Cancer is the second leading cause - دانشکده پزشکی اصفهانmed.mui.ac.ir/basic/imuno/journalclub2D.pdf · 2013-11-04 · • Cancer is the second leading cause of

bull Cancer is the second leading cause

of death worldwide

Despite some successful intervention

strategies in reduction of cancer-related

mortality cancer is still considered a

deadly disease

It is estimated that about one and half

million new cases and half million

deaths from cancer occur in the United

States in 2009 Jemal A Siegel R Ward E Hao Y Xu J Thun MJ

Cancer statistics 2009 CA Cancer J Clin 200959225-49

To overcome cancer mortality the current medical

focus has been centered on the development of novel

biomarkers that can be used for diagnosis prognosis or

therapy of cancer

The immune response targets are revealed to be

promising cancer biomarkers

Cancer patients produce

autoantibodies to proteins

that are mutated misfolded

improperly post-translationally

modified overexpressed

truncated or aberrantly localized

in tumor cells

A large number of tumor antigens that elicit autoantibodies have been identified by different methods

Breast cancer is a major cancer cause and death worldwide

Several breast cancer biomarkers such as BRCA estrogen

receptor and HER2 have been shown to have a diagnostic

prognostic or therapeutic value

However they are reliable just in a proportion of patients and

search for finding novel breast cancer biomarkers is underway

The aim of our study was to identify antigens eliciting a humoral

immune response in HER2+ and HER2 NP breast cancers by

two dimensional electrophoresis (2D) Western blotting and

mass spectrometry

Serum samples Our study groups comprised of 18 breast cancer patients

and 9 normal individuals Breast cancer patients had at least one involved

auxiliary lymph node 9 were HER2 negative and 9 were HER2 positive (3+)

Normal individuals had no history of malignancy

autoimmune disease or severe inflammatory disease

The MCF7 (an ER+ low-expressing HER2 cell line) and SKBR3(an ER- high expressing

HER2 cell line) breast cancer cell lines cultured in culture media contained RPMI 1640

medium 10 fetal bovine serum 100UmL penicillin and 100 mgmL streptomycin

Breast cancer cell line was lysed in a lysis solution (7 M Urea 2 M Thoiurea CHAPS 4 DTT

50 mM IPG buffer 1)

Cell lysates were subjected to 2D-PAGE

2D gels were either stained with blue silver method (Staining solution Coomassie Brilliant

blue MeOH orthophosphoric acid ammonium sulfate) or transferred to PVDF membranes

Immuno-reactive protein spots were punched from the gel

and transferred to a 96 well polypropylene plate

Spots were sent for matrix-assisted laser desorptionionisation time-

of-flighttime-of-flight(MALDI-TOFTOF)mass spectrometry

analysis

2 Dimensional Electrophoresis

Proteomics is the large-scale study of proteins

particularly their structures and functions

The proteome is the entire complement of proteins produced by an organism or system This will vary with time

Proteomics is often considered the next step in the study of biological systems after genomics

Why study Proteomics

First the level of transcription of a gene gives only a rough estimate of its level of

expression into a protein An mRNA produced in abundance may be degraded rapidly

or translated inefficiently resulting in a small amount of protein

Second many proteins experience post-translational modifications that profoundly

affect their activities for example some proteins are not active until they become

phosphorylated Methods such as phosphoproteomics and glycoproteomics are used to

study post-translational modifications

Third many transcripts give rise to more than one protein through alternative splicing

or alternative post-translational modifications

Finally At present there is no other technique that is capable of simultaneously

resolving thousands of proteins in one separation procedure

The core technology of proteomics is 2-DE

To identify as many components of the proteome as possible

Protein Mining catalog all the proteins present in a tissue cell organelle etc

Protein Function

Mapping Protein Post-Translational Modification - Characterization of posttranslational modifications phosphorylation glycosylation oxidation etc

Protein Localization

Protein Expression Studies

Comparative proteomics

- proteomics monitors the expression of a large number of proteins within a cell or tissue and observers quantitatively how the

patte of expression changes under different circumstances (comparative proteomics) such as

bull Healthy Diseased

bull Cancerous Benign

bull Drug resistant Drug susceptible

bull Tissue specific

An interesting use of proteomics is using specific protein biomarkers to diagnose disease

