Quantitative Proteomics and Protein Modification Proteomics

1
Quantitative Proteomics and Protein Modification
Proteomics

• Masaru Miyagi, Ph.D.
• March 5, 2008

2
Outline of Talk

• Introduction to quantitative proteomics
• Introduction to protein modification proteomics

3
Quantitative Proteomics
4
What is Quantitative Proteomic Method?

• Techniques that allow us quantitative
  measurements of individual peptides/proteins/prote
  in modifications in a cell/tissue
• Relative quantification
• Absolute quantification

5
Why Quantitative Proteomics

• Most biological questions cannot be answered by
  simply determining whether a given protein is
  present or absent
• Proteomics technology must include the ability to
  measure the amounts of individual proteins

6
Areas of Quantitative Proteomics

• Expression proteomics
• Identification and quantitation of peptides and
  proteins across samples (diseased vs normal, drug
  treatments, genotypes etc)
• Interaction proteomics
• Characterizing networks of protein-protein
  interactions
• Protein modification proteomics
• Identification and quantitation of
  post-translational modifications (e.g.
  phosphorylation, glycosylation etc.) across
  samples

7
Classifications of Current Relative Quantitative
Proteomic Techniques
Quantitative Proteomic Techniques
Top-down methods
Bottom-up/Shotgun methods
Mass Spectrometry-based on stable isotope
labeling methods
2D-PAGE based methods
Label-free methods
(2D-DIGE etc.)
In vivo metabolic labeling
In vitro labeling
(SILAC etc.)
Proteolytic 18O labeling
Chemical isotope labeling
(iTRAQ etc.)
8
Quantitative Proteomic MethodsGel-based methods
Control
Experimental
Compare spot intensities
Excise spots of interest
digest by a protease
Analyze by tandem mass spectrometry
Search database
Protein id. (Qualitative identification)
9
Quantitative Proteomic MethodsMass
Spectrometry-based on stable isotope labeling
methods
In vivo metabolic labeling
Control (metabolically labeled with light
isotopes)
Experimental (metabolically labeled with heavy
isotopes)
Mix in 11 ratio
digest by a protease
Analyze by LC/MS/MS
Search database
Protein id.
Calculate heavy- and light-peptide ratio
10
Quantitative Proteomic MethodsMass
Spectrometry-based on label free methods
Control
Experimental
digest by a protease
digest by a protease
Analyze by LC/MS/MS
Search database
Protein id.
Compare MS data
Calculate peptide ratio
11
Mass Spectrometry based method- In vivo Metabolic
LabelingStable Isotope Labeling by Amino Acid in
Cell Culture (SILAC)
Ong et al., J Proteome Res. 2003, 2173-181
12
Mass Spectrometry based method- In vitro
LabelingiTRAQ Technique
Ross et al., Mol Cell Proteomics. 2004,
31154-1169
13
Mass Spectrometry based method- In vitro
LabelingProteolytic 18O labeling Technique
Control
Experimental
digest and label by a protease in H216O
digest and label by a protease in H218O
Mix in 11 ratio
Analyze by LC/MS/MS
Search database
Protein id.
Calculate heavy- and light-peptide ratio
Miyagi and Rao, Mass Spectrom Rev. 2007
Jan-Feb26(1)121-36
14
Mass Spectrometry based method- Label free
technique
r.teaftiar
r.teaftiar
r.teaftiar
Identification and quantitation
Sample Preparation
Separation fractionation
Mass spectrometry
15
Absolute Quantification of proteins
Protein extract
Mix with known amount of IS peptide
TLVLVPLLANNR
Digest by a protease
TLVLVPLLANN13C6, 15N4R
LC-MRM
Unpublished data
16
Summary
Mass spectrometry methods
2D-PAGE based methods
Strength
Strength

• High resolving power
• capable of separating several thousand proteins
  in a single gel
• Sensitive in detecting small covalent structural
  changes
• post-translationally modified proteins
• alternatively spliced isoforms

• The data (mass spectra) used to identify proteins
  provide quantitative information
• Capable to analyze membrane proteins

