Functional Proteomics : from molecular machines to protein networks
[
기능단백질체학]
Science 291 (2001) 1221.
WHAT IS PROTEOMICS?
The analysis of complete complements of proteins:
identification and quantification; modifications;
interactions; and activities. FUNCTION.
AND HOW DO THESE CHANGE DURING A BIOLOGICAL
RESPONSE?
Peck SC (2005) Update on Proteomics in Arabidopsis. Where do we go from here? Plant Physiol 138: 591-599
Major Proteomics Directions
Adapted from Human Proteome Organization (www.HUPO.org)
PTMs
Required Reading for Lecture 2: Rose et al.(2004) Plant J 39: 715-733.
“Proteomics is an increasingly
ambiguous term being applied to
almost any aspect of
protein expression,
structure or function.”
How do mass spectrometers get their names?
Types of ion sources:
• Electrospray (ESI)
• Matrix Assisted Laser Desorption Ionization (MALDI)
Types of mass analyzers:
• Quadrupole (Quad, Q)
• Ion Trap
• Time-of-Flight (TOF)
-Either source type can work with either analyzer type: “MALDI- TOF,” “ESI-Quad.”
-Analyzers can be combined to create “hybrid” instruments.
ESI-QQQ, MALDI QQ TOF, Q Trap
What is MSMS?
MS/MS means using two mass analyzers (combined in one instrument) to select an analyte (ion) from a mixture, then generate fragments from it to give structural information.
Ion
source MS-1 MS-2
Mixture of ions
Single ion
Fragments
Tools of Proteomics
1. Protein separation techniques (2-DE) 2. Mass spectrometry
3. Databases (genome/transcriptome analysis)
4. Algorithms to match MS data to proteins
5. Specific proteases (known cut sites)
Solving the Puzzle of Protein Function
Proteomics is a
multipotent tool central to research efforts in many fields and
disciplines. Maximum functional utility will come from joint efforts.
Comparative 2-DE Analysis of Tomato Root Proteins
Figure 3. Rose et al. Plant J. 39: 715
Evaluating the “extractome” rather than the proteome?
Regardless of extraction, 2-DE not good for:
• large proteins
• very hydrophobic proteins
• very basic proteins
Protein Separation Techniques: 2-DE
Copyright ©2003 American Society for Biochemistry and Molecular Biology
Steel, L. F. (2003) Mol. Cell. Proteomics 2: 262-270
2DE of total human serum proteins and proteins fractionated on anti-HSA immunoaffinity resin
How about plant systems?
Enzyme and cleavage rules
Enzyme or Reagent
Cleaves
where? Exceptions Trypsin
(higher specificity)
C-terminal side of K or R
if P is C-term to K or R; after K in CKY, DKD, CKH, CKD, KKR; after R in RRH, RRR, CRK, DRD, RRF, KRR
Lys C C-terminal side of K Asp N N-terminal
side of D Glu C
(bicarbonate)
C-terminal
side of E if P is C-term to E, or if E is C-term to E Glu C
(phosphate)
C-terminal side of D or E
if P is C-term to D or E, or if E is C-term to D or E
ALLOW 1 MISSED CLEAVAGE (1 MC)!
‘Classic’ Combination of 2-DE and MALDI-ToF MS
Spot Picker
Digest with trypsin
“PMF”
Database Search and Protein
Identification
IS THE PROTEIN IDENTIFICATION CONSISTENT WITH THE MIGRATION ON 2-DE?
MALDI-Tof-MS Sample
“Shotgun proteomics”
Two Types of Mass Spectrometers
1. MALDI-ToF (intact peptides) : m/z
2. ESI-MS/MS (peptide fragmentationsequence)
SAMPLE MASS
ANALYZER
DETECTOR
ions resolved ions
(MALDI) (TOF)
LASER
For a peptide of mass 1032, addition of a proton increases the mass to 1033.
m/z = 1033 for the [M+H]+ ion in MALDI.
