# US 8,771,969 B2 · Peptide antibody depletion and its application to mass spectrometry sample preparation

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- Number: US 8,771,969 B2
- Document ID: US8771969B2
- Status: Granted
- Kind: B2
- Application number: 13/536,047
- Filed: 2012-06-28
- Published: 2012-11-01
- Granted: 2014-07-08
- Earliest claimed: 2006-07-03
- Assignees: The Johns Hopkins University
- Applicants: Jennifer E. Van Eyk; David Raymond Graham; Rebekah Lynn Gunrdy
- Inventors: Jennifer E. Van Eyk; David Raymond Graham; Rebekah Lynn Gunrdy
- Web page: https://patentgrep.com/patent/US8771969B2

## CPC

- G01N 33/6842: {Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins}
- G01N 33/6848: {Methods of protein analysis involving mass spectrometry}
- Y10T 436/25125: Digestion or removing interfering materials
- 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)
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

The present invention relates, e.g., to a method for pre-processing a sample for mass spectral analysis, comprising cleaving proteins in the sample to peptides and immunodepleting highly abundant and/or well-ionizing and/or proteotypic peptides from the sample. Also described are methods for identifying well-ionizing peptides for use in this and other methods; analytic (diagnostic) methods using antibodies against highly ionizable peptides from a protein target of interest; and compositions, kits and devices comprising antibodies of the invention.

## Claims

### Claim 1

A method for pre-processing a sample for electrospray ionization mass spectroscopy (ESI) analysis, comprising cleaving proteins in the sample to peptides and immunodepleting highly abundant and/or well-ionizing and/or proteotypic peptides from the sample.

### Claim 2 (depends on 1)

The method of claim 1, wherein the cleaving comprises digesting the proteins with a protease.

### Claim 3 (depends on 2)

The method of claim 2, wherein the protease is trypsin, chymotrypsin, and/or Lys-C.

### Claim 4 (depends on 1)

The method of claim 1, wherein peptides from between about 1-20 proteins are removed from the sample.

### Claim 5 (depends on 1)

The method of claim 1, wherein peptides from between about 8-12 proteins are removed from the sample.

### Claim 6 (depends on 1)

The method of claim 1, wherein the sample is from plasma/serum, and the high abundant and/or well-ionizing and/or proteotypic peptides are from one or more of the proteins listed in Table 5.

### Claim 7 (depends on 1)

The method of claim 1, wherein the highly abundant and/or well-ionizing and/or proteotypic peptides comprise one or more of the peptides listed in Table 3 and/or Table 6 and/or Table 7.

### Claim 8 (depends on 1)

The method of claim 1, wherein the sample contains keratins and/or trypsin, and the high abundant and/or well-ionizing and/or proteotypic peptides are selected from one or more of the peptides listed in Table 2 and/or Table 4.

### Claim 9 (depends on 4)

The method of claim 4, wherein about 3-5 peptides from each of the proteins are removed.

### Claim 10 (depends on 1)

The method of claim 1, wherein the immunodepletion is carried out by contacting the sample comprising cleaved proteins with one or more antibodies that are specific for highly abundant and/or well-ionizing and/or proteotypic peptides in the sample, under conditions that are effective for the antibodies to bind specifically to their cognate peptides, and
separating the resulting antibody/peptide complexes from the sample.

### Claim 11 (depends on 10)

The method of claim 10, further wherein the well-ionizing peptides are identified by
a) cleaving a protein known or suspected to be a contaminant with a protease;
b) subjecting the resulting peptides to mass spectrometry; and
c) ranking the peptides in order of degree of ionization.

### Claim 12 (depends on 10)

The method of claim 10, wherein one or more of the antibodies are monoclonal antibodies.

### Claim 13 (depends on 10)

The method of claim 10, wherein all of the antibodies are monoclonal antibodies.

### Claim 14 (depends on 10)

The method of claim 10, wherein one or more of the antibodies are polyclonal antibodies.

### Claim 15 (depends on 10)

The method of claim 10, wherein all of the antibodies are polyclonal antibodies.