of the invention and/or embodiments thereof the at least two SIL peptides have the same amino acid sequence but have a different isotope labelling. Suitable isotopes include 2H, "B, C, N, ^O, 0 , S, S, S, 4Se, Se, Se, Se, and 2Se. It is to be understood that the method of the invention comprises the use of two or more signal peptides as internal references. Each signal peptide has then a different sequence and for at least one selected signal peptide at least one homopeptide variant will be used. Preferably for each selected different signal peptide a homopeptide is used. Preferably also at least one SIL peptide of at least one selected signal peptide is used. Preferably for each selected different signal peptide a SIL peptide is used. The SIL peptide may comprise one or more stable isotopes, and may comprise at least 2, 3, 4, 5,6 ,7, 8, 9, or 10 stable isotopes, or is even uniformly stable isotope labelled. For example all nitrogen in a SIL peptide are 1 N isotopes. In a preferred embodiment of the invention and/or embodiments thereof, the sample is digested with a selected protease to release one or more specific signal peptides from each protein to be quantitated into a digested sample. In one embodiment the sample is reduced and alkylated prior to the digestion. The digested sample comprises signal peptides and other peptides released by the protease. The reduction, alkylation and digestion of said sample and/or digested sample may be performed by any method known in the art. Peptides may e.g. be reduced using dithiothreitol (DTT) and subsequently alkylated with e.g. 4- vinylpyridine, iodoacetamide or iodoacetic acid. The sample, in which one wishes to measure the one or more selected protein (s) of interest, is preferably digested essentially to completion or partially digested, with the appropriate protease such as trypsin to yield peptides (including the selected signal peptide (s)). The digestion may be carried out by first denaturing the protein sample (e. g., with urea or guanidine HCI), reducing the disulfide bonds in the proteins (e.g with dithiothreitol or mercaptoethanol), alkylating the cysteines (e. g. , by addition of iodoacetamide), and finally (after removal or dilution of the denaturant) addition of the selected proteolytic enzyme such as trypsin, followed by incubation to allow digestion. In one preferred embodiment the denaturing does not result in chemical modification of the proteins. The denaturing may be performed by use of one or more detergents that are MS compatible. In one embodiment RapiGest™ SF Surfactant (Waters) is used to enhance enzymatic digestion of the proteins and as a replacement of urea or guanidine HCL as denaturant during reduction and alkylation. Following incubation, the action of the protease (e.g. trypsin) is preferably terminated, either by addition of a chemical inhibitor (e. g. DFP or PMSF) or by denaturation (through heat or addition of denaturants, or both), by acidification, or removal (e.g. if the protease on a solid support) of the protease. The destruction of the protease activity may be important in order to avoid damage by residual proteolytic activity in the sample. The digestion may be performed by any protease including trypsin, chymotrypsin, Asp-N, Glu-C, Lys-C, lys-N and Arg-C. More than one protease may be used such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more than 10 proteases. In a preferred embodiment of aspects and/or embodiments of the present invention, the digestion is performed essentially to completion. In one embodiment the digestion may be performed by chemical degradation. In a preferred embodiment of aspects and/or embodiments of the present invention, said method further comprises a step of reduction and/or alkylation prior to the digestion step i). In a preferred embodiment of aspects and/or embodiments of the present invention, the reduction is performed with an agent selected from the group consisting of dithiothreitol (DTT), or mercaptoethanol. In a preferred embodiment of aspects and/or embodiments of the present invention, the alkylation is performed with an agent selected from the group consisting of 4-vinylpyridine, iodoacetamide, iodoacetic acid Prior to and/or after reduction, alkylation and/or digestion, internal reference homopeptides for each of the signal peptides of the target protein are added to the first sample or to the digested second sample in a known quantity, allowing subsequent absolute quantitation. Since the internal reference is added at a known concentration, the ratio between the amounts of the natural signal peptide and the homo-variant of the internal reference peptide detected by the final MS analysis allows the concentration of the signal peptide in the sample mixture to be calculated. Similarly when two or more internal reference peptides are used, they may be added prior to and/or after reduction, alkylation and/or digestion in a known quantity. The two or more internal reference peptides may be added simultaneously or at different time points. In embodiments where a SIL peptide is used the SIL peptide may be added prior to and/or after reduction, alkylation and/or digestion. The SIL peptide may be added simultaneously with the homopeptide or separately at a different time point from the addition of the homopeptide. In a preferred embodiment of aspects and/or embodiments of the present invention, the one or more internal reference peptides are generated