Patent Application
20110177491
The present invention relates to a peptide for use in simultaneous quantification of metabolizing enzyme proteins in humans or other mammals with a mass spectrometer and a method of using the same. More specifically, the present invention relates to a peptide consisting of a partial amino acid sequence of a human metabolizing enzyme protein or the like for use in simultaneous quantification of absolute amounts of two or more of human or mammal metabolizing enzyme proteins in a biological sample with amass spectrometer at high sensitivity and a method of using the same.
Metabolizing enzymes occurring in mammals including humans include drug metabolizing enzymes, steroid metabolizing enzymes, amino acid metabolizing enzymes, and nucleic acid metabolizing enzymes. Among these metabolizing enzymes, drug metabolizing enzymes are important enzymes that are involved in effectiveness and occurrence of toxicities of a drug by modifying the drug by metabolism. Examples of such metabolizing enzymes include CYP (P-450), glucuronic acid conjugating enzymes, sulfuric acid conjugating enzymes, and glucuronide conjugating enzymes. These enzyme molecules are subject to induction and inhibition of expressions thereof due to various factors, and such variations in the expressions result in varied effectiveness and toxicities of a drug. As expression profiles of drug-metabolizing enzymes thus determine how a drug is metabolized, these expression profiles are very important information in development of a drug.
An expression profile of a drug metabolizing enzyme can be analyzed at an mRNA level by utilizing PCR, DNA chips, and the like. However, mRNA expression is not necessarily consistent with expression of a protein, which is actual manifestation of an activity of the enzyme. Furthermore, one drug is often metabolized by more than one metabolizing enzyme. In such a case, the magnitude of contribution of each metabolizing enzyme cannot be analyzed by mRNA expression. Achievement of comparison between different metabolizing enzymes by analyzing metabolizing enzymes with a protein expression level and further obtaining an absolute expression level has been awaited.
One example of methods for detecting a metabolizing enzyme protein is a method using an antibody. Although a protein can be detected and quantified by Western blot in this method, it is not only very difficult to prepare a specific antibody, but also it requires considerable time and labor to construct profiles of two or more molecules.
Meanwhile, mass spectrometry techniques have been dramatically advanced, investigated, and utilized to detect and measure various biomaterials in recent years. Mass spectrometers having various functions have been developed, including a mass spectrometer in use for electrosprayionization (ESI), a mass spectrometer in use for liquid chromatography-mass spectrometry (LC-MS), and an MS/MS or tandem mass (tandem MS) spectrometer, which consists of two connected mass spectrometers. Mass spectrometers having these functions in combination are used in detection, measurement, or quantification of biomaterials (Patent Documents 1 to 3).
The inventors of the present invention have invented a method for simultaneous quantification of absolute expression levels of 20 or more membrane proteins with amass spectrometer (Patent Document 4). This invention is a method for obtaining an absolute expression level of a target protein by selecting a highly sensitive peptide from peptides obtained by digesting the target protein with trypsin and using a stable-isotope-labeled peptide that has the same amino acid sequence as the amino acid sequence of the selected peptide to quantify an absolute expression level of a trypsin-digested peptide to be quantified in a biological sample. It is therefore critical to select a highly sensitive peptide in a target protein to achieve quantification at high sensitivity, precision, and reliability.
However, the above-mentioned invention described in Patent Document 4 is an invention related to a method for quantifying an absolute expression level of a cell membrane protein and an amino acid sequence of a peptide of the cell membrane protein, and information on a peptide that can be used for quantification of a metabolizing enzyme, an intracellular protein, was unknown. As an enormous amount of proteins exist in the cell as compared with the cell membrane, quantification of a metabolizing enzyme, an intracellular protein, involves quantification of trace amounts of proteins in a more complicated protein sample, and it was anticipated to be more difficult to select a peptide to be quantified for quantification of a metabolizing enzyme in an intracellular protein sample as compared with selection of a peptide to be quantified in a cell membrane protein.
An object of the present invention is to provide a peptide consisting of an amino acid sequence that can simultaneously quantify absolute protein amounts of metabolizing enzymes in a biological sample at high sensitivity and a method of using the same.
The inventors of the present invention conducted various researches to achieve the foregoing object and found that the presence of a specific amino acid suppresses ionization of peptides and lowers sensitivity, and that the presence of a different specific amino acid increases efficiency of collision-induced dissociation and facilitates ionization of peptides, resulting in high sensitivity. The inventors of the present invention selected a peptide that can be detected with amass spectrometer at high sensitivity and can simultaneously quantify metabolizing enzymes, intracellular proteins, at high sensitivity, identified an amino acid sequence of the peptide by scoring peptides according to criteria and narrowing down a candidate from two or more peptides by total scores, and thus accomplished the present invention.
Specifically, the present invention relates to (1) a peptide for use in simultaneous protein quantification of metabolizing enzymes with a mass spectrometer, consisting of the partial amino acid sequence of a human metabolizing enzyme protein set forth in any one of SEQ ID NOS: 1 to 412, (2) a peptide for use in simultaneous protein quantification of metabolizing enzymes with a mass spectrometer, consisting of the partial amino acid sequence of a mouse metabolizing enzyme protein set forth in any one of SEQ ID NOS: 413 to 695, (3) a peptide for use in simultaneous protein quantification of metabolizing enzymes with a mass spectrometer, consisting of an amino acid sequence including deletion, substitution, or addition of one or two amino acids in the amino acid sequence set forth in any one of SEQ ID NOS: 1 to 695, and (4) a stable-isotope-labeled peptide, wherein one or more of peptide-constituting amino acids contain any one or more of 15N, 13C, 18O, and 2H in the peptide according to any one of the above (1) to (3).
Furthermore, the present invention relates to (5) a kit for simultaneous protein quantification of metabolizing enzymes, comprising the peptide according to any one of the above (1) to (3) and the stable-isotope-labeled peptide according to the above (4), and (6) a method of using the peptide according to any one of the above (1) to (3) and the stable-isotope-labeled peptide according to the above (4) as a probe for simultaneous protein quantification of metabolizing enzymes.
Furthermore, the present invention relates to (7) a method for simultaneous protein quantification of metabolizing enzymes with a liquid chromatograph-tandem mass spectrometer (LC-MS/MS) using a stable-isotope-labeled peptide, comprising the steps of: (a) performing a mass spectrometry by liquid chromatograph-tandem mass spectrometer (LC-MS/MS) using the peptide according to any one of the above (1) to (3) and the stable-isotope-labeled peptide according to the above (4) at predetermined concentration levels of these peptides to create a calibration curve; (b) performing a mass spectrometry by LC-MS/MS by adding the stable-isotope-labeled peptide according to the above (4) to peptide fragments obtained by fragmenting metabolizing enzyme proteins to be quantified in a sample with trypsin to calculate a mass spectrum area ratio of a metabolizing enzyme protein peptide to be quantified and the stable-isotope-labeled peptide; and (c) obtaining a quantitative value from the area ratio using the calibration curve.
