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WO 2016/032319 Al © (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, © v © (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.
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