# US 2021/0239708 A1 · Method to Map Protein Landscapes

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- Number: US 2021/0239708 A1
- Document ID: US20210239708A1
- Status: Published application
- Kind: A1
- Application number: 17/231,977
- Filed: 2021-04-15
- Published: 2021-08-05
- Earliest claimed: 2017-05-25
- Assignees: Wisconsin Alumni Research Foundation
- Applicants: Wisconsin Alumni Research Foundation
- Inventors: Joshua COON; Harald MARX
- Web page: https://patentgrep.com/patent/US20210239708A1

## CPC

- G01N 33/6848: {Methods of protein analysis involving mass spectrometry}
- G01N 33/6824: {involving N-terminal degradation, e.g. Edman degradation}
- G01N 33/6842: {Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins}
- G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids
- G16B 30/20: Sequence assembly
- G16B 40/00: ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
- G16B 40/10: Signal processing, e.g. from mass spectrometry [MS] or from PCR
- G16B 40/30: Unsupervised data analysis
- H01J 49/0036: {Step by step routines describing the handling of the data generated during a measurement}
- H01J 49/004: {Combinations of spectrometers, tandem spectrometers, e.g. MS/MS, MSn}
- G01N 2570/00: Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- H01J: ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS (spark-gaps H01T; electric arc lamps H05B31/00; particle accelerators H05H)

## Abstract

In shotgun proteomics, generally only a fraction of peptides from a parent protein are actually detected. Because a large portion of the protein sequence is not detected, it is often impossible to determine whether the expressed protein is present in a modified, spliced, or truncated form. Provided herein are methods and systems for analyzing polypeptides which allow for the increase of the mean sequence coverage of a protein concomitant with bioinformatics analysis in order to distinguish putative proteoforms with improved amino acid resolution. Aspects of the invention include (1) a deep sequencing strategy to provide more protein sequence coverage than is typically achieved, and (2) a computational approach to view protein expression across its full length and identify regions of the protein that are potentially subject to such regulation. This technology has global utility in proteomics and will be of particular use for the analysis of biosimilar protein drug therapeutics.

## Claims

### Claim 1

A method for analyzing a polypeptide having an amino acid sequence comprising the steps of:
a) obtaining a first and second sample of the polypeptide from a cell;
b) digesting the first sample of the polypeptide with a first protease or chemical agent;
c) digesting the second sample of the polypeptide with a second protease or chemical agent;
d) generating tandem mass spectrometry data on each digested polypeptide sample comprising the steps of: generating a distribution of precursor ions during MS1 stage ionization, fragmenting precursor ions having a mass-to-charge ratio (m/z) within a selected target m/z range during MS2 stage fragmentation, thereby generating a plurality of product ions wherein the product ions correspond to portions of the amino acid sequence of the polypeptide, measuring m/z of the product ions, obtaining peptide extracted-ion chromatogram (XIC) intensities of the product ions from each digested polypeptide sample, and normalizing the measured XIC intensities using quantile normalization, thereby generating mass spectrometry data for each digested polypeptide sample;
e) combining mass spectrometry data from each digested polypeptide sample to generate comprehensive mass spectrometry data on the polypeptide, wherein the comprehensive mass spectrometry data provides sequence coverage for at least 20% of the full length amino acid sequence of the polypeptide; and
f) identifying a portion of the product ions as corresponding to one or more known amino acid sequence fragments of said polypeptide, and calculating abundances of amino acids for one or more selected portions of the polypeptide from the comprehensive mass spectrometry data, wherein the amino acid abundances are calculated as a mean of the XIC peptide intensities matching to the amino acid positions in the polypeptide sequence.

### Claim 2 (depends on 1)

The method of claim 1 further comprising generating at least a partial consensus amino acid sequence for the polypeptide from the comprehensive mass spectrometry data.

### Claim 3 (depends on 1)

The method of claim 1 wherein an isotopic or chemical label is not attached to the polypeptide in order to calculate the abundances of amino acids.

### Claim 4 (depends on 3)

The method of claim 3 wherein the one or more selected portions comprises the N-terminus of the polypeptide.

### Claim 5 (depends on 1)

The method of claim 1 further comprising normalizing the measured intensities of the product ions from each digested polypeptide sample during generation of the comprehensive mass spectrometry data.

### Claim 6 (depends on 5)

The method of claim 5 further comprising performing k-means clustering analysis on normalized intensity data during generation of the comprehensive mass spectrometry data.

### Claim 7 (depends on 1)

The method of claim 1 further comprising digesting one or more additional samples of the polypeptide with one or more additional proteases or chemical agents, wherein the protease or chemical agent used for each sample is a different protease or chemical agent used to digest any other sample.