Quantification of metabolizing enzyme proteins, which was difficult by conventional methods, can be achieved conveniently and rapidly at high precision by a method for quantification of metabolizing enzyme proteins by mass spectrometry using a peptide consisting of the amino acid sequence of the present invention. Furthermore, as metabolizing enzyme proteins can be quantified by the method for quantification of metabolizing enzyme proteins of the present invention without using an antibody, the step of preparing an antibody, which conventionally required the longest time, can be omitted, and further metabolizing enzymes for which an antibody cannot be prepared can be quantified. Therefore, a method of wide application for highly precise quantification of metabolizing enzyme proteins, intracellular proteins, can be provided. Accordingly, a method for quantification of metabolizing enzyme proteins utilizing the present invention can be expected to contribute greatly to understanding of drug interactions, individual variations, and toxicities. Particularly in development of a novel drug, information on metabolizing enzymes of which expressions are changed by a novel drug candidate substance and the extents to which these expressions are changed has a very important significance to predict interactions and toxicities of the substance. The method of the present invention can therefore be expected to contribute greatly to promotion of development of a novel drug.
The peptide of the present invention is a peptide for use in simultaneous protein quantification of metabolizing enzymes with a mass spectrometer, consisting of a partial amino acid sequence of a human metabolizing enzyme protein. Specific examples of the human metabolizing enzyme protein can include CYP1A1 (SwissProt accession number: P04798), CYP1A2 (SwissProt accession number: P05177), CYP1B1 (SwissProt accession number: Q16678), CYP2A6 (SwissProt accession number: P11509), CYP2A7 (SwissProt accession number: P20853), CYP2A13 (SwissProt accession number: Q16696), CYP2B6 (SwissProt accession number: P20813), CYP2C8 (SwissProt accession number: P10632), CYP2C9 (SwissProt accession number: P11712), CYP2C18 (SwissProt accession number: P33260), CYP2C19 (SwissProt accession number: P33261), CYP2D6 (SwissProt accession number: P10635), CYP2E1 (SwissProt accession number: P05181), CYP2F1 (SwissProt accession number: P24903), CYP2J2 (SwissProt accession number: P51589), CYP2R1 (SwissProt accession number: Q6VVX0), CYP2S1 (SwissProt accession number: Q96SQ9), CYP2W1 (SwissProt accession number: Q8TAV3), CYP3A4 (SwissProt accession number: P08684), CYP3A5 (SwissProt accession number: P20815), CYP3A7 (SwissProt accession number: P24462), CYP3A43 (SwissProt accession number: Q9HB55), CYP4A11 (SwissProt accession number: Q02928), CYP4B1 (SwissProt accession number: P13584), CYP4F2 (SwissProt accession number: P78329), CYP4F3 (SwissProt accession number: Q08477), CYP4F8 (SwissProt accession number: P98187), CYP4F11 (SwissProt accession number: Q9HBI6), CYP4F12 (SwissProt accession number: Q9HCS2), CYP4F22 (SwissProt accession number: Q6NT55), CYP4V2 (SwissProt accession number: Q6ZWL3), CYP4X1 (SwissProt accession number: Q8N118), CYP4Z1 (SwissProt accession number: Q86W10), CYP7A1 (SwissProt accession number: P22680), CYP7B1 (SwissProt accession number: 075881), CYP8B1 (SwissProt accession number: Q9UNU6), CYP11B1 (SwissProt accession number: P15538), CYP11B2 (SwissProt accession number: P19099), CYP17A1 (SwissProt accession number: P05093), CYP19A1 (SwissProt accession number: P11511), CYP21A2 (SwissProt accession number: P08686), CYP24A1 (SwissProt accession number: Q07973), CYP26A1 (SwissProt accession number: 043174), CYP26B1 (SwissProt accession number: Q9NR63), CYP26C1 (SwissProt accession number: Q6VOLO), CYP27A1 (SwissProt accession number: Q02318), CYP27B1 (SwissProt accession number: 015528), CYP27C1 (SwissProt accession number: Q4GOS4), CYP39A1 (SwissProt accession number: Q9NYL5), CYP46A1 (SwissProt accession number: Q9Y6A2), CYP51A1 (SwissProt accession number: Q16850), UGT1A1 (SwissProt accession number: P22309), UGT1A3 (SwissProt accession number: P35503), UGT1A4 (SwissProt accession number: P22310), UGT1A5 (SwissProt accession number: P35504), UGT1A6 (SwissProt accession number: P19224), UGT1A7 (SwissProt accession number: Q9HAW7), UGT1A10 (SwissProt accession number: Q9HAW8), UGT2A1 (SwissProt accession number: Q9Y4X1), UGT2B4 (SwissProt accession number: P06133), UGT2B7 (SwissProt accession number: P16662), UGT2B10 (SwissProt accession number: P36537), UGT2B11 (SwissProt accession number: 075310), UGT2B17 (SwissProt accession number: 075795), UGT2B28 (SwissProt accession number: Q9BY64), GSTA1 (SwissProt accession number: P08263), GSTA2 (SwissProt accession number: P09210), GSTA3 (SwissProt accession number: Q16772), GSTA4 (SwissProt accession number: 015217), GSTA5 (SwissProt accession number: Q7RTV2), GSTK1 (SwissProt accession number: Q9Y2Q3), GSTM1 (SwissProt accession number: P09488), GSTM2 (SwissProt accession number: P28161), GSTM3 (SwissProt accession number: P21266), GSTM4 (SwissProt accession number: Q03013), GSTM5 (SwissProt accession number: P46439), GSTP1 (SwissProt accession number: P09211), GSTT1 (SwissProt accession number: P30711), GSTT2 (SwissProt accession number: P30712), MGST1 (SwissProt accession number: P10620), MGST2 (SwissProt accession number: Q99735), MGST3 (SwissProt accession number: 014880), SULT1A2 (SwissProt accession number: P50226), SULT1A3 (SwissProt accession number: P50224), SULT1B1 (SwissProt accession number: 043704), SULT1C2 (SwissProt accession number: P000338), SULT1C3 (SwissProt accession number: Q6IMI6), SULT1C4 (SwissProt accession number: 075897), SULT1E1 (SwissProt accession number: P49888), SULT2A1 (SwissProt accession number: Q06520), SULT2B1 (SwissProt accession number: 000204), SULT4A1 (SwissProt accession number: Q9BR01), SULT4S6 (SwissProt accession number: Q7LFX5), P450R (SwissProt accession number: P16435), MGMT (SwissProt accession number: P16455), dCK (SwissProt accession number: P27707), CDA (SwissProt accession number: P32320), cN-IA (SwissProt accession number: Q9BX13), cN-IB (SwissProt accession number: Q96P26), cN-II (SwissProt accession number: P49902), cN-III (SwissProt accession number: Q9H0P0), dNT-1 (SwissProt accession number: Q8TCD5), dNT-2 (SwissProt accession number: Q9NPB1), Ecto-5′-NT (SwissProt accession number: P21589), RRM1 (SwissProt accession number: P23921), RRM2 (SwissProt accession number: P31350), UMP/CMPK (SwissProt accession number: P30085), dCMPDA (SwissProt accession number: P32321), CTP synthase 1 (SwissProt accession number: P17812), and CTP synthase 2 (SwissProt accession number: Q9NRF8).