Effect of Mass Accuracy and Mass Tolerance on PMF Search Results (ARATH)
Search m/z Mass tolerance (Da) # Hits
1529 1 >100
1529.73 0.01 84
1529.734 0.001 10
Peptide Matches for PMF of m/z 1529.73 Peptide
Peptide Sequence Identification Matched
m/z difference FQTCDTGKEYPLK expressed pro 1528.722
(0.008)
SVGSDSEFQQISR hypothetical pro 1528.715 (0.015)
TDWSKAPFTASYR Glucanase 1528.73
(0.000)
Peptide Resolution by MALDI-TOF
Resolution = Peak mass
Half max peak width (e.g., 1245/045 = 2766)
FOR MOST APPLICATIONS, THE EFFECTIVE MAXIMUM SIZE OF A PEPTIDE THAT FLIES IN MALDI IS BETWEEN 500 and 3,000 Da. PEPTIDE SIZE MATTERS.
C = 12.000
C = 12.011
Bottom-Up versus Top-Down Approaches
IEF-2DE Analysis and Protein Identification
Authors: Xia Wu, Steven J. Clough, Steven C. Huber and Man-Ho Oh*
[Abstract] Isoelectric focusing followed by SDS-PAGE (IEF-2DE) separates proteins in a two- dimensional matrix of protein pI (Protein Isoelectric Point) and molecular weight (MW). The technique is particularly useful to distinguish protein isoforms (Radwan et al., 2012) and proteins that contain post-translational modifications such as phosphorylation (Oh et al., 2012) and lysine acetylation (Wu et al., 2011). Proteins that are separated by IEF-2DE can be identified by
immunoblot analysis using sequence-specific antibodies or by mass spectrometry.
*Corresponding author: Man-Ho Oh, Department of Biological Sciences, Chungnam National University, Daejeon 305-764, Korea
Xia et al., (2013). Bio-Protocol
100
75
50
37 250 150
25
pI 3.0
100
75
50
37 250 150
25
0 h (IPTG) 16 h (IPTG)
IB : anti-pY IB : anti-pY
+
pI 10.0 -
pI 3.0
+ -
pI 10.0
A B
Flag-BRI1
1
2
Spot 1: maltose transporter Periplasmic protein Spot 2: chain A of OmpF porin protein
Flag-BRI1 cytoplasmic protein
Oh et al. Frontiers in Plant proteomics 2012
Brassinosteroid signaling
A nice overview
?
Now, one piece at a time…..
Reprinted from Gudesblat, G.E., and Russinova, E. (2011). Plants grow on brassinosteroids. Curr. Opin. Plant Biol. 14:530-537with permission from Elsevier.
…and proteomic approaches
Kim et al., Science 321:557-560(2008)
BL-treated plants Proteins labeled
with Cy5
Control plants Proteins labeled
with Cy3
Red proteins (black
arrows) are BR-induced, green proteins (white arrows) are BR-repressed
BSKs P
det2-1 plants (7 d on agar)
Isolate PM fraction Treat 2 h ±
BL
2-DE with amazing resolution!
det2-1 plants (7 d on agar)
Isolate PM fraction Treat 2 h ± BL
2-DE with amazing resolution!
…and proteomic approaches
Gene ontology categorization of biological processes for identified proteins with potential LysAc sites from LC-MS analysis.
Wu X et al. Plant Physiol. 2011;155:1769-1778
Lysine Acetylation is a Widespread Protein
Modification for Diverse Proteins in Arabidopsis
Xia Wu, Man-Ho Oh, Eliezer M. Schwarz, Clayton T. Larue, Mayandi Sivaguru, Brian S. Imai, Peter M. Yau, Donald R. Ort and Steven C. Huber
Screening of LRR-RLKs interacting with BAK1
Kinetic Characterization : label- free technique
Small molecule and small peptides
Qualitative Kinetics Assays
Label-Free, Real Time Results
Higher Throughput
Simple and fast
Surface Plasmon Resonance (SPR) has been used to monitor macromolecular interactions in real time
The BIAcore 3000 system is an instrument that uses SPR technology for measuring the interactions of macromolecules with each other, and with small ligands
BIAcore 3000 system
measuring the binding constants of macromolecular interactions
- protein-protein
-nucleic acids – protein - protein-ligand
- antibody affinities
- characterization of mutant proteins
Leucine-rich repeat receptor-like kinases (LRR-RLKs) are widespread
LRR-RLKs are abundant in plant and animal cells and bind a wide variety of
signaling molecules
In Arabidopsis, more than 200 different LRR-RLKs are involved in developmental signaling and pathogen responses
PLANT ANIMAL
Bishop, G.J., and Koncz, C. (2002). Brassinosteroids and plant steroid hormone signaling. Plant Cell 14:S97-110.