by post-synthetic labelling. In a preferred embodiment of aspects and/or embodiments of the present invention, the one or more internal reference peptides are generated by chemical synthesis. In a preferred embodiment of aspects and/or embodiments of the present invention, the one or more internal reference peptides are generated in live cells through metabolic incorporation. In a preferred embodiment of aspects and/or embodiments of the present invention, said method further comprises a step of concentrating of said signal peptides and/or said internal reference peptides. In a preferred embodiment of aspects and/or embodiments of the present invention, the signal peptides and/or internal reference peptides are concentrated using a technique selected from the group consisting of solid- phase extraction, liquid-chromatography, gas-chromatography, ion- exchange-based separation, anion- and cation-chromatography, reversed- phased-based separation, hydrophilic -interaction-based separation, hydrophobic-interaction-based separation, size-exclusion-based separation, affinity-based separation, or immunoaffinity-based separation . In a preferred embodiment the method to quantitate the target protein is a method without the use of antibodies. In a preferred embodiment the method to quantitate the target protein is SRM, MRM, PRM or CRM, more preferably SRM or MRM. In a preferred embodiment of aspects and/or embodiments of the present invention, the method further comprises obtaining a standard curve of the signal peptide or target protein. Obtaining a standard curve provides even more accurate quantitation of the target protein. The standard curve is obtained by repeating step i) to iv) with a corresponding reference peptide preparation of known quantity /added into said sample to obtain a reference peptide standard curve and then comparing the quantity of the homopeptide obtained in step iv) to the reference peptide curve obtained in step v). After which the quantitation of said target protein in said sample is obtained. The amount of reference peptide is then correlated to the amount of target protein in the sample. The reference peptide for the standard curve may be a SIL peptide or a homopeptide, preferably a homopeptide. The quantitation of the target protein may be done by SRM, CRM, PRM or MRM, or by any other quantitation method known to a skilled person. In a preferred embodiment of aspects and/or embodiments of the present invention, the mass spectrometry comprises a technique selected from the group comprising fourier-transform ion cyclotron resonance (FTICR), TOF, Q-TOF triple quadrupole based methods, MALDI-TOF, LC- MS. LC-MS/MS, LC-ESI-MS/MS, LC-MALDI-MS/MS including FT mass spectrometry based devices, MRM, SRM, CRM, and PRM based methods and combinations thereof In a preferred embodiment of aspects and/or embodiments of the present invention, the mass spectrometry comprises ionization by ESI or MALDI. In a preferred embodiment of aspects and/or embodiments of the present invention, the mass spectrometry comprises LC-MS analysis preferably comprising two or more dimensions of chromatographic fractionation, or preferably comprising multidimensional chromatography, or preferably comprising a single dimension of LC separation, or preferably comprising a two or more dimensions of LC separation. In a preferred embodiment of aspects and/or embodiments of the present invention, the mass spectrometry comprises fourier-transform ion cyclotron resonance (FTICR), Q-TOF or triple quadrupole based methods. In a preferred embodiment of aspects and/or embodiments of the present invention, the mass spectrometry comprises LC-MS/MS, LC-ESI- MS/MS, LC-MALDI-MS/MS, multiple reaction monitoring (MRM), selected reaction monitoring (SRM), consecutive reaction monitoring (CRM), and/or parallel reaction monitoring (PRM) based methods. In a preferred embodiment of aspects and/or embodiments of the present invention, the mass spectrometry comprises extracted ionchr om atogram s. In a preferred embodiment of aspects and/or embodiments of the present invention, the mass spectrometry comprises ion trap based methods, ESI-triple quadrupole based method, Orbitrap based methods, ESI-TOF based methods, ESI-Q-TOF based methods and Maldi based methods. In principle any kind of sample that comprises a protein that is to be quantified may be used in the methods of the invention. In a preferred embodiment of aspects and/or embodiments of the present invention, the sample is a sample from a patient. In a preferred embodiment of aspects and/or embodiments of the present invention, the sample is a sample selected from the group consisting of serum, tissue, urine or other body fluids such as amniotic fluid, aqueous humour and vitreous humour, bile, blood serum, breast milk, cerebrospinal fluid, cerumen, chyle, chyme, endolymph and perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus, nasal drainage, phlegm, pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, tears, sweat, vaginal secretion, vomit. The sample may also be any a cell culture aliquot, a test sample in a experiment, an extract of an process, an extract of a plant, an extract of a microorganism culture, an extract of a cell culture, or any other kind of industrial protein mixture or extract such as a food sample, or an environmental sample. In a preferred embodiment of aspects and/or embodiments of the present invention, the sample is a serum