The peptide of the present invention is a peptide consisting of any of partial amino acid sequences of the above-mentioned human metabolizing enzyme proteins. Specific examples of the peptide include peptides each consisting of any of amino acid sequences of SEQ ID NOS: 1 to 5, which are partial sequences of CYP1A1; amino acid sequences of SEQ ID NOS: 6 to 8, which are partial sequences of CYP1A2; and amino acid sequences of SEQ ID NOS: 9 to 14, which are partial sequences of CYP1B1. CYP1A1, CYP1A2, and CYP1B1 can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of any of amino acid sequences of SEQ ID NOS: 15 to 17, which are partial sequences of CYP2A6; amino acid sequences of SEQ ID NOS: 18 and 19, which are partial sequences of CYP2A7; amino acid sequences of SEQ ID NOS: 20 and 21, which are partial sequences of CYP2A13; amino acid sequences of SEQ ID NOS: 22 to 25, which are partial sequences of CYP2B6; amino acid sequences of SEQ ID NOS: 26 to 29, which are partial sequences of CYP2C8; amino acid sequences of SEQ ID NOS: 30 and 31, which are partial sequences of CYP2C9; amino acid sequences of SEQ ID NOS: 32 to 34, which are partial sequences of CYP2C18; amino acid sequences of SEQ ID NOS: 35 to 37, which are partial sequences of CYP2C19; amino acid sequences of SEQ ID NOS: 38 and 39, which are partial sequences of CYP2D6; amino acid sequences of SEQ ID NOS: 40 to 46, which are partial sequences of CYP2E1; an amino acid sequence of SEQ ID NO: 47, which is a partial sequence of CYP2F1; amino acid sequences of SEQ ID NOS: 48 to 55, which are partial sequences of CYP2J2; amino acid sequences of SEQ ID NOS: 56 to 63, which are partial sequences of CYP2R1; amino acid sequences of SEQ ID NOS: 64 to 72, which are partial sequences of CYP2S1; and amino acid sequences of SEQ ID NOS: 73 to 79, which are partial sequences of CYP2W1, are also the peptides of the present invention. CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2R1, CYP2S1, and CYP2W1 can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of any of amino acid sequences of SEQ ID NOS: 80 and 81, which are partial sequences of CYP3A4; amino acid sequences of SEQ ID NOS: 82 to 84, which are partial sequences of CYP3A5; amino acid sequences of SEQ ID NOS: 85 to 88, which are partial sequences of CYP3A7; and amino acid sequences of SEQ ID NOS: 89 to 96, which are partial sequences of CYP3A43, are also the peptides of the present invention. CYP3A4, CYP3A5, CYP3A7, and CYP3A43 can be quantified using any the corresponding peptides.
Similarly, peptides each consisting of any of amino acid sequences of SEQ ID NOS: 97 to 101, which are partial sequences of CYP4A11; amino acid sequences of SEQ ID NOS: 102 to 111, which are partial sequences of CYP4B1; amino acid sequences of SEQ ID NO: 112 to 114, which are partial sequences of CYP4F2; an amino acid sequence of SEQ ID NO: 115, which is a partial sequence of CYP4F3; amino acid sequences of SEQ ID NOS: 116 to 119, which are partial sequences of CYP4F8; amino acid sequences of SEQ ID NOS: 120 to 122, which are partial sequences of CYP4F11; amino acid sequences of SEQ ID NOS: 123 to 127, which are partial sequences of CYP4F12; amino acid sequences of SEQ ID NOS: 128 to 136, which are partial sequences of CYP4F22; amino acid sequences of SEQ ID NOS: 137 to 144, which are partial sequences of CYP4V2; amino acid sequences of SEQ ID NOS: 145 to 154, which are partial sequences of CYP4X1; and amino acid sequences of SEQ ID NOS: 155 to 165, which are partial sequences of CYP4Z1, are also the peptides of the present invention. CYP4A11, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4V2, CYP4X1, and CYP4Z1 can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of any of amino acid sequences of SEQ ID NOS: 166 to 171, which are partial sequences of CYP7A1; amino acid sequences of SEQ ID NOS: 172 to 178, which are partial sequences of CYP7B1; amino acid sequences of SEQ ID NOS: 179 to 185, which are partial sequences of CYP8B1; amino acid sequences of SEQ ID NOS: 186 to 191, which are partial sequences of CYP11B1; amino acid sequences of SEQ ID NOS: 192 to 195, which are partial sequences of CYP11B2; amino acid sequences of SEQ ID NOS: 196 to 200, which are partial sequences of CYP17A1; and amino acid sequences of SEQ ID NOS: 201 to 207, which are partial sequences of CYP19A1, are also the peptides of the present invention. CYP7A1, CYP7B1, CYP8B1, CYP11B1, CYP11B2, CYP17A1, and CYP19A1 can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of any of amino acid sequences of SEQ ID NOS: 208 to 210, which are partial sequences of CYP21A2; amino acid sequences of SEQ ID NOS: 211 to 216, which are partial sequences of CYP24A1; amino acid sequences of SEQ ID NOS: 217 to 226, which are partial sequences of CYP26A1; amino acid sequences of SEQ ID NOS: 227 to 238, which are partial sequences of CYP26B1; amino acid sequences of SEQ ID NOS: 239 to 249, which are partial sequences of CYP26C1; amino acid sequences of SEQ ID NOS: 250 to 256, which are partial sequences of CYP27A1; amino acid sequences of SEQ ID NOS: 257 to 265, which are partial sequences of CYP27B1; and amino acid sequences of SEQ ID NOS: 266 to 275, which are partial sequences of CYP27C1, are also the peptides of the present invention. CYP21A2, CYP24A1, CYP26A1, CYP26B1, CYP26C1, CYP27A1, CYP27B1, and CYP27C1 can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of any of an amino acid sequence of SEQ ID NO: 276, which is a partial sequence of CYP39A1; amino acid sequences of SEQ ID NOS: 277 to 284, which are partial sequences of CYP46A1; and amino acid sequences of SEQ ID NOS: 285 to 296, which are partial sequences of CYP51A1, are also the peptides of the present invention. CYP39A1, CYP46A1, and CYP51A1 can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of any of an amino acid sequence of SEQ ID NO: 297, which is a partial sequence of UGT1A1; amino acid sequences of SEQ ID NOS: 298 and 299, which are partial sequences of UGT1A3; amino acid sequences of SEQ ID NOS: 300 and 301, which are partial sequences of UGT1A4; an amino acid sequence of SEQ ID NO: 302, which is a partial sequence of UGT1A5; amino acid sequences of SEQ ID NOS: 303 to 