Weakness

• Insensitive in detecting small covalent
  structural changes
• such as alternatively spliced isoforms

Weakness

• A problem multiple proteins in a single spot
• Biased toward soluble proteins

17
Expression Proteomics ofPhotoreceptor Outer
Segments - Why Proteomics

• There is no protein synthesis machinery in the
  outer segments
• Full of membrane proteins
• gt 80 of a total protein
• Proteomic approach is the only high-throughput
  method that does not require foreknowledge of
  proteins expressed in the outer segments

Outer Segments
18
Proteomic Study of Photoreceptor Outer Segments
P60 rats
Exposed to green light
Controls No light
1,200-1,400 lux green light (490-580 nm)
1 AM 9 AM
Sacrificed at 9 AM
Sacrificed at 9 AM
Prepared OS by sucrose density ultracentrifugation
method
19
Expression Proteomics of ROS
Control ROS
Light exposed ROS
H216O
H216O
digested by Lys-C or trypsin
digested by Lys-C or trypsin
Alkaline pH
H216O
H218O
16O labeled by Lys-C or trypsin
18O labeled by Lys-C or trypsin
Acidic pH
Analyzed by LC/MS/MS
Inactivate the protease
Search database
Protein id.
Calculate 16O and 18O2 peptide ratio
Mix in 11 ratio
20
Unpublished data
21
Proteins Involved in Phototransduction
Photoreceptor OS tries to desensitize the
phototransduction signaling
Unpublished data
22
Other Proteins
Unpublished data
23
Pathway analysis on apoptotic pathways activated
upon light exposure
24
Missing Information in the Quantitative
Proteomics Methods Discussed so far
Po
Pt
t
Pt Po Psynthesized - Pdegraded
25
Label cells or organism with D2O
Extract proteins
Isolate proteins of Interest
Acid hydrolyze the proteins
Analyze deuterated amino acid by MS
Four healthy subjects were given 70 D2O (50-70
mL) every 3-4 h over 24 h, and then once or twice
a day over 10 weeks.
26
Label cells or organism with D2O
Extract proteins
Isolate proteins of Interest
Digest by a protease
Mass spectra of a peptide from serum albumin.
Analyze deuterated peptide by MS
C57BL/6J mice had free access to 6 D2O over the
course of the experiments.
27
Protein Modification Proteomics
28
Protein Modifications
Textbook of Biochemistry (5th edition), p. 267
29
Classifications of Protein Modifications

• Modifications by cellular systems
• Co-translational modifications
• Post-translational modifications
• Modifications occurs under unusual cellular
  conditions
• Nitration
• 4-Hydroxynonenal (HNE) modification

30
Co- and Post-Translational Modifications

• Co-translational modifications
• occurs during protein synthesis
• N-terminal processing (removal of initiator-Met
  and N-acetylation)
• N-glycosylation
• partial proteolysis
• Post-translational modifications
• occurs after protein synthesis
• phosphorylation
• methylation
• O-glycosylation

31
Why Protein Modification Proteomics?

• Protein modifications are often involved in the
  various functions of proteins
• therefore knowledge of protein modifications
  provide useful information to understand the
  structure-functions of proteins

32
Analytical Challenges

• The degrees of modifications are usually very
  low lt 10
• Diverse chemical properties of modifications

Need to have different strategies for different
modifications!
33
Phosphorylation
Liebler, D. C. (2002) Introduction to Proteomics,
Humana Press
34
Cell. 2006 Nov 3127(3)635-48
35
N-Glycosylation
Kaji et al., Nat Protoc. 20061(6)3019-3027
36
Tyrosine Nitration
SATA N-succinimidyl S-acetylthioacetate
Zhang et al., J Proteome Res. 2007 6, 2257-2268
37
4-Hydroxynonenal (HNE)
Roe et al., Anal Chem. 2007 79, 3747-3756
38
Summary

• For quantitative proteomics
• choose appropriate methods for your own research
  projects
• Depending on proteins of interests, target
  organelles and what kind of quantitative
  information you want to have
• For protein modification proteomics
• Likely to need to employ enrichment methods
• Better to obtain quantitative information as well