sample, plasma sample, tissue sample, cerebrospinal fluid, or urine sample. In a preferred embodiment of aspects and/or embodiments of the present invention, the sample is a cell culture supernatant, or a bioreactor supernatant. The target protein may be a protein that is a marker of a disease, a transgenic protein, a protein that is a reference for a culture growth, a reference protein for a diagnosis, a reference protein for a health check. In a preferred embodiment of aspects and/or embodiments of the present invention, the target protein is a marker. In a preferred embodiment of aspects and/or embodiments of the present invention, the target protein is a marker for a disease. In a preferred embodiment of aspects and/or embodiments of the present invention, the target protein is a marker for bacterial infection(s), viral infection(s), inflammatory diseases, proliferative diseases, cancer, neurological diseases, cardiovascular diseases, diabetes, pulmonary diseases such as COPD, and/or immunological diseases. The method and kits of the present invention is very suitable for quantitation of protein markers of diseases selected from the group consisting of Alzheimer disease, Multiple Sclerosis, Parkinson disease, breast cancer, colorectal cancer, lung cancer, cervical cancer, and pr e eclampsia. Any bloodtest, urinetest, sputumtest, serumtest, that checks for a protein may be performed with the method of the present invention, such as test for C-reactive protein, haemoglobin, TSH, gonadotropin, albumin, creatine kinase, ferritin, FSH, gamma glutamyltransferase, homocysteine, immunoglobulins, lactate dehydrogenase, lipase, parathormone, prolactin, PSA, transferrin, troponin. In a preferred embodiment of aspects and/or embodiments of the present invention, the method is used for pharmacokinetic studies of an individual. In a preferred embodiment of aspects and/or embodiments of the present invention, the method is used for a health check of an individual In a preferred embodiment of aspects and/or embodiments of the present invention, the method is used for diagnosing a disease. In a preferred embodiment of aspects and/or embodiments of the present invention, said disease is selected from the group consisting of bacterial infection, viral infection, cancer, inflammatory diseases, proliferative diseases, neurological diseases, cardiovascular diseases, diabetes, pulmonary diseases such as COPD, and/or immunological diseases. In a preferred embodiment of aspects and/or embodiments of the present invention, said disease is selected from the group consisting of Alzheimer disease, Multiple Sclerosis, Parkinson disease, breast cancer, colorectal cancer, lung cancer, cervical cancer, pre-eclampsia . Preferably said disease is selected from cervical cancer, or pre-eclampsia. In a preferred embodiment of aspects and/or embodiments of the present invention the method is a method of diagnosing a disease comprising the steps according to the first aspect of the invention and/or embodiments thereof. In a preferred embodiment of aspects and/or embodiments of the present invention the method is a method of diagnosing a disease comprising a method for quantitation of a target protein in a sample by mass spectrometric analysis wherein a homopeptide of a signal peptide of said target protein is used as internal reference peptide. In preferred embodiment, the target protein is a protein marker for said disease. In a preferred embodiment of aspects and/or embodiments of the present invention the method is a method of diagnosing a disease comprising a method for quantitation of a protein marker in a sample of a patient by mass spectrometric analysis wherein a homopeptide of a signal peptide of said protein marker is used as internal reference peptide. In a preferred embodiment of aspects and/or embodiments of the present invention the method is a method of treatment of a disease according to the first aspect of the invention and/or embodiments thereof and administering an effective amount of a medicine for said disease. In preferred embodiment, the target protein is a protein marker for said disease. In a preferred embodiment of aspects and/or embodiments of the present invention the method is a method of treating a disease in a patient comprising a method for quantitation of a protein marker for said disease in a sample from said patient by mass spectrometric analysis wherein a homopeptide of a signal peptide of said protein marker is used as internal reference peptide, and administering an effective amount of a medicine for said disease to said patient. In a second aspect the invention is directed to a kit for carrying out anyone of the methods according to the first aspect of the invention and/or embodiments thereof, the kit comprising at least one homopeptide of a signal peptide of a target protein. The signal peptide or homopeptide of the target protein may be selected as indicated above. Suitable target proteins are serum proteins, tissue proteins, and cancer tissue proteins. In a preferred embodiment of aspects and/or embodiments of the present invention the target protein is selected form the group consisting of C- reactive protein, haemoglobin, TSH, gonadotropin, albumin, creatine kinase, ferritin, FSH, gamma glutamyltransferase, homocysteine, immunoglobulins, lactate dehydrogenase, lipase, parathormone, prolactin, PSA, transferrin, troponin. The sequence of the homopeptide may be selected