### Claim 8 (depends on 7)

The method of claim 7 further comprising digesting a third sample of the polypeptide with a third protease, digesting a fourth sample of the polypeptide with a fourth protease, digesting a fifth sample of the polypeptide with a fifth protease, and digesting a sixth sample of the polypeptide with a sixth protease.

### Claim 9 (depends on 7)

The method of claim 7 wherein the proteases or chemical agents are proteases selected from the group consisting of trypsin, Lys-N, Lys-C, Glu-C, chymotrypsin, Asp-N, and combinations thereof, wherein each selected protease is different for each sample.

### Claim 10 (depends on 1)

The method of claim 1 wherein the comprehensive mass spectrometry data provides sequence coverage for at least 50% of the full length amino acid sequence of the polypeptide.

### Claim 11 (depends on 1)

The method of claim 1 wherein the comprehensive mass spectrometry data provides sequence coverage for at least 80% of the full length amino acid sequence of the polypeptide.

### Claim 12 (depends on 1)

The method of claim 1 wherein the polypeptide is an antibody, antibody-drug conjugate, or a therapeutic protein.

### Claim 13

A method for analyzing two or more polypeptides comprising the steps of:
a) independently digesting a first sample of a first polypeptide and a first sample of a second polypeptide with a first protease or chemical agent;
b) independently digesting a second sample of the first polypeptide and a second sample of the second polypeptide with a second protease or chemical agent;
c) generating tandem mass spectrometry data on each digested polypeptide sample comprising the steps of: generating a distribution of precursor ions during MS1 stage ionization, fragmenting precursor ions having a mass-to-charge ratio (m/z) within a selected target m/z range during MS2 fragmentation, thereby generating a plurality of product ions wherein the product ions correspond to amino acid sequences of the polypeptides, and measuring m/z and intensity of the product ions, thereby generating mass spectrometry data for each digested polypeptide sample; and
d) for each polypeptide, combining mass spectrometry data from each digested polypeptide sample of that polypeptide to generate comprehensive mass spectrometry data, wherein the comprehensive mass spectrometry data provides sequence coverage for at least 20% of the full length amino acid sequence for that polypeptide; and
e) generating at least a partial consensus amino acid sequence for each polypeptide from the comprehensive mass spectrometry data or calculating abundances of amino acids for selected portions of each polypeptide from the comprehensive mass spectrometry data.

### Claim 14 (depends on 13)

The method of claim 13 where the first polypeptide is a control polypeptide and the second polypeptide is the same polypeptide which has undergone a suspected modification, splice, truncation, polymorphism or mutation.

### Claim 15 (depends on 13)

The method of claim 13 where the first polypeptide is a control polypeptide and the second polypeptide is produced by a cell which has been administered a treatment.

### Claim 16 (depends on 13)

The method of claim 13 where the first polypeptide is a control therapeutic polypeptide, antibody, or antibody-drug conjugate, and the second polypeptide is a production therapeutic polypeptide, antibody, or antibody-drug conjugate made during a biochemical process or manufacturing process.

### Claim 17 (depends on 13)

The method of claim 13 further comprising independently digesting one or more additional samples of the first polypeptide and one or more additional samples of the second polypeptide proteases with one or more additional proteases or chemical agents, wherein the one or more additional proteases or chemical agents used for each additional sample is a different protease or chemical agent.

### Claim 18 (depends on 13)

The method of claim 13 wherein the comprehensive mass spectrometry data provides sequence coverage for at least 50% of the amino acid sequence of the polypeptide.

### Claim 19 (depends on 13)

The method of claim 13 further comprising comparing the consensus amino acid sequences of each polypeptide or the abundances of amino acids of each polypeptide, and identifying differences in amino acid sequence or amino acid abundance between the polypeptides.

### Claim 20

A system for analyzing a polypeptide having an amino acid sequence comprising:
a) an ion source for generating ions from a plurality of digested samples of the polypeptide;
b) ion fragmentation optics in communication with the ion source for generating product ions;
c) an ion detector in communication with the ion fragmentation optics for detecting ions according to their mass-to-charge ratios;
d) a mass analyzer in communication with the ion detector, wherein the mass analyzer comprises a software program enabling the mass analyzer to:
i) measure mass-to-charge ratios and intensity of the detected ions, thereby generating mass spectrometry data for each digested polypeptide sample;
ii) normalize the measured intensities of the product ions from each digested polypeptide sample; and
iii) combine mass spectrometry data from each digested polypeptide sample to generate comprehensive mass spectrometry data on the polypeptide, wherein the comprehensive mass spectrometry data provides sequence coverage for at least 20% of the full length amino acid sequence of the polypeptide.