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Quantification of Membrane and MembraneBound Proteins in Normal and Malignant
Breast Cancer Cells Isolated from the Same
Patient with Primary Breast Carcinoma
Liang, Zhao et. Al, 2006
Presented by:
Richard Pelikan
October 27, 2006
BioInf 2032
Motivation

How do you affect cells?
Motivation

How do you affect cells?
 Target
the cell membrane
Motivation

How do you affect cells?
 Target
the cell membrane
 What do you target?
Here goes
nothin’!
Objective

SILAC = Stable Isotope Labeling with
Amino acids in cell Culture

Determine if SILAC can effectively
determine changes in the protein
expression levels on cell membranes

Determine if these measurements are
related to disease
Outline
Introduction to Proteomics
 Experiments
 Results
 Discussion

Outline
Introduction to Proteomics
 Experiments
 Results
 Discussion

Introduction to Proteomics

Proteomics: The study of proteins
 How
do proteins interact?
 What effect can we have on proteins?
 How are proteins related to states of health?

More difficult than genomics
 Differences
between cells, organisms, etc.
Introduction to Proteomics

Which proteins can we monitor to measure
health?
 Biomarkers:
biological entities which shows
information

How do you find biomarkers?
 Develop
multiple protein assays
 Use high-throughput protein measurement
systems
Mass Spectrometry (MS)

Zap proteins with lasers, generating a
unique signature for the protein mixture
Mass Spectrometry (MS)

Zap proteins with lasers, generating a
unique signature for the protein mixture
1) Collect biofluids
Mass Spectrometry (MS)

Zap proteins with lasers, generating a
unique signature for the protein mixture
1) Collect biofluids
2) Zap with lasers
Mass Spectrometry (MS)
Zap proteins with lasers, generating a
unique signature for the protein mixture
1) Collect biofluids
2) Zap with lasers
3) Analyze data
100
90
80
70
60
Intensity

50
40
30
20
10
0
0
500
1000
1500
2000
2500
3000
mass/charge
3500
4000
4500
5000
Mass Spectrometry (MS)
Heavy molecules move slower
Amount

Mass / Charge
Mass Spectrometry (MS)
Heavy molecules move slower
7 Daltons
Amount

16 Daltons
(I’m tall for my
weight!)
Mass / Charge
Mass Spectrometry (MS)
Heavy molecules move slower
Amount

Mass / Charge
Mass Spectrometry (MS)
Heavy molecules move slower
Amount

Mass / Charge
Mass Spectrometry (MS)
Heavy molecules move slower
Amount

Mass / Charge
Mass Spectrometry (MS)
Heavy molecules move slower
Amount

Mass / Charge
Mass Spectrometry (MS)
Heavy molecules move slower
Amount

7
Mass / Charge
Mass Spectrometry (MS)
Heavy molecules move slower
Amount

7
Mass / Charge
Mass Spectrometry (MS)
Heavy molecules move slower
Amount

7
16
Mass / Charge
Mass Spectrometry (MS)

There is a tradeoff between the simplicity
of data production and quality of data

(In general) you don’t know which peak
corresponds to which protein

There are ways to control which proteins
you expect to see
Example of protein control
Introduction
Quantities of proteins can be measured
using MS technology
 It is necessary to have control over what
you see in the data to be able to identify
proteins
 Verification is still important!

Outline
Introduction to Proteomics
 Experiments
 Results
 Discussion

Experiments

Biofluid: Cells taken from a 74-year old
patient with breast cancer
 Some
cells are healthy, some are from the
tumor itself

Technology: MS instrumentation is
relatively standard
 Details
results
are only for performing replication of
Experiments – Protein Control
Normal cells
Tumor cells
+ Light tag solution
+ Heavy tag solution
Cells produce
tagged proteins
Mass spectra of the mixture
shows uneven proportions of
light and heavy proteins
m/z
Experiments – measuring ratios

A protein is broken down into peptides by
trypsin digestion

Each peptide generates a light-heavy pair

The ratio of each pair is averaged to
achieve the assumed ratio of the parent
protein
Figure 2 – Peptide Ratios
Experiments

Force cells to produce proteins with
differently weighted tags

Measure the differences in amounts of
proteins with light or heavy tags

Identify and study which proteins are
differentially expressed
Outline
Introduction to Proteomics
 Experiments
 Results
 Discussion

Results

997 proteins identified through the SILAC
technique
 830
of which are actually membrane proteins
 Only 35 were found to be “differentially
expressed”
 Many of these are reported in literature as
cancer biomarkers

Immunohistochemistry seems to reflect
the results seen in the MS data
Results - Immunohistochemistry
Normal

Cancer
Normal
Cancer
Staining seems to reflect regulation
observed in MS data (for this individual)
Outline
Introduction to Proteomics
 Experiments
 Results
 Discussion

Discussion

SILAC seems to be effective for
characterizing changes in the membrane
proteome

Didn’t detect one of the most prominent
membrane tumor markers
Discussion

Issues exist with this approach
 How
can it be high-throughput?
 To what degree is the differential expression
ratio significant to disease?
 Why the reliance on biopsy material?

Suggestions
 Do
a classification study
 Explain their independent tests better
Thank you!!

Forget proteomics, look forward to: