Quantitation of insulin-like growth factor-i and insulin-like growth factor-ii with high-resolution mass spectrometry

Abstract

Methods are provided for determining the amount of an IGF-I and/or IGF-II protein in a sample using high resolution/high accuracy mass spectrometry. The methods generally comprise enriching an IGF-I and/or IGF-II protein in a sample, ionizing an IGF-I and/or IGF-II protein from the sample to generate IGF-I and/or IGF-II protein ions, and determining the amount of IGF-I and/or IGF-II protein ions with high resolution/high accuracy mass spectrometry.

1 . A method for determining the amount of an insulin-like growth factor-I (IGF-I) protein in a sample, the method comprising:
 a. subjecting IGF-I protein in the sample to ionization under conditions suitable to produce one or more ions detectable by mass spectrometry; and   b. determining the amount of said one or more ions by high resolution/high accuracy mass spectrometry;   
       wherein said high resolution/high accuracy mass spectrometry is conducted with a mass analyzer capable of a FWHM of greater than or equal to about 10,000 and an accuracy of less than or equal to about 50 ppm; and wherein the amount of the determined ion or ions is related to the amount of the IGF-I protein in the sample. 
     
     
         2 . The method of  claim 1 , wherein said IGF-I protein is a human IGF-I protein. 
     
     
         3 . (canceled) 
     
     
         4 . The method of  claim 1 , wherein said IGF-I protein is an intact IGF-I protein. 
     
     
         5 . The method of  claim 1 , wherein said IGF-I protein is intact long R3 IGF-I or a fragment thereof. 
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 1 , wherein the IGF-I protein is chemically modified prior to ionization. 
     
     
         8 . The method of  claim 7 , wherein the chemical modification comprises reduction of one or more disulfide bridges in the IGF-I protein. 
     
     
         9 . The method of  claim 7 , wherein the chemical modification comprises alkylation of one or more cysteines in the IGF-I protein. 
     
     
         10 . The method of  claim 1 , wherein said IGF-I protein from said sample is purified with solid phase extraction (SPE) prior to ionization. 
     
     
         11 . The method of  claim 1 , wherein said IGF-I protein from said sample is purified by high performance liquid chromatography (HPLC) prior to ionization. 
     
     
         12 . The method of  claim 10 , wherein said IGF-I protein from said sample is further purified by high performance liquid chromatography (HPLC) prior to ionization and said SPE and HPLC are conducted with on-line processing. 
     
     
         13 . The method of  claim 1 , wherein said high resolution/high accuracy mass spectrometry is conducted with an orbitrap mass spectrometer. 
     
     
         14 . The method of  claim 1 , wherein the high resolution/high accuracy mass spectrometry is conducted with a time of flight (TOF) mass spectrometer. 
     
     
         15 . The method of  claim 1 , wherein said high resolution/high accuracy mass spectrometry is conducted with an orbitrap or time of flight mass analyzer capable of a FWHM of greater than or equal to about 20,000 and an accuracy of less than or equal to about 10 ppm. 
     
     
         16 . The method of  claim 1 , wherein said high resolution/high accuracy mass spectrometry is conducted with an orbitrap or time of flight mass analyzer capable of a FWHM of greater than or equal to about 20,000 and an accuracy of less than or equal to about 5 ppm. 
     
     
         17 . The method of  claim 1 , wherein said step of determined the amount of one or more ions comprises collecting spectrometric data from one or more peaks with each peak resulting from an isotopic form a said one or more ions. 
     
     
         18 . The method of  claim 17 , wherein two or more peaks each resulting from a different isotopic form of an ion are used to confirm the identity of an IGF-I protein. 
     
     
         19 . The method of  claim 17 , wherein spectrometric data from a peak resulting from a single isotopic form of an ion is used to determine the amount of an IGF-I protein in said sample. 
     
     
         20 . The method of  claim 17 , wherein spectrometric data from two or more peaks each resulting from a single isotopic form of an ion are used to determine the amount of an IGF-I protein in said sample. 
     
     
         21 . The method of  claim 1 , wherein said sample comprises a biological sample. 
     
