Non-signal imidazole reagents for mass spectrometry analysis of phosphomonoesters
Analytical chemical reagents termed non-signal imidazoles and a method for their use that provide a host of advantages for analysis of phosphomonoesters are described. The method and compounds of the invention provide a host of advantages for the analysis of phosphomonoester-containing compounds, namely characteristic, multi-analyte detection with high sensitivity and specificity of known and unknown phosphomonoester-compounds simultaneously.
1. A method for detecting a phosphomonoester-containing compound, said method comprising the steps of:
providing a sample to be assayed for the presence of a phosphomonoester-containing compound;
providing a non-signal imidazole reagent, said reagent having the structure:
wherein either R 1 or R 2 is hydrogen (H) or deuterium (D), B comprises one to eight carbon atoms, A comprises an aryl group, and at least one of A or B is substituted with one or more atoms or groups other than H when A is phenyl and B comprises an amide group; and wherein the imidazole reagent has no ionic group;
combining said sample and said non-signal imidazole reagent, wherein said non-signal imidazole reagent forms a product comprising a phosphorimidazolide anion signal group with any reactable phosphomonoester-containing compound in said sample; and
detecting said product as a phosphorimidazolide anion by mass spectrometry.
2. The method of claim 1 , wherein said reagent comprises a mixture of two or more different non-signal imidazole reagents.
3. The method of claim 1 , wherein said phosphomonoester-containing compound is a nucleotide, dinucleotide or trinucleotide.
4. The method of claim 1 , wherein said phosphomonoester-containing compound is a phosphopeptide.
5. The method of claim 1 , wherein said non-signal imidazole reagent is N-[2-(1H-imidazol-4-yl)ethyl]benzamide.
6. The method of claim 1 , wherein said non-signal imidazole reagent is selected from the group consisting of N-[2-(1H-imidazol-4-yl)ethyl-d 4 ]benzamide, 4-chloro-N-[2-(1H-imidazol-4-yl)ethyl]benzamide, 4-bromo-N-[2-(1H-imidazol-4-yl)ethyl]benzamide, and N-[2-(1H-imidazol-,4-yl)ethyl]benz-d 5 -amide.
7. The method of claim 1 , wherein said product is detected by matrix-assisted laser desorption ionization mass spectrometry.
8. The method of claim 1 , wherein B is substituted with four deuterium atoms.
9. The method of claim 1 , wherein A is substituted with a bromine atom (Br), a chlorine atom (Cl), or one or more deuterium atoms.
10. The method of claim 1 , wherein A comprises a naphthyl or biphenyl group.
11. The method of claim 1 , wherein A is substituted with one or more fluorine atoms, alkyl groups or alkoxy groups.
12. The method of claim 1 , wherein B comprises an amide group or an ether group.
13. The method of claim 1 , wherein B is —CD 2 CD 2 — and A is R 3 C 6 H 4 CONH—, wherein R 3 is Br or Cl.