305, which are partial sequences of UGT1A6; amino acid sequences of SEQ ID NOS: 306 and 307, which are partial sequences of UGT1A7; and an amino acid sequence of SEQ ID NO: 308, which is a partial sequence of UGT1A10, are also the peptides of the present invention. UGT1A1, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT1A7, and UGT1A10 can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of any of amino acid sequences of SEQ ID NOS: 309 and 310, which are partial sequences of UGT2A1; an amino acid sequence of SEQ ID NO: 311, which is a partial sequence of UGT2B4; amino acid sequences of SEQ ID NOS: 312 and 313, which are partial sequences of UGT2B7; an amino acid sequence of SEQ ID NO: 314, which is a partial sequence of UGT2B10; an amino acid sequence of SEQ ID NO: 315, which is a partial sequence of UGT2B11; an amino acid sequence of SEQ ID NO: 316, which is a partial sequence of UGT2B17; and an amino acid sequence of SEQ ID NO: 317, which is a partial sequence of UGT2B28, are also the peptides of the present invention. UGT2A1, UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B17, and UGT2B28 can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of any of an amino acid sequence of SEQ ID NO: 318, which is a partial sequence of GSTA1; an amino acid sequence of SEQ ID NO: 319, which is a partial sequence of GSTA2; an amino acid sequence of SEQ ID NO: 320, which is a partial sequence of GSTA3; amino acid sequences of SEQ ID NOS: 321 to 323, which are partial sequences of GSTA4; and an amino acid sequence of SEQ ID NO: 324, which is a partial sequence of GSTA5, are also the peptides of the present invention. GSTA1, GSTA2, GSTA3, GSTA4, and GSTA5 can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of any of an amino acid sequence of SEQ ID NO: 325, which is a partial sequence of GSTK1; an amino acid sequence of SEQ ID NO: 326, which is a partial sequence of GSTM1; amino acid sequences of SEQ ID NOS: 327 and 328, which are partial sequences of GSTM2; an amino acid sequence of SEQ ID NO: 329, which is a partial sequence of GSTM3; an amino acid sequence of SEQ ID NO: 330, which is a partial sequence of GSTM4; an amino acid sequence of SEQ ID NO: 331, which is a partial sequence of GSTM5; an amino acid sequence of SEQ ID NO: 332, which is a partial sequence of GSTP1; amino acid sequences of SEQ ID NOS: 333 and 334, which are partial sequences of GSTT1; and an amino acid sequence of SEQ ID NO: 335, which is a partial sequence of GSTT2, are also the peptides of the present invention. GSTM1, GSTM2, GSTM3, GSTM4, GSTM5, GSTT1, and GSTT2 can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of any of an amino acid sequence of SEQ ID NO: 336, which is a partial sequence of MGST1; an amino acid sequence of SEQ ID NO: 337, which is a partial sequence of MGST2; and an amino acid sequence of SEQ ID NO: 338, which is a partial sequence of MGST3, are also the peptides of the present invention. MGST1, MGST2, and MGST3 can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of any of an amino acid sequence of SEQ ID NO: 339, which is a partial sequence of SULT1A2; an amino acid sequence of SEQ ID NO: 340, which is a partial sequence of SULT1A3; an amino acid sequence of SEQ ID NO: 341, which is a partial sequence of SULT1B1; an amino acid sequence of SEQ ID NO: 342, which is a partial sequence of SULT1C2; an amino acid sequence of SEQ ID NO: 343, which is a partial sequence of SULT1C3; an amino acid sequence of SEQ ID NO: 344, which is a partial sequence of SULT1C4; an amino acid sequence of SEQ ID NO: 345, which is a partial sequence of SULT1E1; an amino acid sequence of SEQ ID NO: 346, which is a partial sequence of SULT2A1; an amino acid sequence of SEQ ID NO: 347, which is a partial sequence of SULT2B1; an amino acid sequence of SEQ ID NO: 348, which is a partial sequence of SULT4A1; and an amino acid sequence of SEQ ID NO: 349, which is a partial sequence of SULT4S6, are also the peptides of the present invention. SULT1A2, SULT1A3, SULT1B1, SULT1C2, SULT1C3, SULT1C4, SULT1E1, SULT2A1, SULT2B1, SULT4A1, and SULT4S6 can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of any of amino acid sequences of SEQ ID NOS: 350 to 359, which are partial sequences of P450R; amino acid sequences of SEQ ID NOS: 360 and 361, which are partial sequences of MGMT; amino acid sequences of SEQ ID NOS: 362 and 363, which are partial sequences of dCK; and an amino acid sequence of SEQ ID NO: 364, which is a partial sequence of CDA, are also the peptides of the present invention. P450R, MGMT, dCK, and CDA can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of amino acid sequences of SEQ ID NOS: 365 and 366, which are partial sequences of cN-IA; amino acid sequences of SEQ ID NOS: 367 to 374, which are partial sequences of cN-IB; an amino acid sequence of SEQ ID NO: 375, which is a partial sequence of cN-II; and amino acid sequences of SEQ ID NOS: 376 to 382, which are partial sequences of cN-III, are also the peptides of the present invention. cN-IA, cN-IB, cN-II, and cN-III can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of any of an amino acid sequence of SEQ ID NO: 383, which is a partial sequence of dNT-1; amino acid sequences of SEQ ID NOS: 384 and 385, which are partial sequences of dNT-2; amino acid sequences of SEQ ID NOS: 386 to 388, which are partial sequences of Ecto-5′-NT; amino acid sequences of SEQ ID NOS: 389 to 392, which are partial sequences of RRM1; amino acid sequences of SEQ ID NOS: 393 and 394, which are partial sequences of RRM2; an amino acid sequence of SEQ ID NO: 395, which is a partial sequence of UMP/CMPK; and an amino acid sequence of SEQ ID NO: 396, which is a partial sequence of dCMPDA, are also the peptides of the present invention. dNT-1, dNT-2, Ecto-5′-NT, RRM1, RRM2, UMP/CMPK, and dCMPDA can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of any of amino acid sequences of SEQ ID NOS: 397 to 405, which are partial sequences of CTP Synthase 1; and amino acid sequences of SEQ ID NOS: 406 to 412, which are partial sequences of CTP Synthase 2, are also the peptides of the present invention. CTP Synthase 1 and CTP Synthase 2 can be quantified using any of the respective peptides.
Furthermore, the peptide of the present invention is a peptide consisting of any of partial amino acid sequences of mouse metabolizing enzyme proteins set forth in SEQ ID NOS: 413 to 695. Specific examples of the mouse metabolizing enzyme protein include Cyp11a1 (SwissProt accession number: Q9QZ82), Cyp17a1 (SwissProt accession number: P27786), Cyp19a1 (SwissProt accession number: P28649), Cyp1a1 (SwissProt accession number: P00184), Cyp1a2 (SwissProt accession number: P00186), Cyp21 (SwissProt accession number: P03940), Cyp24a1 (SwissProt accession number: Q64441), Cyp26a1 (SwissProt accession number: 055127), Cyp27a1 (SwissProt accession number: Q9 DBG1), Cyp27b1 (SwissProt accession number: 035084), Cyp2a4 (SwissProt accession number: P15392), Cyp2a5 (SwissProt accession number: P20852), Cyp2b19 (SwissProt accession number: 055071), Cyp2c29 (SwissProt accession number: Q64458), Cyp2c39 (SwissProt accession number: P56656), Cyp2c70 (SwissProt accession number: Q91W64), Cyp2d10 (SwissProt accession number: P24456), Cyp2d26 (SwissProt accession number: Q8CIM7), Cyp2d9 (SwissProt accession number: P11714), Cyp2e1 (SwissProt accession number: Q05421), Cyp2f2 (SwissProt accession number: P33267), Cyp2j5 (SwissProt accession number: 054749), Cyp2r1 (SwissProt accession number: Q6VVW9), Cyp2s1 (SwissProt accession number: Q9 DBX6), Cyp39a1 (SwissProt accession number: Q9JKJ9), Cyp3a11 (SwissProt accession number: Q64459), Cyp3a13 (SwissProt accession number: Q64464), Cyp3a16 (SwissProt accession number: Q64481), Cyp3a25 (SwissProt accession number: 009158), Cyp46a1 (SwissProt accession number: Q9WVK8), Cyp4a14 (SwissProt accession number: 035728), Cyp4b1 (SwissProt accession number: Q64462), Cyp4f14 (SwissProt accession number: Q9EP75), Cyp4v3 (SwissProt accession number: Q9 DBW0), Cyp5a (SwissProt accession number: P36423), Cyp8b1 (SwissProt accession number: 088962), GSTO1 (SwissProt accession number: 009131), ST2B1 (SwissProt accession number: 035400), ST3A1 (SwissProt accession number: 035403), CHST3 (SwissProt accession number: 088199), SNAT (SwissProt accession number: 088816), UD2B5 (SwissProt accession number: P17717), GSTP1 (SwissProt accession number: P19157), GSTM4 (SwissProt accession number: P19639), GSTA4 (SwissProt accession number: P24472), GSTA3 (SwissProt accession number: P30115), GSTM5 (SwissProt accession number: P48774), ST1E1 (SwissProt accession number: P49891), ARY1 (SwissProt accession number: P50294), ARY2 (SwissProt accession number: P50295), ARY3 (SwissProt accession number: P50296), ST1A1 (SwissProt accession number: P52840), UD12 (SwissProt accession number: P70691), CGT (SwissProt accession number: Q64676), UD17C (SwissProt accession number: Q6ZQM8), ST1C1 (SwissProt accession number: Q80VR3), CHST2 (SwissProt accession number: Q80WV3), MGST3 (SwissProt accession number: Q9CPU4), ST1C2 (SwissProt accession number: Q9D939), GSTK1 (SwissProt accession number: Q9DCM2), CHST7 (SwissProt accession number: Q9EP78), CHST1 (SwissProt accession number: Q9EQC0), CHST5 (SwissProt accession number: Q9QUP4), NAT6 (SwissProt accession number: Q9R123), CHST4 (SwissProt accession number: Q9R111), and MAAI (SwissProt accession number: Q9WVL0).
The peptide of the present invention is a peptide consisting of any of partial amino acid sequences of the above-listed proteins. Specific examples of the peptide include peptides each consisting of any of amino acid sequences of SEQ ID NOS: 413 to 422, which are partial sequences of Cyp11a1; amino acid sequences of SEQ ID NOS: 423 to 429, which are partial sequences of Cyp17a1; amino acid sequences of SEQ ID NOS: 430 to 436, which are partial sequences of Cyp19a1; amino acid sequences of SEQ ID NOS: 437 and 438, which are partial sequences of Cyp1a1; and amino acid sequences of SEQ ID NOS: 439 to 443, which are partial sequences of Cyp1a2. Cyp11a1, Cyp17a1, Cyp19a1, Cyp1a1, and Cyp1a2 can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of any of amino acid sequences of SEQ ID NOS: 444 to 448, which are partial sequences of Cyp21; amino acid sequences of SEQ ID NOS: 449 to 455, which are partial sequences of Cyp24a1; amino acid sequences of SEQ ID NOS: 456 to 464, which are partial sequences of Cyp26a1; amino acid sequences of SEQ ID NOS: 465 to 470, which are partial sequences of Cyp27a1; and amino acid sequences of SEQ ID NOS: 471 to 478, which are partial sequences of Cyp27b1, are also the peptides of the present invention. Cyp21, Cyp24a1, Cyp26a1, Cyp27a1, and Cyp27b1 can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of any of an amino acid sequence of SEQ ID NO: 479, which is a partial sequence of Cyp2a4; an amino acid sequence of SEQ ID NO: 480, which is a partial sequence of Cyp2a5; amino acid sequences of SEQ ID NOS: 481 to 483, which are partial sequences of Cyp2b19; an amino acid sequence of SEQ ID NO: 484, which is a partial sequence of Cyp2c29; amino acid sequences of SEQ ID NOS: 485 to 488, which are partial sequences of Cyp2c39; amino acid sequences of SEQ ID NOS: 489 to 492, which are partial sequences of Cyp2c70; an amino acid sequence of SEQ ID NO: 493, which is a partial sequence of Cyp2d10; amino acid sequences of SEQ ID NOS: 494 to 497, which are partial sequences of Cyp2d26; amino acid sequences of SEQ ID NOS: 498 and 499, which are partial sequences of Cyp2d9; an amino acid sequence of SEQ ID NO: 500, which is a partial sequence of Cyp2e1; amino acid sequences of SEQ ID NOS: 501 to 505, which are partial sequences of Cyp2f2; amino acid sequences of SEQ ID NOS: 506 to 511, which are partial sequences of Cyp2j5; amino acid sequences of SEQ ID NO: 512 and 513, which are partial sequences of Cyp2r1; and amino acid sequences of SEQ ID NOS: 514 to 526, which are partial sequences of Cyp2s1, are also the peptides of the present invention. Cyp2a4, Cyp2a5, Cyp2b19, Cyp2c29, Cyp2c39, Cyp2c70, Cyp2d10, Cyp2d26, Cyp2d9, Cyp2e1, Cyp2f2, Cyp2j5, Cyp2r1, and Cyp2s1 can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of any of an amino acid sequence of SEQ ID NO: 527, which is a partial sequence of Cyp39a1; an amino acid sequence of SEQ ID NO: 528, which is a partial sequence of Cyp3a11; amino acid sequences of SEQ ID NOS: 529 to 534, which are partial sequences of Cyp3a13; amino acid sequences of SEQ ID NOS: 535 and 536, which are partial sequences of Cyp3a16; and amino acid sequences of SEQ ID NOS: 537 and 538, which are partial sequences of Cyp3a25, are also the peptides of the present invention. Cyp39a1, Cyp3a11, Cyp3a13, Cyp3a16, and Cyp3a25 can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of any of amino acid sequences of SEQ ID NOS: 539 to 545, which are partial sequences of Cyp46a1, are also the peptides of the present invention. Cyp46a1 can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of any of amino acid sequences of SEQ ID NOS: 546 to 550, which are partial sequences of Cyp4a14; amino acid sequences of SEQ ID NOS: 551 to 554, which are partial sequences of Cyp4b1; amino acid sequences of SEQ ID NOS: 555 to 558, which are partial sequences of Cyp4f14; amino acid sequences of SEQ ID NOS: 559 to 566, which are partial sequences of Cyp4v3; amino acid sequences of SEQ ID NOS: 567 to 570, which are partial sequences of Cyp5a; and amino acid sequences of SEQ ID NOS: 571 to 576, which are partial sequences of Cyp8b1, are also the peptides of the present invention. Cyp4a14, Cyp4b1, Cyp4f14, Cyp4v3, Cyp5a, and Cyp8b1 can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of any of amino acid