     
         22 . The method of  claim 1 , wherein said sample comprises plasma or serum. 
     
     
         23 . The method of  claim 1 , wherein said one or more ions detectable by mass spectrometry comprise one or more IGF-I ions in a 6+, 7+, 8+, or 9+ charge state. 
     
     
         24 . The method of  claim 1 , wherein said one or more ions detectable by mass spectrometry comprise one or more ions selected from the group consisting of ions with mass to charge ratios within ranges of 850.8±2, 957.1±2, 1093.7±2, and 1275.8±2. 
     
     
         25 . The method of  claim 1 , wherein said one or more ions detectable by mass spectrometry comprise one or more ions selected from the group consisting of ions with mass to charge ratios within ranges of 850.8±1, 957.1±1, 1093.7±1, and 1275.8±1. 
     
     
         26 . The method of  claim 1 , wherein said one or more ions detectable by mass spectrometry comprise one or more ions selected from the group of ions with a mass to charge ratio of 1091.9447±0.1, 1092.8031±0.1, 1092.9445±0.1, 1093.0881±0.1, 1093.2308±0.1, 1093.3740±0.1, 1093.5167±0.1, 1093.6597±0.1, 1093.8028±0.1, 1093.9458±0.1, 1094.0889±0.1, 1094.2319±0.1, 1094.3754±0.1, 1094.5185±0.1, 1094.6606±0.1, and 1095.3717±0.1. 
     
     
         27 . (canceled) 
     
     
         28 . A method for determining the amount of native intact insulin-like growth factor (IGF-I) in a biological fluid sample, said method comprising:
 a. subjecting the intact IGF-I native to the sample, purified by solid phase extraction (SPE) and high performance liquid chromatography (HPLC), to ionization under conditions suitable to produce one or more ions detectable by mass spectrometry; wherein said one or more ions detectable by mass spectrometry comprise one or more IGF-I ions in a 6+, 7+, 8+, or 9+ charge state;   b. determining the amount of said one or more ions by high resolution/high accuracy mass spectrometry; and   c. using the amount of said one or more ions to determine the amount of native intact IGF-I in the sample,   
       wherein said high resolution/high accuracy mass spectrometry is conducted with an orbitrap or time of flight mass analyzer capable of a FWHM of greater than or equal to about 10,000 and an accuracy of less than or equal to about 50 ppm. 
     
     
         29 . The method of  claim 28 , wherein said high resolution/high accuracy mass spectrometry is conducted with an orbitrap or time of flight mass analyzer capable of a FWHM of greater than or equal to about 20,000 and an accuracy of less than or equal to about 10 ppm. 
     
     
         30 . The method of  claim 28 , wherein said high resolution/high accuracy mass spectrometry is conducted with an orbitrap or time of flight mass analyzer capable of a FWHM of greater than or equal to about 20,000 and an accuracy of less than or equal to about 5 ppm. 
     
     
         31 . The method of  claim 28 , wherein said biological fluid comprises plasma or serum. 
     
     
         32 . The method of  claim 28 , wherein said one or more of said ions comprise one or more IGF-I ions a 7+ or 8+ charge state. 
     
     
         33 . The method of  claim 28 , wherein one or more of said ions are selected from the group of ions with a mass to charge ratio within the mass to charge ratio ranges of 957.1±2 and 1093.7±2. 
     
     
         34 . The method of  claim 28 , wherein one or more of said ions are selected from the group of ions with a mass to charge ratio within the mass to charge ratio ranges of 957.1±1 and 1093.7±1. 
     
     
         35 . The method of  claim 28 , wherein said one or more ions detectable by mass spectrometry comprise one or more ions selected from the group consisting of ions with mass to charge ratios of 1091.9447±0.1, 1092.8031±0.1, 1092.9445±0.1, 1093.0881±0.1, 1093.2308±0.1, 1093.3740±0.1, 1093.5167±0.1, 1093.6597±0.1, 1093.8028±0.1, 1093.9458±0.1, 1094.0889±0.1 1094.2319±0.1, 1094.3754±0.1, 1094.5185±0.1, 1094.6606±0.1, and 1095.3717±0.1. 
     
     
         36 - 99 . (canceled)