Quantitative peptide or protein assay
Peptide and/or protein quantitation methods, kits, and compositions, particularly useful for mass spectrometry, are provided herein based on a bathocuproine-based composition complex such as bathocuproinedisulfonic acid disodium salt hydrate complex. The methods are one-step rapid absorbance methods using small sample volumes. They produce a robust signal with high signal to background ratio and accurately quantitate even complex peptide mixtures with low variability and high sensitivity.
1. A kit for protein or peptide quantitation of a sample comprising:
(a) a reagent composition comprising sodium carbonate;
(b) a reagent composition comprising acetonitrile and a complex having the formula:
wherein
each of R 1 and R 2 is independently chosen from C 1 -C 6 straight and branched alkyls, C 6 -C 20 aryl, alkylaryl, and arylalkyls;
each of R 3 and R 4 is independently chosen from the group consisting of hydrogen; sulfonate salts chosen from sodium, potassium, and lithium; phosphonate salts chosen from sodium, potassium, and lithium; and carboxylate salts chosen from sodium, potassium, and lithium; and
each of R 5 and R 6 is
wherein R 7 is independently chosen from the group consisting of hydrogen; sulfonate salts chosen from sodium, potassium, and lithium; phosphonate salts chosen from sodium, potassium, and lithium; and carboxylate salts chosen from sodium, potassium, and lithium;
with the proviso that at least one of R 3 , R 4 , and R 7 is not H;
(c) a reagent composition comprising copper sulfate; and
(d) instructions for the peptide quantitation determination, wherein the determination comprises combining the components (a), (b), and (c) with the sample to form a colored complex, wherein acetonitrile is present in the combination at a concentration ranging from about 10% to about 50%, wherein the kit does not contain an additional reducing agent to reduce excess Cu 2+ to Cu 1+ , and wherein the concentration of peptide or protein in said sample is directly proportional to the amount of colored complex that forms once the sample and components are mixed.
2. The kit of claim 1 , wherein the complex is present in the combination at a concentration ranging from about 0.04 M to about 0.1 M.
3. The kit of claim 1 , wherein each of R 1 and R 2 is independently chosen from methyl, ethyl, propyl, butyl, and phenyl.
4. The kit of claim 1 , wherein the kit further comprises (e) an internal standard chosen from peptides, peptide mixtures, and protein digests.
5. The kit of claim 1 , wherein the complex has the formula:
6. The kit of claim 5 , wherein the acetonitrile is present at a concentration ranging from about 10% to about 30%.
7. The kit of claim 1 , wherein the copper sulfate is present at a concentration ranging from about 0.25 M to about 0.5 M.
8. The kit of claim 1 , wherein the reagent composition of (a) further comprises tartrate.
9. The kit of claim 8 , wherein the tartrate is present at a concentration ranging from about 5.7 mM to about 22.7 mM.
10. The kit of claim 8 , where the tartrate is sodium tartarate.
11. The kit of claim 1 , wherein the reagent composition of (a) further comprises sodium bicarbonate.