*Finding Needles in Haystacks: The Use of Quantitative Proteomics for the Early Detection… DOI: http://dx.doi.org/10.5772/intechopen.80942*

*Advances in the Molecular Understanding of Colorectal Cancer*

[39]

**140**

**Protein**

HSP70

Histological: 81 CRC

HSP 110

samples

Cell culture lines

IHC

BCL

HSP90

Α

Histological samples:

Immunoprecipitation with

MALDI-TOF

LC-MS/MS

IHM-2 antibody

Immunoblotting or MS

analysis

RT-PCR

S100 A9

Histological samples:

2DGE, spot excision, trypsin

ESI-TRAP

1200 protein spots. 45 proteins

15 proteins validated with

[81]

IHC. The most significantly

overexpressed were HSP60,

S100A9 and TCTP. 4-3-3b and

aldehyde dehydrogenase 1 were

identified as having prognostic

benefit.

Immunoblot and IHC for GSN

[90]

overexpressed.

LC-MS/MS

Dukes stage B (n = 28)

digestion.

TMA with normal colon

mucosa (n = 50), primary

(n = 515) and metastatic

CRC (n = 224)

GSN

Histological samples:

Protein digestion, iTRAQ

Q-Star Pulsar

802 proteins identified, 82 with

differential expression.

MMP-1 immunoreactivity only

N/A

[96]

LC-MS/MS

labeling, fractionation

IHC, in-situ hybridization,

N/A

5 CRC

MMP-1

Histological samples:

20 adenoma, 142

RT-PCR

CRC.

with paired normal

tissue

TCTP

56 CRC

cell culture lines

**Sample type**

**Workflow** SDS-PAGE

Immunoblot analysis

**MS platform**

N/A

**Proteins identified**

Cell culture: HSP110, HSP70 elevated

in highly metastatic cell lines. HSP 90,

HSP60, HSP27 variable expression.

BCL preferentially elevated in weakly

metastatic cell lines.

Histological IHC: HSP70, HSP110,

Bcl-2 corresponded to cell line results.

Bcl-2 positive staining correlated to less

invasive cancer and earlier clinical stage.

Tumor HSP90α overexpression was

N/A

correlated with the metastasis and poor

prognosis of CRC patients.

**Validation set**

N/A

**References**

[88]


**143**

**Protein**

PRTN3 ATM

C3

Fecal samples:

Protein extraction, GE,

LTQ-FT

834 proteins identified, 29 statistically

N/A

[118]

increased in CRC including ATM,

S100A8, S100A9, and C9

Hybrid MS

LC-MS/MS

in-gel tryptic digestion. FIT

analysis using antibody-

based assays.

(n = 315). Three

series of patients with

CRC, nonadvanced

adenomas, advanced

adenomas and healthy

normal.

C9

Plasma: 69 CRC and

Assay for 187 biomarkers

6490 triple

15 transition, 13 protein cross classifier.

MS-MRM assay. Identification

[23]

of over 50% of the proteins

from the discovery set. Proteins:

Alpha1-acid glycoprotein,

Alpah1-Antitrypsin, Alpha-Amylase 2B, Clusterin, Complement component C9,

Mitochondrial Delta(3,5)-

Delta(2,4)-dienoyl-CoA

isomerase, Ferritin light chain,

Gelsolin, Metalloproteinase

inhibitor 1, Osteopontin,

Selenium-binding protein 1,

Seprase, Spondin-2.

quadrupole

mass

spectrometer

LC-MS/MS

identified from a literature

search.

Sample preparation and

trypsin digestion.

Targeted MS analysis using

MRM.

69 healthy control.

Stool samples: 12 CRC, 10 healthy controls

**Sample type**

**Workflow** 1D-SDS GE. Tryptic peptides excised for MS

**MS platform**

LTQ-FT MS LC-MS/MS

**Proteins identified**

830 proteins, 134 increased in CRC (78 significantly more enriched than FIT negative samples).

Validation set identification of 63 of the proteins from discovery set, 33 selected for further validation using SRM.

Differentially expressed proteins: Complement component C4B, Glucose-

6-phosphate isomerase, Proteinase 3 (PRTN3), Alpha-2-micorglobulin (A2M), A100A8, S100A9, Azurocidin, Ceruloplasmin

**Validation set**

**References**

[119]

*Finding Needles in Haystacks: The Use of Quantitative Proteomics for the Early Detection…*

*DOI: http://dx.doi.org/10.5772/intechopen.80942*


### *Finding Needles in Haystacks: The Use of Quantitative Proteomics for the Early Detection… DOI: http://dx.doi.org/10.5772/intechopen.80942*

*Advances in the Molecular Understanding of Colorectal Cancer*

**142**

**Protein** TIMP-2

Histological and

Serum samples: 72

CRC patients, 68

healthy controls

TIMP-3

Histological samples:

TMA

N/A

> IHC staining of MMP-1,2,7,13 using a commercial

kit and TIMP-1,2,3,4 hand

stained.

A2

Serum samples: 100

ELISA

N/A

A2 levels significantly lower in patients

N/A

[113]

See also [90]

with colon cancer when compared to

control subjects

patients CRC, 70

healthy controls

A3

Serum and cell

725 Candidate proteins

Q Exactive

356 EV proteins from Shotgun analysis.

SRM analysis: 22 proteins

[26]

A11, A3, A4, Tenascin-N,

Transferring receptor protein

1, GLUT-1, C9, CD88 antigen,

78 kDa glucose-regulation

protein, Alpha-1-acid

glycoprotein, MMP-9,

Angiopoietin-1, CD67 antigen,

Mucin-5B, Adapter protein

GRB2, A5, Olfactomedin-4,

Neutral amino acid transport

B(0), Tripeptidyl-peptidase

1, Heat shock-related 70 kDa

protein 2, Proteasome subunit

alpha type-5, Neutrophil

gelatinase-associated lipocalin.

46 proteins selected for SRM analysis for

target peptide selection.

LC-MS/MS

identified by Literature

search, Shotgun proteomics

for Extracellular Vesicle

(EV) Proteins

culture supernatants.

Training set: 51 CRC,

26 healthy controls.

A4

A11

351 CRC patients.

**Sample type**

**Workflow** Serum: ELISA for TIMP-2

N/A

and MMP-2

Histological: IHC

**MS platform**

**Proteins identified**

Serum levels of MMP-2 and TIMP-2

were significantly lower in CRC.

IHC demonstrated overexpression for

both, immunoreactivity correlated with

tumor grade.

TIMP-3 the only marker of independent

N/A

[111]

prognostic value

**Validation set**

N/A

**References**

[110]


*Synopsis of biomarker identification including sample type, pre-analytic workflow and MS technique (where applicable).*