Sample Preparation 2-D Electrophoresis Spot Detection amp Image Analysis

Enzymatic Digestion Peptide-Mass Fingerprinting

Peptide Sequencing via MS Database Search

Protein Identification

(I) (III)

(IV) (V) (VI)

(II)

Sample preparation buffer composition IPG strip rehydration 1st dimension IEF run IPG strip wash Equilibration buffer Equilibration buffer 2nd dimension SDS-PAGE Gel staining Immunoblot Imaging

Sample preparation

buffer composition

IPG strip rehydration

1st dimension IEF run

IPG strip wash

Equilibration buffer I

Equilibration buffer II

2nd dimension SDS-PAGE

Gel staining

Imaging

2-D Electrophoresis

Consistent protocol Limit sample degradation

bull fresh cellstissue bull protease inhibitors bull keep sample cold bull long term storage at ndash80degC

Limit sample contamination

bull gloves (keratin)

SolubilisationDenaturation buffer

bull separate proteins into individual components

bull reliable running in the IEF

Gentle lysis method 1 Osmotic lysis (cultured cells)

Suspend cells in hypoosmotic solution

2 Repeated freezing and thawing (bacteria)

Freeze using liquid nitrogen

3 Detergent lysis (yeast and fungi)

Lysis buffer (containing urea and detergent)

SDS (have to be removed before IEF)

4 Enzymatic lysis (plant bacteria fungi)

Lysomzyme (bacteria)

Cellulose and pectinase (plant)

Lyticase (yeast)

Vigorous lysis method

1 Sonication probe (cell suspension)

Avoid overheat cool on ice between burst

2 French pressure (microorganism with cell wall)

Cells are lysed by shear force

3 Mortar and pestle (solid tissue microorganism)

Grind solid tissue to fine powder with liquid nitrogen

4 Sample grinding kit (for small amount of sample)

For precious sample

5 Glass bead (cell suspension microorganism)

Using abrasive vortexed bead to break cell walls

Chaotrope

Urea (up to 9 M)

Thiourea (up to 2 M)

bull Disrupt of hydrogen and hydrophobic bonds

Note Urea (if tdeggt37degC) cyanate (HN=C=O)

carbamylation (Lys Arg)

(R-NH2 R-NH-CO-NH2)

carbamylation trains

Reductants

b-mercaptoethanol

DTT(dithiothreitol)

bull Break disulfide bridge

(within or between protein)

Detergents ndash surfactants

CHAPS (up to 4)

bull Disrupt membranes

bull Break hydrophobic interactions

bull Solubilise lipids

bull Release membrane bound proteins

Note No net charge for IEF (no SDS)

Soluble in urea

Isoelectric Focusing

Measure the protein concenteration in you samples

1 Biuret

2 Lowry methods

3 Bradford methods

4 UV methods

5 Special methods

6 Other commercial methods

BCA assay (bicinchoninic acid assay Pierce)

DC protein assay (detergent compatible Bio-rad)

DCRC protein assay (detergentreducing agent compatible

Bio-rad)

Before runninng IEF you shouldhellip

Introduced by Gorg A

Ref Gorg A (1994) Westermeier (2001)

Dried gel strips can be stored at -20 to -

80 from months to years

Amersham Biosciences Bio-Rad

IPG strips (3 mm x 18 cm x 05 mm)

Narrow range

Medium range

Broad range

4 7

35 45 55 67

40 50 60

3 10

6 11

Load 400 ml sample per groove (no bubbles)

Place IPG strip gel facing down in groove

Add ~25 ml non conductive oil per groove

Peel off protective film from strip

Rehydrate overnight (~22 hrs) at room temperature

Program power supply

bull Number of gels (1-10)

bull Max voltage 5000 V

bull Vhold 125 V

bull Duration 24 hrs 00 min

bull Max current 80 mAstrip

bull Volt hours 80000 Vh

pI

+

+ - - - - - -

+ + + + + + + -

pH 4 pH 7

Cathode Anode

Place wet wicks (DH2O) under each end of strip

Set chiller temperature for 20degC (setting ~25)

Remove IPG strip from tray

Let oil drip off the strip

Place IPG strip gel facing up in equilibration tray

+ -

Add 10 ml equilibration buffer 1 per tray