sequences of SEQ ID NOS: 577 and 578, which are partial sequences of GSTO1; an amino acid sequence of SEQ ID NO: 579, which is a partial sequence of ST2B1; amino acid sequences of SEQ ID NOS: 580 to 585, which are partial sequences of ST3A1; amino acid sequences of SEQ ID NOS: 586 to 595, which are partial sequences of CHST3; an amino acid sequence of SEQ ID NO: 596, which is a partial sequence of SNAT; and an amino acid sequence of SEQ ID NO: 597, which is a partial sequence of UD2B5, are also the peptides of the present invention. GSTO1, ST2B1, ST3A1, CHST3, SNAT, and UD2B5 can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of any of an amino acid sequence of SEQ ID NO: 598, which is a partial sequence of GSTP1; an amino acid sequence of SEQ ID NO: 599, which is a partial sequence of GSTM4; amino acid sequences of SEQ ID NOS: 600 to 603, which are partial sequences of GSTA4; amino acid sequences of SEQ ID NOS: 604 and 605, which are partial sequences of GSTA3; amino acid sequences of SEQ ID NOS: 606 to 609, which are partial sequences of GSTM5; and amino acid sequences of SEQ ID NOS: 610 to 613, which are partial sequences of ST1E1, are also the peptides of the present invention. GSTP1, GSTM4, GSTA4, GSTA3, GSTM5, and ST1E1 can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of any of amino acid sequences of SEQ ID NOS: 614 and 615, which are partial sequences of ARY1; amino acid sequences of SEQ ID NOS: 616 to 620, which are partial sequences of ARY2; amino acid sequences of SEQ ID NOS: 621 to 624, which are partial sequences of ARY3; amino acid sequences of SEQ ID NOS: 625 and 626, which are partial sequences of ST1A1; amino acid sequences of SEQ ID NOS: 627 to 629, which are partial sequences of UD12; amino acid sequences of SEQ ID NOS: 630 to 638, which are partial sequences of CGT; amino acid sequences of SEQ ID NOS: 639 to 641, which are partial sequences of UD17C; and amino acid sequences of SEQ ID NOS: 642 and 643, which are partial sequences of ST1C1, are also the peptides of the present invention. ARY1, ARY2, ARY3, ST1A1, UD12, CGT, UD17C, and ST1C1 can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of any of amino acid sequences of SEQ ID NOS: 644 to 650, which are partial sequences of CHST2; an amino acid sequence of SEQ ID NO: 651, which is a partial sequence of MGST3; amino acid sequences of SEQ ID NOS: 652 to 655, which are partial sequences of ST1C2; and amino acid sequences of SEQ ID NOS: 656 to 659, which are partial sequences of GSTK1, are also the peptides of the present invention. CHST2, MGST3, ST1C2, and GSTK1 can be quantified using any of the corresponding peptides.
Similarly, peptides each consisting of any of amino acid sequences of SEQ ID NOS: 660 to 671, which are partial sequences of CHST7; amino acid sequences of SEQ ID NOS: 672 to 676, which are partial sequences of CHST1; amino acid sequences of SEQ ID NOS: 677 to 683, which are partial sequences of CHST5; amino acid sequences of SEQ ID NOS: 684 to 686, which are partial sequences of NAT6; amino acid sequences of SEQ ID NOS: 687 to 691, which are partial sequences of CHST4; and amino acid sequences of SEQ ID NOS: 692 to 695, which are partial sequences of MAAI, are also the peptides of the present invention. CHST7, CHST1, CHST5, NAT6, CHST4, and MAAI can be quantified using any of the corresponding peptides.
(Peptides Derived from Homologues or Peptides Corresponding to Human SNPs)
Examples of the peptide of the present invention can further include peptides for use in simultaneous protein quantification of metabolizing enzymes with a mass spectrometer, consisting of an amino acid sequence including deletion, substitution, or addition of one or two amino acids in any one of amino acid sequences of SEQ ID NOS: 1 to 695. Specific preferred examples of these peptides can include peptides derived from homologues (homologous proteins) of mammals other than humans or mice, consisting of an amino acid sequence including deletion, substitution, or addition of one or two (preferably one) amino acids in any one of amino acid sequences of SEQ ID NOS: 1 to 695, and peptides corresponding to SNPs in human metabolizing enzyme proteins, consisting of an amino acid sequence including deletion, substitution, or addition of one or two (preferably one) amino acids in any one of partial amino acid sequences of a human metabolizing enzyme protein set forth in SEQ ID NOS: 1 to 413. Examples of the above-mentioned mammals other than human or mouse can include primates other than human (for example, monkey, baboon, and chimpanzee), swine, bovine, equine, goat, sheep, dog, feline, rabbits, guinea pig, gerbil, hamster, and rat.
The above-mentioned peptides consisting of partial amino acid sequences of human metabolizing enzyme proteins, the above-mentioned peptides consisting of partial amino acid sequences of mouse metabolizing enzyme proteins, and the above-mentioned peptides derived from homologues or peptides corresponding to human SNPs (hereinafter, these peptides of the present invention may be collectively referred to as “unlabeled peptide of the invention”) of the present invention can be produced by common chemical synthesis methods. As such a peptide synthesis method, a stepwise elongation method of extending a chain by binding amino acids one by one based on the amino acid sequence information or a fragment condensation method of synthesizing fragments consisting of several amino acids beforehand and then coupling the fragments can be employed.
Furthermore, when the labeled peptide of the present invention is a stable-isotope-labeled peptide in which one or more amino acids constituting the above-mentioned unlabeled peptide of the invention contain any one or more of 15N, 13C, 18O, and 2H, the type, the position, and the number of the amino acids are not particularly limited so long as the peptides have differences in mass that can be separated by a liquid chromatograph-tandem mass spectrometer (LC-MS/M). Above all, leucine labeled with 13C at six sites is more preferably included. Such a stable-isotope-labeled peptide can be chemically synthesized by appropriate measures such as an F-moc method (Amblard M, Fehrentz J A, Martinez J, Subra G. Methods Mol Biol. 298: 3-24 (2005)) using amino acids labeled with a stable isotope. Furthermore, as a stable-isotope-labeled peptide is chemically identical to a peptide to be quantified and shows an identical behavior in LC-MS/MS measurement except that the mass of a labeled amino acid is different, the stable-isotope-labeled peptide can be used as an internal standard peptide in simultaneous protein quantification of metabolizing enzymes with a mass spectrometer.
The kit of the present invention for simultaneous protein quantification of metabolizing enzymes is not particularly limited so long as the kit comprises the unlabeled peptide of the invention and the stable-isotope-labeled peptide of the present invention. The using method of present invention is not particularly limited so long as the method uses the unlabeled peptide of the invention and the stable-isotope-labeled peptide of the present invention as probes for simultaneous protein quantification of metabolizing enzymes. The unlabeled peptide of the invention, which is also a peptide to be quantified, is used to create a calibration curve for quantification, and the stable-isotope-labeled peptide is used to create a calibration curve for quantification and as an internal standard peptide.
