Sergio Melgar

1
Sergio Melgar

• Fall 2006
• Gen 875 Class Presentation

2

• 2 D gel Electrophoresis
• Mass Spectrometry

3
Steps in 2D GE

• Sample preparation
• Isoelectric focusing (first dimension)
• SDS-PAGE (second dimension)
• Visualization of proteins spots
• Identification of protein spots
• Spot pattern evaluation/annotation

4
Steps in a 2D-gel experiment
5
Isoelectric point (pI)

• Separation by charge

Protein stops migrating where there is no net
charge
6
SDS-PAGE Principles
Loading Gel
Running Gel
7
Mass Spec Principles
Sample

_
Mass Analyzer Time of Flight (TOF)
Detector
Ionizer
8
Soft Ionization Methods
337 nm UV laser
Fluid (no salt)

_
Gold tip needle
cyano-hydroxy cinnamic acid
MALDI
ESI
Matrix-Assisted Laser Desorption Ionization
Electrospray Ionization
9
Generalized Protein Identification by MS
Spectrum of fragments generated
MATCH
Library
Peptide Mass Fingerprinting
Database of sequences (i.e. SwissProt)
10
MS-MS Proteomics
11
How Tandem MS sequencing works

• Use Tandem MS two mass analyzers in series with
  a collision cell in between
• Collision cell a region where the ions collide
  with a gas (He, Ne, Ar) resulting in
  fragmentation of the ion
• Fragmentation of the peptides occur in a
  predictable fashion, mainly at the peptide bonds
• The resulting daughter ions have masses that are
  consistent with known molecular weights of
  dipeptides, tripeptides, tetrapeptides

Ser-Glu-Leu-Ile-Arg-Trp
Collision Cell
Ser-Glu-Leu-Ile-Arg
Ser-Glu-Leu-Ile
Ser-Glu-Leu
Etc
12
Two dimentional human urine proteomic map

• Oh, J. et al

13

• Clinical diagnosis depends on identification of
  known disease associated proteins.
• One marker techniques RIA, ELISA
• Radioimmuno assay (RIA) enzyme-linked
  immunosorbent assay enzyme-linked immunosorbent
  assay (ELISA)

14

• Disadvantages of one marker techniques

15

• Some diseases are complex and might require
  multiple biomarkers
• Multiple biomarkers can improve diagnosis or
  prognosis
• Urine is the most easily accessible body fluid
• Urine filtered blood

16

• Liquid Chromatography coupled with MS/MS
• High throughput
• Relative abundance difficult to evaluate
• 2D gel MS
• Relative abundance
• pI and molecular weights
• Up to 10,000 proteins can be analyzed

17
Problems with 2D gel MS

• 2D gel MS
• Low abundance of proteins
• Complex nature of urine
• Low reproducibility
• Strategy
• Elimination of
• Interfering small molecules
• Abundant proteins (albumin)

18

• Sample preparation

19

• Low recovery after second dialysis suggests 70
  low weight proteins.

20

• TCA precipitation weakens interactions with
  interfering molecules.

21
Elimination of interfering molecules

• 2D PAGE

22
Elimination of interfering molecules

• 2D PAGE

Human, no dialisis Human, dialisis
Bovine, no dialisis
23
Variation among individuals
24
Pooling the urine
25
Identification of protein Peptide Mass
Fingerprinting
26
Generalized Protein Identification by MS
Spectrum of fragments generated
MATCH
Library
Peptide Mass Fingerprinting
Database of sequences (i.e. SwissProt)
27
Identification of protein Peptide Mass
Fingerprinting

• Image analysis
• Peptide mass fingerprinting (PMF) by MALDI-TOF-MS
• Match to human proteins in Swiss-Prot database

28
Identification of protein Peptide Mass
Fingerprinting

• 157 identified spots
• 113 annotated proteins (80)
• Different results from previous studies

29
Removal of albumin (Affi gel blue)

• Enhances low abundance proteins

30
Conclusions

• Reproducible results after double dialysis and
  TCA precipitation
• Pooled samples
• Results do not overlap with previous studies

31
Conclusions

• Needs complementation with multi-HPLC-MS/MS
• Removal of albumin enhances low abundance protein
  detection

32

• How useful can be this technique for prognosis or
  diagnosis?