High sensitivity quantitation of peptides by mass spectrometry
AssigneeAnderson Forschung Group, Inc.
InventorNORMAN L. ANDERSON
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Abstract
The instant invention provides an economical flow-through method for determining amount of target proteins in a sample. An antibody preparation (whether polyclonal or monoclonal, or any equivalent specific binding agent) is used to capture and thus enrich a specific monitor peptide (a specific peptide fragment of a protein to be quantitated in a proteolytic digest of a complex protein sample) and an internal standard peptide (the same chemical structure but including stable isotope labels). Upon elution into a suitable mass spectrometer, the natural (sample derived) and internal standard (isotope labeled) peptides are quantitated, and their measured abundance ratio used to calculate the abundance of the monitor peptide, and its parent protein, in the initial sample
Claims14
-26. (canceled)
A method for capturing similar amounts of 2 or more different monitor peptides from a sample containing widely varying concentrations of the different monitor peptides comprising the steps of:
- a. fragmenting the proteins of the samples,
- b. producing labeled monitor peptides,
- c. adding the product of step a to the product of step b containing a known amount of labeled monitor peptide,
- d. preparing at least one support having at least one binding agent which binds a small fraction of the more abundant peptide in the sample and at least one binding agent which binds higher fraction of the less abundant peptide in the sample,
- e. loading the product of step c onto the support prepared in step d,
- f. washing the support of step e, then
- g. eluting the peptides from the support to obtain the monitor peptides, and
- h. subjecting the eluate of step g to mass spectrometry.
A support system for collecting more than one monitor peptide from a peptide sample wherein at least one monitor peptide is present in the sample in much greater concentration than at least one other monitor peptide, said support having attached thereto a first binding agent being applied to said support to bind only a small fraction of the monitor peptide of higher concentration and a second binding agent being applied to said support to bind a higher fraction of the monitor peptide of low concentration in the sample.
A kit comprising at least one labeled monitor peptide wherein the kit further comprises at least one binding agent for capture of at least one monitor peptide.
A method of measuring the progress of proteolytic digestion of a sample comprising the steps of:
- a. selecting at least two monitor peptides derived from the sequence of one known protein in the sample, wherein at least one of said monitor peptides is known to be released earlier in the time course of a reference digest relative to at least one other monitor peptide,
- b. measuring the amount of said monitor peptides at one or more time points during digestion, and
- c. comparing the relative amounts of said monitor peptides in a mathematical process to compute an index reflecting the degree to which digestion has progressed relative to the time course of said reference digest.
A method of quantifying the amount of at least one target protein in a sample comprising the steps of:
- a. digesting the sample to provide peptides,
- b. preparing at least one labeled synthetic monitor peptide comprising a sequence of said target protein(s) to provide an internal standard,
- c. adding an aliquot of the product of step b containing a known amount of labeled monitor peptide to the product of step,
- d. loading the product of step c onto at least one support which has attached thereto at least one binding agent which binds to at least one monitor peptide,
- e. washing the support(s) of step d to remove unbound peptides,
- f. eluting the monitor peptide(s) from the binding agent(s) bound to the support(s) of step d,
- g. loading the monitor peptides eluted in step f onto a second support which has attached thereto at least one binding agent different from the binding agent(s) of step d and which binds to at least one monitor peptide,
- h. washing the support(s) of step g to remove unbound peptides,
- i. eluting the monitor peptides from the binding agent(s) bound to the support(s) in step g, and
- j. subjecting the eluate obtained in step i to mass spectrometry to determine the amount of monitor peptide(s) in the eluate in comparison to the labeled synthetic standard.
A method of characterizing the structure of a target protein in a sample, said method comprising the steps of:
- a. subjecting an aliquot of said sample to a denaturing process,
- b. digesting the product of step a,
- c. digesting a second aliquot of said sample without denaturing,
- d. preparing at least one labeled synthetic monitor peptide comprising a sequence of the target protein to provide an internal standard,
- e. to the product of step b add a known amount of the product of step d,
- f. to the product of step c add the same known amount of the product of step d,
- g. load each of the products of steps e and f onto separate supports which have attached thereto at least one binding agent which binds to the monitor peptide,
- h. wash each support prepared in step g,
- i. elute the peptides form each support,
- j. subject each eluate obtained in step i to mass spectrometry, and
- k. compare the amount of monitor peptide in each of the eluates.
-41. (canceled)
Dependencies are inferred from the claim text.