as indicated above. In a preferred embodiment of aspects and/or embodiments of the present invention said homopeptide comprises at least one homo amino acid, preferably at least 2, 3, 4, 5, or 6 homo amino acids. Preferably said homo amino acid is selected from the group consisting of homo-arginine, homo- lysine, homo-histidine, homo-aspartic acid, homo-glutamic acid, homo- serine, homo-threonine, homo-asparagine, homo-glutamine, homo-cysteine, homo-glycine , homo-proline, homo-alanine, homo-valine, homo-isoleucine, homo-leucine, homo-methionine, homo-phenylalanine, homo-tryptophan, homo-selenocysteine. The homo amino acid may be an alpha amino acid or a beta amino acid. Preferably said homo amino acid is an a amino acid. Preferred embodiments of the invention with regard to the signal peptide, homopeptide, homo amino acids, and target protein indicated above are likewise relevant for the kit of the invention. In a preferred embodiment of aspects and/or embodiments of the present invention the kit additionally comprises at least one SIL peptide of said signal peptide. Preferably said SIL peptide and said homopeptide have the same amino acid sequence, Preferably said SIL peptide and said homopeptide have the same amino acid sequence as a signal peptide, Preferred embodiments of the invention with regard to the SIL peptide indicated above for the method are likewise relevant for the SIL peptide in the kit of the invention. In a preferred embodiment of aspects and/or embodiments of the present invention the kit comprises at least 2 homopeptides and/or at least 2 SIL peptides. In preferred embodiments, the homopeptide and SIL peptide have the same sequence as the signal peptide but differ in their mass or mass fragmentation pattern from each other and from said signal peptide. The homopeptide and SIL peptide have the same sequence as a signal peptide, however the mass of the homopeptide is different from the mass of the SIL peptide and the mass of the signal peptide and the mass of the SIL peptide is different from the mass of the signal peptide. Due to the differences in mass the homopeptide, SIL peptide and signal peptide will have a different peak in the mass spectrum and may therefore be separately identified. The homopeptide has a more hydrophobic character than the corresponding signal peptide due to its additional methylene group. The increased hydrophobicity will lead to a different retention time on a column than the corresponding signal peptide and corresponding SIL peptide. In a preferred embodiment of aspects and/or embodiments of the present invention the kit comprises MS matrix material. Appropriate matrix materials for each type of laser used in MALDI are well known in the art and the term "MALDI matrix material" or "MS matrix material" will be clearly understood by one of skill in the art. Without limiting the present invention, examples of commonly used matrix materials include sinapinic acid (SA), a- cyano-4-hydroxycinnamic acid (HCCA), 2,5-dihydroxybenzoic acid (DHB), 7-hydroxy-4-(trifluoromethyl)coumarin (HFMC), 3-Hydroxy Picolinic Acid (3- HPA), 5-(trifluoro-methyl)uracil, caffeic acid, succinic acid, anthranilic acid, 3- aminopyrazine-2-carboxylic acid, tetrakis(pentafluorfenyl)porfyrine and ferulic acid. Matrices are suitably dissolved in acetonitrile/water/formic acid (500:500:1; v/v/v), or other suitable ratio's depending on the matrix used. In a preferred embodiment of aspects and/or embodiments of the present invention the kit comprises instructions for use. The instruction may comprise instructions for the homopeptide, the SIL peptide, the matrix material, the protease, alkylating agent, reducing agent. The instruction may comprising instructions regarding the digestion step, alkylation step, reduction step, sample handling, instructions for Mass spectrometer. In a preferred embodiment of aspects and/or embodiments of the present invention the kit comprises a protease. Suitable protease for the kit of the invention include trypsin, chymotrypsin, Asp-N, Glu-C, Lys-C, lys-N and Arg-C. The kit of the invention may comprise more than one protease such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more than 10 proteases. In a preferred embodiment of aspects and/or embodiments of the present invention the kit comprises further chemicals such as alkylating agents, digesting agents, protease denaturing agents, protease inhibitors, and reducing agents. Suitable reducing agents for the kit of the present invention comprise dithiothreitol (DTT), mercaptoethanol Tris (2-carboxyethyl) phosphine, Tris (2-carboxyethyl) phosphine hydrochloride, 2- aminoethanethiol (2-MEA)-HCl, Tributylphosphine (TBP), cysteine hydrochloride. Suitable digesting agents for the kit of the present invention comprise urea or guanidine HCI. Suitable alkylating agents comprise 2— vinylpyridine, 4-vinylpyridine, iodoacetamide, or iodoacetic acid. Suitable denaturing agents comprise detergents, acids, and urea. Suitable protease inhibitor are diisopropylfluorophosphate (DFP), phenylmethanesulfonylfluoride (PMSF), aprotinin, bestatin, chymostatin, E- 64, leupeptin, pefabloc SC, peptstatin, N-alpha-tosyl-L-lysinyl- chloromethylketone (TLCK), N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK), ovomucin, and alpha-2-Macroglobulin antitrypsin. For the purpose of clarity and a concise description features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described. Experimental section