The method for simultaneous protein quantification of metabolizing enzymes by LC-MS/MS using the stable-isotope-labeled peptide of the present invention is not particularly limited so long as the method comprises the steps of: (a) performing a mass spectrometry by LC-MS/MS using the unlabeled peptide of the invention and the stable-isotope-labeled peptide of the present invention at predetermined concentration levels to create a calibration curve; (b) performing a mass spectrometry by LC-MS/MS by adding the stable-isotope-labeled peptide of the present invention to peptide fragments obtained by fragmenting metabolizing enzyme proteins to be quantified in a sample with trypsin to calculate a mass spectrum area ratio of a metabolizing enzyme protein peptide to be quantified and a stable-isotope-labeled peptide; and (c) obtaining quantitative values from the area ratio using the calibration curve. Examples of the above-mentioned sample as a source of proteins to be quantified can include various tissues such as the liver and cultured cells derived from various tissues. Furthermore, when the amounts of proteins to be quantified contained in a sample are not more than the measurement limit of a mass spectrometer, a sample fractioned by a method of filling a high pressure nitrogen gas or the like is preferably used in measurement. Furthermore, when two or more peptides to be quantified are identified for one target metabolizing enzyme protein, simultaneous protein quantification of metabolizing enzymes at higher precision and reliability can be achieved by quantification using two or more peptides to be quantified.
Furthermore, when a protein to be quantified and a subtype thereof have very similar amino acid sequences, the protein to be quantified can also be quantified by obtaining a difference between a quantitative value of a peptide common to these proteins and a quantitative value of a peptide specific to one protein. For example, the quantitative value of a metabolizing protein CYP3A4 can be obtained by (a quantitative value of a peptide common to CYP3A4 and CYP3A5)—(a quantitative value of a peptide specific to CYP3A5).
Hereafter, the present invention will be specifically described with reference to the following Examples. However, the technical scope of the present invention is not limited to these examples.
Measurement by nanoLC-MS/MS was performed using peptides to be quantified, which are partial sequence peptides of human metabolizing enzymes CYP and P450R (unlabeled peptides: CYP1A2, YLPNPALQR (SEQ ID NO: 7); CYP2A6, GTGGANIDPTFFLSR (SEQ ID NO: 15); CYP2B6, GYGVIFANGNR (SEQ ID NO: 25); CYP2C8, GNSPISQR (SEQ ID NO: 28); CYP2C9, GIFPLAER (SEQ ID NO: 30); CYP2C18, IAENFAYIK (SEQ ID NO: 32); CYP2C19, GHFPLAER (SEQ ID NO: 35); CYP2D6, DIEVQGFR (SEQ ID NO: 39); CYP2E1, GIIFNNGPTWK (SEQ ID NO: 44); CYP2J2, EENGQPFDPHFK (SEQ ID NO: 52); CYP3A4, LQEEIDAVLPNK (SEQ ID NO: 81); CYP3A5, DTINFLSK (SEQ ID NO: 83); CYP3A7, FNPLDPFVLSIK (SEQ ID NO: 85); CYP3A43, YIPFGAGPR (SEQ ID NO: 91); CYP4A11, IPIPIAR (SEQ ID NO: 100); CYP51A1, FAYVPFGAGR (SEQ ID NO: 290); and P450R, FAVFGLGNK (SEQ ID NO: 355)) and synthesized stable-isotope-labeled peptides [isotope-labeled 13C6, peptides: CYP1A2, YLPNPAL (13C6, (SEQ ID NO: 7); CYP2A6, GTGGANIDPTFFL (13C6,15N) SR (SEQ ID NO: 15); CYP2B6, GYGVIFA(13C6,15N)NGNR (SEQ ID NO: 25); CYP2C8, GNSPI (13C6,15N) SQR (SEQ ID NO: 28); CYP2C9, GIFPL(13C6,15N)AER (SEQ ID NO: 30); CYP2C18, IAENFAYI (13C6,15N)K (SEQ ID NO: 32); CYP2C19, GHFPL(13C6,15N) AER(SEQ ID NO: 35); CYP2D6, DIEVQGF (13C6,15N)R (SEQ ID NO: 39); CYP2E1, GIIFNNGP(13C6,15N)TWK (SEQ ID NO: 44); CYP2J2, EENGQPFDPHF(13X6,15N)K (SEQ ID NO: 52); CYP3A4, LQEEIDAVLP(13C6, ‥N)NK (SEQ ID NO: 81); CYP3A5, DTINFL (13C6,15N)SK (SEQ ID NO: 83); CYP3A7, FNPLDPFVLSI (13C6,15N)K (SEQ ID NO: 85); CYP3A43, YIPFGAGP(13C6,15N)R (SEQ ID NO: 91); CYP4A11, IPIPIA(13C6, 1-5N) R (SEQ ID NO: 100); CYP51A1, FAYVPFGA (13C6,15N)GR (SEQ ID NO: 290); and P450R, FAVFGL(13C6,15N)GNK (SEQ ID NO: 355)] under the following conditions.
50 fmol each of 13C6,15N-labeled peptides were added to 1 fmol of unlabeled peptides for measurement by nanoLC-MS/MS. A reverse phase C18 150 μm I.D.×40 mm was used as a column. 0.1% formic acid and 0.1% formic acid/acetonitrile were used as mobile phases. Analysis was performed with a linear gradient of increasing the concentration of 0.1% formic acid/acetonitrile to 45% over 35 minutes. 4000 Q Trap of Applied Biosystems was used as amass spectrometer. A peptide was identified when a peptide to be quantified and a labeled peptide were eluted with the same time. The results are shown in
A human liver tissue was shredded with scissors and then suspended in 10 mM. Tris-HCl, 10 mM NaCl, and 1.5 mM MgCl2. The suspension was homogenized with a glass homogenizer, and the solution was centrifuged at 8000×g for 10 minutes. Further, the supernatant was centrifuged at 100,000×g for 60 minutes to obtain a crude membrane fraction. The obtained crude membrane fraction was denatured in a buffer containing 7 M guanidine hydrochloride, 0.1 M Tris-HCl, and 10 mM EDTA (pH 8.5) and subjected to reduction with DTT and carbamide methylation with iodoacetamide to protect the SH group of a cysteine residue. Concentration and desalting were performed by a methanol chloroform precipitation method. The fraction was suspended in 1.2 M urea and 10 mM Tris-HCl, then trypsin was added in an amount corresponding to 1/100 of the protein weight, and proteins were digested with the enzyme at 37° C. for 16 hours to obtain a peptide sample.
50 fmol of 13C6,15N-labeled peptides were added to 5 μg of the obtained crude membrane fraction peptide sample, and peptides were measured by LC-MS/MS (
A calibration curve was created using a selected peptide to be quantified to examine linearity. 50 fmol each of 13C6,15N-labeled peptides were added to each of 1, 5, 10, 50, and 100 fmol of an unlabeled peptide, and peptides were similarly measured by nanoLC-MS/MS. MS spectrum area ratios (unlabeled peptide/13C6,15N-labeled peptide) were calculated to create a calibration curve.
The quantification results CYP and P450R are shown in Table 1.
