US2015362504A1PendingUtilityA1
Quantitative peptide or protein assay
Est. expiryJun 11, 2034(~7.9 yrs left)· nominal 20-yr term from priority
G01N 33/6839G01N 2458/30G01N 2560/00G01N 33/6833
53
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Claims
Abstract
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.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A direct method for determining peptide or protein concentration in a sample, the method comprising
(a) combining the sample with a quantitation assay reagent composition comprising a complex to form a mixture where the complex contains bathocuproinedisulfonic acid disodium salt hydrate shown below:
(b) incubating the mixture under conditions sufficient to form a colored complex; and
(c) measuring absorbance of the colored complex at 450 nm to 500 nm as a direct indicator of peptide or protein concentration in the sample.
2 . The method of claim 1 further comprising, after (c), determining the peptide or protein concentration in the sample by comparing the directly measured absorbance with the absorbance of at least one sample containing a known concentration of a standard.
3 . The method of claim 2 where the standard is selected from the group consisting of a peptide, a peptide mixture, or a protein digest.
4 . The method of claim 1 performed prior to mass spectrometry analysis of the sample.
5 . The method of claim 1 where the reagent composition in (a) further comprises tartrate and copper sulfate.
6 . The method of claim 1 where the incubation temperature is selected from the group consisting of room temperature to about 45° C., from about 19° C. to about 22° C., about 37° C., and about 45° C.
7 . The method of claim 1 where the sample volume is selected from the group consisting of about 5 μl to about 20 μl, about 5 μl, from about 10 μl to 20 μl, from about 15 μl to from 20 μl, and not exceeding about 200 μl.
8 . The method of claim 1 where the sample comprises a plurality of peptides.
9 . The method of claim 1 where absorbance is determined by an automated microplate reader.
10 . The method of claim 1 where the standard comprises a peptide, peptide mixture, or peptide digest at a known concentration.
11 . The method of claim 1 where the sample is in a solvent selected from the group consisting of an aqueous solvent, an organic solvent, and combinations thereof.
12 . The method of claim 1 further comprising, after (c), analyzing the peptide(s) by mass spectrometry.
13 . The method of claim 1 where an assay component contains at least one of an organic solvent or a detergent to improve peptide or protein solubility.
14 . The method of claim 1 wherein the reagent composition is bathocuproinedisulfonic acid disodium salt hydrate with about 50% acetonitrile.
15 . A direct method for determining peptide or protein concentration in a sample, the method comprising
(a) combining the sample with a quantitation assay reagent composition comprising a complex with 50% acetonitrile to form a mixture, where the complex contains bathocuproinedisulfonic acid disodium salt hydrate shown below:
(b) incubating the mixture under conditions sufficient to form a colored complex; and
(c) measuring absorbance of the colored complex at 450 nm to 500 nm as a direct indicator of peptide or protein concentration in the sample.
16 . The method of claim 15 further comprising, after (c), determining the peptide or protein concentration in the sample by comparing the directly measured absorbance with the absorbance of at least one sample containing a known concentration of a standard.
17 . The method of claim 15 where the reagent composition in (a) further comprises tartrate and copper sulfate.
18 . The method of claim 15 further comprising, after (c), analyzing the peptide(s) by mass spectrometry.
19 . A peptide quantitation reagent composition comprising at least one excipient and
(a) a complex at a concentration ranging from about 0.04 M to about 0.1 M where the complex contains bathocuproinedisulfonic acid disodium salt hydrate shown below:
(b) tartrate at a concentration ranging from about 5.7 mM to about 22.7 mM, and
(c) copper sulfate at a concentration ranging from about 0.25 mM to about 0.5 mM, resulting in a peptide quantitation reagent composition.
20 . A peptide quantitation reagent composition comprising at least one excipient and (a) a complex at a concentration ranging from about 0.04 M to about 0.1 M in 50% acetonitrile, where the complex contains bathocuproinedisulfonic acid disodium salt hydrate shown below:
(b) tartrate at a concentration ranging from about 5.7 mM to about 22.7 mM, and
(c) copper sulfate at a concentration ranging from about 0.25 mM to about 0.5 mM, resulting in a peptide quantitation reagent composition.
21 . A peptide quantitation reagent kit comprising instructions for mass spectroscopy quantitation of peptides using the kit, and reagents comprising
(a) a complex containing bathocuproinedisulfonic acid disodium salt hydrate shown below:
(b) tartrate, and
(c) copper sulfate,
the composition having pH ranging from about pH 12 to about pH 13.
22 . A peptide quantitation reagent kit comprising instructions for mass spectroscopy quantitation of peptides using the kit, and reagents comprising
(a) a complex in 50% acetonitrile, where the complex contains
bathocuproinedisulfonic acid disodium salt hydrate shown below:
(b) tartrate, and
(c) copper sulfate,
the composition having pH ranging from about pH 12 to about pH 13.
23 . A direct method for determining peptide or protein concentration in a sample, the method comprising
(a) combining the sample with a quantitation assay reagent composition comprising a complex to form a mixture where the complex contains the following general formula
where
each of R 1 and R 2 is independently alkyl including but not limited to a C 1 -C 6 straight or branched alkyl or a C 6 -C 20 aryl, alkylaryl, or arylalkyl such as methyl (—CH 3 ), ethyl (—CH 2 CH 3 ), propyl (—CH 2 CH 2 CH 3 ), butyl (—CH 2 CH 2 CH 2 CH 3 ) or phenyl (—C 6 H 5 );
each of R 3 and R 4 is independently selected from the group consisting of hydrogen (H), sulfonate (—SO 3 − ) salt of sodium (Na + ), potassium (K + ) or lithium (Li + ); phosphonate (—PO 3 − ) salt of sodium (Na + ), potassium (K + ) or lithium (Li + ); and carboxylate (—CO 2 − ) salt of sodium (Na + ), potassium (K + ) or lithium (Li + ); and
each of R 5 and R 6 is
where R 7 is independently selected from the group consisting of hydrogen (H), sulfonate (—SO 3 − ) salt of sodium (Na + ), potassium (K + ) or lithium (Li + ); phosphonate (—PO 3 − ) salt of sodium (Na + ), potassium (K + ) or lithium (Li + ); and carboxylate (—CO 2 − ) salt of sodium (Na + ), potassium (K + ) or lithium (Li + );
with the proviso that at least one of R 3 , R 4 , and R 7 is not H;
(b) incubating the mixture under conditions sufficient to form a colored complex; and
(c) measuring absorbance of the colored complex at 450 nm to 500 nm as a direct indicator of peptide or protein concentration in the sample.Join the waitlist — get patent alerts
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