Example 1: Retention time: Methods used are standard procedures for instance described in

Mustafa et al. Mol Cell Proteomics. 2012; 1: Table 1

tR= retention time Variation in quantitation between samples when using only the endogenous signal peptide (figure 1); SIL peptide (figure 2), homopeptide (figure 3).

Selected Reaction Monitoring. Digested sera were spiked with stable isotope-labelled (SIL) and homopeptides of HSP90-alpha protein (YIDQEELNK) and the proteins listed in Table 1. The homopeptides contain homo-lysine and the SIL is labelled with 1 N and 13 C and has a mass difference of 10 dalton compared to the non-labelled variant. Subsequently, samples were desalted using 96-well SPE C18 plates (Sigma Aldrich DSC- C18; 25 mg) and off-line fractionated with a Luna 5 µιη, 150 x 2 mm SCX column using the following conditions: buffer A (14 mM KH2PO4, pH 2.5, adjusted with 37% (w/w) HC1) in water/acetonitrile (75:25); buffer B (buffer A containing 350 mM KCl); linear gradient from 0% buffer A to 40% (v/v) buffer B in buffer A in 40 min followed a wash with 100% buffer B until 45 min and equilibration of the column in buffer A for 17 min. Fractionated peptides were measured using a nanoACQUITY LC system equipped with a BEH300 C18, 1.7 µηι, 75 µιη x 200 mm column connected to a Xevo TQ-S (Waters Corp., Milford, MA) triple quadrupole mass spectrometer in positive electrospray ionization mode. SRM signals were recorded for doubly-charged peptide precursor ions as well as for stable-isotope-labelled and homopeptides. The following parameters were set using a nanoflow Zspray ion source: capillary voltage 3000 V, nebulizer gas (nitrogen) 0.15 bar, collision gas flow 0.15 niL/min (argon), source temperature 70°C. The nomenclature of the peptide fragments depends on whether it originates from the N or C terminus. Fragments from the N-terminus are labelled b, and fragments from the C terminus are labelled y. The number then indicates the length of the fragment. For example b3 is a peptide fragment from the N terminus with a length of 3 amino acids. As can be seen, the variations between samples in determined quantity when only using the endogenous signal peptide (figure 1) is large, the amounts of y2, y7 and b2 fragments of the peptide vary considerably. The determination of the quantity is not much improved when using the SIL peptide (figure 2). The variation is still large. However when one uses the homopeptide (figure 3) one can see that variations between samples is very much reduced. Through all samples the determined quantity of a specific peptide/transition is more or less the same having only a small variation.. Claims

1. Method for quantitation of a target protein in a sample by mass spectrometric analysis wherein a homopeptide of a signal peptide of said target protein is used as internal reference peptide.

