Lecture 10. Proteomics II
**Lewis TS, Hunt JB, Aveline LD, Jonscher KR, Louie DF, Yeh JM,
Nahreini TS, Resing KA, Ahn NG. 2000. Identification of novel
MAP kinase pathway signaling targets by functional proteomics
and mass spectrometry. Mol Cell 6:1343-1354
Zhu H, Bilgin M, Bangham R, Hall D, Casamayor A, Bertone P, Lan
N, Jansen R, Bidlingmaier S, Houfek T, Mitchell T, Miller P, Dean
RA, Gerstein M, Snyder M. 2001. Global analysis of protein
activities using proteome chips. Science. 293:2101-2105.
Paper 1.
Lewis et al. 2000. Identification of novel MAP kinase pathway
signaling targets by functional proteomics and mass spectrometry.
Mol Cell 6:1343-1354
Two parts:
1. 2D-gels to Display the Proteome
and Potential Erk1/2 Targets
2. MS to Identify the Erk1/2 Targets
The Mitogen Activated Protein Kinase (MAPKK) Family
MAPKKK
MAPKK
MAPK
Raf
MKK1
MKK4
MEK1/2
JEK1/2
MKK6
ERK1/2
JNK1/2
Growth
Differentiation
Apoptosis
Stress Responses
p38
Phorbol Ester (PMA)
ras
Protein Kinase C (PKC)
raf
MEK1/2
Erk1/2
Differentiation K562 cells
To Megakaryocytes
(Platelets)
Part 1. 2D Gels to Compare the Proteome
in PMA treated K562 cells
3500 proteins resolved: detection from 5000-107 copies per cell
91 proteins found different in PMS treated cells (30 down, 61 up):
magnitude change >1.5-fold;
reproducible in 3 gels from at least 2 experiments
Kinetic Analysis of PMA-dependent changes in expression
Down
Up
Mobility
Altered
Cluster Analysis of the 91 Changes Reveals
4 General Classes
Question: Are these proteins regulated by Erks or PKC?
Phorbol Ester (PMA)
ras
raf
MEK1/2
Erk1/2
Protein Kinase C (PKC)
1. Pharmacological Inhibitor
U0126 Should Inhibit Changes
Differentiation K562 cells
To Megakaryocytes
(Platelets)
1. Pharmacological Inhibitor Should Inhibit Changes
Question: Are these proteins regulated by Erks or PKC?
No Phorbol Ester (PMA)
ras
raf
MEK1/2*
Erk1/2
2. Dominant Signaling Mutations
Should Cause Changes in Absence
of PMA
MEK1 Dominant Positive Mutation
Differentiation K562 cells
To Megakaryocytes
(Platelets)
2. Dominant Signaling Mutations Should Cause
Changes in Absence of PMA
Correspondence
Between U0126
Inhibition and
MKK1* Stimulation
Conclusion:
66% of Changes
Are DIRECT Targets
of Erk1/2
MALDI (Matrix Assisted Laser Desorption Ionization) Mass Spec
Used to Fingerprint the 91 proteins
Peptide Mixture
HPLC coupled with nanospray MS Used to
Confirm Identity of the Proteins
-Result: 41 proteins account for the 91 Changes
-25 are Direct Targets of Erks
-20/25 Represent newly Defined Targets
Product:Precursor Relationships
Paper 2. Protein Chips to Study Protein:Protein
or Protein:Lipid Interactions
Zhu H, et al 2001 Global analysis of protein
activities using proteome chips. Science.
293:2101-2105.
Yeast Proteome on a Single Glass Slide:
5800 GST Proteins Purified and Placed on the Chip
Probed with anti-GST as a CONTROL
Finding All Protein Partners for Calmodulin:
14/39 Contain a Conserved Calmodulin Motif
Biotinylated Calmodulin Used to Probe Chip
Identification of Lipid Binding Proteins
PI3
Interacting
Proteins
Specific
fir PI3
Bind PI3
and PC
Conventional Techniques Confirm
Lipid Binding by Proteins Identified
with the CHIP