Immunoprecipitation
• Optimizing IP protocols with BRI1-Flag and BAK1-GFP
– Agarose beads vs Magnetic beads – Incubation time
– Detergent levels at incubation
• Large amount of protein is key
• Good antibody
T A
G
Orbitrap Elite Hybrid Ion Trap Mass Spectrometer
http://www.thermoscientific.com/ecomm/servlet/productsdetail_1 1152_L10727_87170_13901130_-1?ca=orbitrapelite
Protein Phosphorylation
• One of the most common intracellular protein modification
• Approximately 5 % of the Arabidopsis thaliana genome encodes for protein kinases & phosphatases (H. sapiens:
5 %)
• Approximately 30 % of all human proteins are
phosphorylated. How about plants, e.g., A. thaliana?
• Protein phosphorylation is of special importance in the regulation of functions, e.g. metabolism or cell signaling
In order to better understand the molecular mechanisms
where phosphorylation is involved this modification has
to be analyzed.
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Current results generated from Orbitrap (both CID and ETD were performed)
• Total 25 RLKs were run on the Orbitrap (February 16, 2012)
• pS/pT sites identified in 18 RLKs, pY sites also identified in 5 of them
• Spectra data not curated yet
An example of Orbitrap data (BAM1, CID)
31
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Another example (HAESA-like 1, ETD)
Experimental Procedure
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1500 µg of total membrane proteins (microsomal fraction) from Col-0 seedlings treated with or without BL
Tryptic digestion
Peptides desalting
IMAC IP: Anti-pY Poly-MAC
LC/MS/MS (Orbitrap)
LC/MS/MS (Orbitrap)
LC/MS/MS (Orbitrap)
Identified in vitro phosphorylation sites in At1g72300
1 MIDEKMRSKS IGPFVRQVKP LSPHMVLFVL LYVLSISVFF LTVSEAVCNL 51 QDRDSLLWFS GNVSSPVSPL HWNSSIDCCS WEGISCDKSP ENRVTSIILS 101 SRGLSGNLPS SVLDLQRLSR LDLSHNRLSG PLPPGFLSAL DQLLVLDLSY 201 ……
751 SENAELEINS NGSYSEVPPG SDKDISLVLL FGNSRYEVKD LTIFELLKAT
801 DNFSQANIIG CGGFGLVYKA TLDNGTKLAV KKLTGDYGMM EKEFKAEVEV 851 LSRAKHENLV ALQGYCVHDSARILIYSFME NGSLDYWLHE NPEGPAQLDW 901 PKRLNIMRGA SSGLAYMHQI CEPHIVHRDI KSSNILLDGN FKAYVADFGL 951 SRLILPYRTH VTTELVGTLG YIPPEYGQAW VATLRGDVYS FGVVMLELLT 1001 GKRPMEVFRP KMSRELVAWV HTMKRDGKPE EVFDTLLRES GNEEAMLRVL 1051 DIACMCVNQN PMKRPNIQQV VDWLKNIEAE KNQNNREEPE EEEET
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Red: in vitro phosphorylation sites Blue: identified phosphopetides
Using Mass Spec Data to Identify Proteins
www.matrixscience.com, and select “MASCOT”
Identify the protein from which these tryptic
peptides were derived
Note that only 5 of the 6 queried peptide masses matched. If the
‘unmatched list’ was longer you could rerun it without the matched masses.
Click here to see which peptides matched and coverage.
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Entered values
