Methods for quantifying activity of protein modifying enzymes
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Publication Number
US-9879304-B2
Publication Date
2018-01-30
Expiration Date
Abstract
The present invention provides a method of quantifying the activity of a protein modifying enzyme in a sample, comprising: (i) grouping modified peptides from a first sample and modified peptides from a second sample into a single group according to one of the following parameters: (a) modified peptides having a modification site that is modified by the same protein modifying enzyme; or (b) modified peptides having a modification site that is part of the same modification motif; (ii) calculating enrichment of the modified peptides from the first sample compared to the modified peptides from the second sample in the group; and (iii) calculating the statistical significance of said enrichment; wherein a statistically significant enrichment is indicative of a protein modifying enzyme being activated in the first sample compared to the second sample. In some embodiments, the method further comprises identifying modified peptides in a first sample and a second sample using mass spectrometry (MS) prior to step (i).
Core Innovation
The invention provides a mass spectrometry method for identifying an activated protein modifying enzyme in a sample. The method compares a first sample and a second sample by using peptides and reference modified peptides to generate mixtures for independent MS measurements, then compares mass spectrometry data with data in a database of modified peptides to identify modified peptides present in both samples.
After identifying modified peptides, the method groups modified peptides from the first and second samples into a single group based on a shared biological parameter. The grouping is according to modified peptides having a modification site that is modified by the same protein modifying enzyme or modified peptides having a modification site that is part of the same modification motif.
The method calculates enrichment of the modified peptides from the first sample compared to the second sample within each group and then calculates the statistical significance of the enrichment. Statistically significant enrichment is indicative of a protein modifying enzyme being activated in the first sample compared to the second sample, and differential enrichment of grouped modified peptides is used to identify activated protein modifying enzymes from MS-derived peptide modification information.
In embodiments described in the document context, modified peptides are quantified in an MS workflow for protein kinases by grouping modified peptides with shared modification sites or shared phosphorylation motifs. The workflow supports using reference modified peptides or internal standards, including stable heavy isotope labeling approaches, and includes options for modified-peptide enrichment and identification.
Claims Coverage
Independent claim clm-00001 covers a two-sample MS workflow that identifies modified peptides using reference modified peptides, groups them by shared modification-enzyme or modification motif, and computes enrichment and statistical significance to indicate activation. One inventive feature is captured in the supplied independent claim content.
Activated protein modifying enzyme identification from two-sample MS enrichment
A method of identifying an activated protein modifying enzyme in a sample using mass spectrometry, comprising adding reference modified peptides to peptides obtained from a first sample and a second sample to produce a first mixture of peptides and reference modified peptides and a second mixture of peptides and reference modified peptides, carrying out mass spectrometry on the first mixture and on the second mixture, comparing the data relating to peptides from the first and second samples with data in a database of modified peptides to identify modified peptides in the first and second samples, grouping the modified peptides into a single group according to a shared modification-enzyme parameter or a shared modification-motif parameter, calculating enrichment of modified peptides from the first sample compared to the second sample in the group, and calculating the statistical significance of said enrichment, wherein statistically significant enrichment is indicative of a protein modifying enzyme being activated in the first sample compared to the second sample.
The claim coverage centers on MS-based identification of modified peptides using reference modified peptides, grouping by shared modification-enzyme or modification motif, and using enrichment and statistical significance to indicate activation of the relevant protein modifying enzyme in the first sample relative to the second sample.
Stated Advantages
A statistically significant enrichment is indicative of a protein modifying enzyme being activated in the first sample compared to the second sample.
Documented Applications
Application to protein kinases using a Kinase Substrate Enrichment Analysis (KSEA) workflow to quantify activity by grouping modified peptides and using enrichment with statistical significance.
Validation context using AML cell line comparisons, including enrichment outcomes and Western blot confirmation, and motif-based analyses and heterogeneity across primary AML cases [procedural detail omitted for safety].
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