2. Method according to claim 1 wherein two or more internal reference peptides are used.

3. Method according to claim 2 wherein at least one of the reference peptides is a homopeptide and at least one of the reference peptides is a stable-isotope-labelled (SIL) peptide.

4. Method according to any of the preceding claims wherein the homopeptide and SIL peptide have the same amino acid sequence as a signal peptide of said target protein but differ in mass and/or mass fragmentation pattern from each other and from said signal peptide.

5. Method according to any of the preceding claims wherein said homopeptide comprises at least two homo amino acids.

6. Method according to any of the preceding claims wherein the homo amino acids in said homopeptide can be different or identical.

7. Method according to anyone of the preceding claims wherein said homo amino acid is selected from the group consisting of homo- arginine, homo-lysine, homo-histidine, homo-aspartic acid, homo-glutamic acid, homo-serine, homo-threonine, homo-asparagine, homo-glutamine, homo-cysteine, homo-glycine , homo-proline, homo-alanine, homo-valine, homo-isoleucine, homo-leucine, homo-methionine, homo-phenylalanine, homo-tryptophan, homo-selenocysteine.

8. Method according to claim 7 wherein said homo amino acid preferably is an a amino acid.

9. Method according to anyone of the preceding claims comprising the following steps: i) Digesting of a sample suspected to comprise said target protein to provide a signal peptide of said target protein; ii) Addition of one or more homopeptides of said signal peptide with a known quantity; iii) Optionally addition of one or more SIL peptides of said signal peptide in a known quantity; iv) Quantitation of the target protein by mass spectrometric analysis.

10. Method according to claim 9 wherein the homopeptide and/or the SIL peptide is added before the digesting step.

11. Method according to anyone of the preceding claims further comprising the steps v) Repeat step i) to iv) with a corresponding reference peptide preparation of known quantity spiked into said sample to obtain a target protein standard curve; vi) Quantity of said target protein obtained in step iv) is compared to the protein standard curve obtained in step v); vii) Quantitation of said target protein in said sample is obtained. 12. Method according to anyone of the preceding claims wherein the method to quantitate the target protein is SRM or MRM.

13. Method according to anyone of the preceding claims wherein the mass spectrometry comprises a technique selected from the group comprising fourier-transform ion cyclotron resonance (FTICR), TOF, Q-TOF triple quadrupole based methods, MALDI-TOF, LC-MS. LC-MS/MS, , LC-ESI-MS/MS, LC-MALDI-MS/MS including FT mass spectrometry based devices, MRM, SRM, CRM, and PRM based methods and combinations thereof.

14. Method according to anyone of the preceding claims wherein the sample is a sample selected from the group consisting of blood, serum, plasma, or tissue.

15. Method according to anyone of the preceding claims wherein said target protein is selected from the group consisting of serum proteins, tissue proteins, cancer tissue proteins.

16. Method according to anyone of the preceding claims wherein the target protein is a marker for a disease selected from the group consisting of bacterial infection(s), for viral infection(s), inflammatory diseases, proliferative diseases, cancer, neurological diseases.

17. Method according to anyone of the preceding claims wherein the method is used for diagnosing a disease.

18. Method according to claim 17 wherein said disease is selected from the group consisting of bacterial infection, viral infection, cancer, neurological diseases. 19. Kit for carrying out anyone of the methods according to anyone of claims 1-18 comprising i) at least one homopeptide of a signal peptide of a target protein, ii) optionally at least one SIL peptide of said signal peptide, wherein said SIL peptide and said homopeptide have the same amino acid sequence, iii) optionally MS matrix material, and iv) optionally instructions for use.

