Method for diagnosing cancer through detection of deglycosylation of glycoprotein

Patent metadata

Field	Value
Publication number	US-9938561-B2
Application number	US-201414906799-A
Country	US
Kind code	B2
Filing date	Jul 17, 2014
Priority date	Jul 25, 2013
Publication date	Apr 10, 2018
Grant date	Apr 10, 2018

Abstract

Provided is a method for diagnosing cancer through a difference with a control group in view of the ratio of a deglycosylated peptide fragment and a non-glycosylated peptide fragment in a protein including an N-linked glycosylation motif. Further provided is a method for diagnosing cancer through the detection of the glycosylation ratio in the protein according to the subject matter enables the diagnosis of cancer with high specificity and sensitivity using at least one existing marker, and can be useful in discovering new markers for diagnosing cancer.

First claim

What is claimed is:

1. A method of diagnosing a liver cancer in a subject or a sample in need thereof comprising the steps of:
providing a biological sample from the subject comprising proteins having a N-linked glycosylation motif;
de-glycosylating the proteins comprised in the sample;
fragmenting the de-glycosylated proteins to obtain de-glycosylated peptides comprising the N-linked motif, and non-glycosylated peptides comprising a non-glycosylated motif and which do not comprise the N-linked motif;
determining in the fragmented proteins an amount of the de-glycosylated peptide at the N-linked motif and the amount of a non-glycosylated peptide which does not contain the N-linked motif and a ratio of the amount of the de-glycosylated peptide to the non-glycosylated peptide; and
diagnosing the subject or the sample as the cancer or susceptible to the cancer if the ratio is changed in the subject or in the sample compared to that of a control,
wherein the proteins, the peptide fragments from the proteins which are de-glycosylated at the N-linked glycosylation motif, the de-glycosylated peptide and the non-glycosylated peptide of the proteins are at least one as listed in Table 1; and the amount is determined using a LC-MS obtained by SIM (Selected Ion Monitoring) or MRM (Multiple reaction monitoring).

2. The method of claim 1 , wherein the de-glycosylation is performed by using a PNGase-F.

3. The method of claim 1 , wherein the fragmentation is performed by treating the sample with at least one of a trypsin, a lysine-C, an arginine-C or an aspartic acid N.

4. The method of claim 1 , wherein the sample is selected from the group consisting of a cell, a whole blood, a serum, a plasma, a saliva, a urine, a follicular fluid, a breast milk and a pancreatin.

5. The method of claim 1 , wherein the protein having an N-linked motif is an AFP (alpha feto protein), the de-glycosylated peptide is VDFTEIQK (SEQ ID NO:9), and the non-glycosylated peptide is GYQELLEK (SEQ ID NO:10) in Table 1.

6. The method of claim 1 , wherein the determination of the amount using the MRM is performed by monitoring a m/z value and optimized collision energy as described in the table below:

Optimized

collision

Protein
Peptide
Precursor ion
Product ion
energy
Ion

name
sequence
(m/z)
(m/z)
(volt)
type

Alpha-
VDFTEIQK
490.258382
880.441074
13.3
y7

fetoprotein
(SEQ ID
490.258382
765.414131
18.3
y6

(AFP)
NO: 9)
490.258382
818.845717
15.3
y5

490.258382
517.298038
11.3
y4

490.258382
388.255445
21.3
y3

490.258382
275.171381
21.3
y2

490.258382
362.171047
9.3
b3

490.258382
468.218725
9.3
b4

490.258382
592.261319
11.3
b5

490.258382
705.345388
9.8
b6

490.258382
833.40396
7.3
b7

GYQELLEK
490.258382
759.424696
11.9
y6

(SEQ ID
490.258382
631.366118
18.3
y5

NO: 10)
490.258382
502.323525
15.3
y4

490.258382
389.239481
17.3
y3

490.258382
276.155397
21.3
y2

490.258382
221.092088
11.3
b2

490.258382
349.150646
9.3
b3

490.258382
591.277303
11.3
b5

490.258382
704.361367
7.3
b6

490.258382
833.40396
9.3
b7

Assignees

• Seoul Nat Univ R&Db Foundation

Inventors

• Kim Youngsoo
• Yoon Jung-Hwan
• Kim Hyunsoo
• Kim Kyunggon
• Jin Jonghwa

Classifications

• G01N33/5758 Primary

  involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites · CPC title

• C12Q1/34 Primary

  involving hydrolase · CPC title

• G01N2333/98

  acting on amide bonds in linear amides (3.5.1) · CPC title

• G01N2440/38

  addition of carbohydrates, e.g. glycosylation, glycation · CPC title

• G01N2560/00

  Chemical aspects of mass spectrometric analysis of biological material · CPC title