Measurement by conventional LC-MS/MS was performed using peptides to be quantified, which are partial sequence peptides of human Ara-C metabolizing enzymes, (unlabeled peptides: dCK, AQLASLNGK (SEQ ID NO: 362); CDA, AVSEGYK (SEQ ID NO: 364); cN-IA, GFLEALGR (SEQ ID NO: 366); cN-IB, GFLEDLGR (SEQ ID NO: 369); cN-II, VFLATNSDYK (SEQ ID NO: 375); cN-III, GELIHVFNK (SEQ ID NO: 379); dNT-1, TVVLGDLLIDDK (SEQ ID NO: 383); Ecto-5′-NT, VPSYDPLK (SEQ ID NO: 388); RRM1, LNSAIIYDR (SEQ ID NO: 390); RRM2, ENTPPALSGTR (SEQ ID NO: 393); UMP/CMPK, FLIDGFPR (SEQ ID NO: 395); dCMPDA, LIIQAGIK (SEQ ID NO: 396); CTPS1, FVGQDVEGER (SEQ ID NO: 402); CTPS2, ADGILVPGGFGIR (SEQ ID NO: 409)) and synthesized stable-isotope-labeled peptides [isotope-labeled peptides: dCK, AQLASL(13C6,15N)NGK (SEQ ID NO: 362); CDA, AV (13C6,15N)SEGYK (SEQ ID NO: 364); cN-IA, GFLEAL (13C6,15N)GR (SEQ ID NO: 366); cN-IB, GFLEDL (13C6,15N) GR (SEQ ID NO: 369); cN-II, VFLA(13C6,15N) TNSDYK (SEQ ID NO: 375); cN-III, GELIHVF(13C6,15N) NK(SEQ ID NO: 379); dNT-1, TVVLGDLLI (13C6,15N)DDK (SEQ ID NO: 383); Ecto-5′-NT, VPSYDPL(13C6,15N)K (SEQ ID NO: 388); RRM1, LNSAII (13C6, 15N) YDR (SEQ ID NO: 390); RRM2, ENTPPAL (13C6,15N)SGTR (SEQ ID NO: 393); UMP/CMPK, FLIDGFP (13C6,15N)R (SEQ ID NO: 395); dCMPDA, LIIQAGI (13C6,15N)K (SEQ ID NO: 396); CTPS1, FVGQDV(13C6, 5N)EGER (SEQ ID NO: 402); and CTPS2, ADGILVPGGFGI (13C6,15N)R (SEQ ID NO: 409)] under the following conditions.
500 fmol each of 13C6,15N-labeled peptides were added to 10 fmol of unlabeled peptides for measurement by conventional LC-MS/MS. A reverse phase C18 0.5 mm I.D.×150 mm was used as a column. 0.1% formic acid and 0.1% formic acid/acetonitrile were used as mobile phases. Analysis was performed with a linear gradient of increasing the concentration of 0.1% formic acid/acetonitrile to 45% over 60 minutes. API 5000 of Applied Biosystems was used as a mass spectrometer. A peptide was identified when a peptide to be quantified and a labeled peptide are eluted with the same time. The results are shown in
5.0×108 cells of human leukemic cell strain K562 were cultured in an RPMI-1640 medium containing 10% FBS and then crushed by a cavitation method. This solution was centrifuged at 10,000×g for 10 minutes. Then, the supernatant was centrifuged at 100,000×g for 60 minutes to obtain a soluble fraction. The obtained soluble fraction was denatured in a buffer containing 7 M guanidine hydrochloride, 0.1 M Tris-HCl, and 10 mM EDTA (pH 8.5) and subjected to reduction with DTT and carbamide methylation with iodoacetamide to protect the SH group of a cysteine residue. Concentration and desalting were performed by a methanol chloroform precipitation method. The fraction was suspended in 1.2 M urea and 10 mM Tris-HCl, then trypsin was added in an amount of 1/100 corresponding to the protein weight, and proteins were digested with the enzyme at 37° C. for 16 hours to obtain a peptide sample.
500 fmol of 13C6,15N-labeled peptides were added to 50 μg of the obtained crude membrane fraction peptide sample and measured by LC-MS/MS. After measurement, MS spectrum area ratios (13C6,15N-labeled peptide) were calculated, and quantitative values were obtained using the calibration curve.
A calibration curve was created using a selected peptide to be quantified. 500 fmol each of 13C6,15N-labeled peptides were added to 1, 5, 10, 50, 100, 500, and 1000 fmol of an unlabeled peptide and measured by conventional LC-MS/MS in the same manner as in Example 1. The MS spectrum area ratios (unlabeled peptide/13C6,15N-labeled peptide)were calculated to create a calibration curve.
The results of Ara-C metabolizing enzyme quantification are shown in Table 2.
LC-MS/MS measurement was performed under the following conditions using a peptide to be quantified (unlabeled peptide: ENT2, VALTLDLDLEK (SEQIDNO: 696)), which is apartial sequence peptide of a human transporter ENT2 (SLC29A2), and a synthesized stable-isotope-labeled peptide [isotope-labeled peptide: ENT2, VALTLDLDL (13C6,15N)EK (SEQ ID NO: 696)], but the peptide could not be quantified due to the low detection sensitivity.
500 fmol of the 13C6,15N-labeled peptide was added to 500 fmol of the unlabeled peptide and measured by conventional LC-MS/MS. A reverse phase C18 0.5 mm I.D.×150 mm was used as a column. 0.1% formic acid and 0.1% formic acid/acetonitrile were used as mobile phases. Analysis was performed with a linear gradient of increasing the concentration of 0.1% formic acid/acetonitrile to 45% over 60 minutes. API 5000 of Applied Biosystems was used as a mass spectrometer. The peptide was identified under a condition that the peptide to be quantified and the labeled peptide were eluted with the same time, but no peaks were detected for either the peptide to be quantified or the labeled peptide due to the low sensitivity.
LC-MS/MS measurement was performed under the following conditions using a peptide to be quantified (unlabeled peptide: BGT1, QELIAWEK (SEQ ID NO: 697)), which is a partial sequence peptide of a human transporter BGT1 (SLC6A12), and a synthesized stable-isotope-labeled peptide [isotope-labeled peptide: BGT1, QELIA(13C6,15N)WEK (SEQ ID NO: 697)], but the peptide could not be quantified because peptides were not retained on the reverse phase column and therefore the sensitivity was low.
500 fmol of the 13C6,15N-labeled peptide was added to 500 fmol of the unlabeled peptide and measured by conventional LC-MS/MS. A reverse phase C18 0.5 mm I.D.×150 mm was used as a column. 0.1% formic acid and 0.1% formic acid/acetonitrile were used as mobile phases. Analysis was performed with a linear gradient of increasing the concentration of 0.1% formic acid/acetonitrile to 45% over 60 minutes. API 5000 of Applied Biosystems was used as a mass spectrometer. The peptide was identified under a condition that the peptide to be quantified and the labeled peptide were eluted with the same time, but no peaks were detected for either the peptide to be quantified or the labeled peptide.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2008-251212 | Sep 2008 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2009/004948 | 9/28/2009 | WO | 00 | 3/24/2011 |