20. Kit according to claim 19 wherein said homopeptide and optionally said SIL peptide have the sequence of a signal peptide of said target protein but differ in mass and/or mass fragmentation pattern from each other of said signal peptide.

21. Kit according to anyone of claim 19 or 20 wherein said homopeptide comprises at least two homo amino acid.

22. Kit according to anyone of claim 19 to 2 1 wherein said homo amino acid is selected from the group consisting of homo-arginine, homo-lysine, homo-histidine, homo-aspartic acid, homo-glutamic acid, homo serine, homo-threonine, homo-asparagine, homo-glutamine, homo-cysteine, homo-glycine , homo-proline, homo-alanine, homo-valine, homo-isoleucine, homo-leucine, homo-methionine, homo-phenylalanine, homo-tryptophan, homo-selenocysteine.

23. Kit according to any of claim 19 to 22 wherein said homo amino acid preferably is an a amino acid. 24. Kit according to anyone of claim 19 to 23 further comprising a protease.

25. Kit according to anyone of claim 19 to 24 comprising at least 2 homopeptides and/or at least 2 SIL peptides.

International application No PCT/NL2014/05Q587

A. CLASSIFICATION O F SUBJECT MATTER INV. G01N33/68 ADD.

According to International Patent Classification (IPC) or to both national classification and IPC

B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols G01N

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)

EPO-Internal BIOSIS, EMBASE, WPI Data

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

WO 2010/122673 Al (EISAI R&D MAN CO LTD 19 ,20,23 [JP] ; SHINKYO RAKU [JP] ; ISHIDA T0M0MI [JP] j 28 October 2010 (2010-10-28) exampl e s 1-6, claim 11 1-18,21, 22 ,24,25

W0 2011/110341 Al (JPT PEPTIDE 1-25 TECHNOLOGI ES GMBH [DE] ; ZERWECK JOHANNES [DE] ; SCHUTKOWSKI ) 15 September 2011 (2011-09-15) c l aims 1 , 4 , 6

/ -

X Further documents are listed in the continuation of Box C. See patent family annex.

* Special categories of cited documents : "T" later document published after the international filing date or priority date and not in conflict with the application but cited to understand "A" document defining the general state of the art which is not considered the principle or theory underlying the invention to be of particular relevance "E" earlier application or patent but published on or after the international "X" document of particular relevance; the claimed invention cannot be filing date considered novel or cannot be considered to involve an inventive "L" document which may throw doubts on priority claim(s) orwhich is step when the document is taken alone cited to establish the publication date of another citation or other " document of particular relevance; the claimed invention cannot be special reason (as specified) considered to involve an inventive step when the document is "O" document referring to a n oral disclosure, use, exhibition or other combined with one or more other such documents, such combination means being obvious to a person skilled in the art "P" document published prior to the international filing date but later than the priority date claimed "&" document member of the same patent family

Date of the actual completion of the international search Date of mailing of the international search report

19 March 2015 26/03/2015

Name and mailing address of the ISA/ Authorized officer European Patent Office, P.B. 5818 Patentlaan 2 NL - 2280 HV Rijswijk Tel. (+31-70) 340-2040, Fax: (+31-70) 340-3016 Hohwy, Morten International application No PCT/NL2014/05Q587

C(Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

X WENCHU YANG ET AL: "Guani dinated protein 1-25 i nternal standard for immunoaff i ni ty-1 i qui d chromatography/tandem mass spectrometry quanti tati on of protei n therapeuti cs" , RAPID COMMUNICATIONS I N MASS SPECTROMETRY, vol . 28, no. 13, 15 July 2014 (2014-07-15) , pages 1489-1500, XP055177119, ISSN : 0951-4198, D0I : 10. 1002/rcm. 6924 abstract; p . 1496 International application No Information on patent family members PCT/NL2014/05Q587

Patent document Publication Patent family Publication cited in search report date member(s) date

WO 2010122673 Al 28-10-2010 2421883 Al 29-02-2012 2012028284 Al 02-02-2012 2010122673 Al 28-10-2010 2010123085 Al 28-10-2010

W0 2011110341 Al 15-09-2011 2545384 Al 16-01-2013 2013522174 A 13-06-2013 2013095514 Al 18-04-2013 2011110341 Al 15-09-2011