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What happens to proteins after translocation
into ER?
-Protein glycosylation!!!
• Carbohydrate (sugar) molecules are linked
to amino acids in protein
• carried out by enzymes that reside in ER
• sugar molecules are further modified in Golgi
Most proteins that get translocated to ER get
Glycosylated, few cytosolic proteins are glycosylated
Functions of glycosylation (?) :
• inhibitors of glycosylation have little effect on protein functions
• increases resistance to proteases
•some protein-protein interactions depend on glycosylation
Carbohydrate (sugar) molecules :
glucose (Glc)
galactose (Gal)
mannose (Man)
fucose (Fuc)
N-acetyl-glucosamine (GlcNAc)
N-acetyl-galactosamine (GalNAc)
sialic acid (NANA)

Glycoproteins
N-glycosidic linkage
CH2-CO-NH-GlcNAc-GlcNAc-….
peptide
backbone
O-glycosidic linkage
CH2-O-GalNAc-….
peptide
backbone
Typical N-linked oligosaccharide:
Man-GlcNAc-Gal-NANA
CH2-CO-NH-GlcNAc-GlcNAc-Man
Fuc
Man-GlcNAc-Gal-NANA
Typical O-linked oligosaccharide:
CH2-O-GalNAc-GalNAc-NANA
NANA
After translocation into the ER all proteins move on to the Golgi
(both soluble and integral membrane proteins)
• Some proteins function in the ER
and reside there permanently
signal peptidase, glycosylation
enzymes, etc.
• ER resident proteins contain an
ER retention signal, 4 amino acids
at C-terminus of protein
• These proteins move onto Golgi,
then retrieved, brought back to ER,
• How do proteins move between ER and Golgi?
VESICULAR TRANSPORT
Movement between Golgi and ER:
• form two distinct, membrane-bounded compartments
• components are transferred via transport vesicles that bud off from ER
and fuse to the Golgi.
• vesicles contain both lumenal (soluble) and integral membrane proteins
Protein Maturation in Golgi: I. Glycosylation
lysosomal
enzymes
only
Summary of protein trafficking:
FREE
Receptor
Mediated
endocytosis
RER
Signal peptide
● Protein phosphorylation
PhosphoBase v. 2.0
(http://www.cbs.dtu.dk/databases/PhosphoBase/)
Enzymes:
1. Basophilic protein kinases
(e.g. protein kinase C (PKC))
2. Acidophilic protein kinases
(e.g. protein kinase CK2)
3. Proline-directed protein kinases
(e.g. protein kinase cdc2)
4. Protein tyrosine kinases
(e.g. epidermal growth factor
receptor, EGFR)
5. Protein serine/threonine kinases
Source: Lehninger pg 1053
● Protein methylations
Protein methylation:
1. Increases hydrophobicity
2. May alter the charge of the
protein ( e.g. if a carboxyl
group of Glu is methylated)
Source: Lehninger pg 1053
Proteomics and posttranslational
modifications
Proteomics and posttranslational
modifications
Eukaryotic cell.
Examples of protein
properties are shown,
including the
interaction of
proteins and protein
modifications.
protein-ligand
interactions
protein
complexes
(machines)
Patterson and
Aebersold, Nature
Genetics (supp.),
33, 311 (2003)
post-translational
modified proteins
protein families
(activity or structural)
Complexity of the Proteome
• Protein processing and modification comprise an
important third dimension of information, beyond those of
DNA sequence and protein sequence.
• Complexity of the human proteome is far beyond the
more than 30,000 human genes(20,000-25,000).
• The thousands of component proteins of a cell and their
post-translational modifications may change with the cell
cycle, environmental conditions, developmental stage,
and metabolic state.
• Proteomic approaches that advance beyond identifying
proteins to elucidating their post-translational
modifications are needed.
Proteomic Analysis of Posttranslational Modifications
–Mass spectrometry and other
biophysical methods can be
used to determine and localize
potential PTMs. However,
PTMs are still challenging
aspects of proteomics with
current methodologies
Isolation of modified proteins:
Modification analysis is usually done by comparison of
experimental data to a known amino acid sequence.
A central consideration in the characterization of
Modifications is the need for as large an amount of the
protein as possible. Why?
-typically not homogeneous, a very small fraction of total…
● Recombinantly expressed proteins or recombinantly
expressed proteins that are modified in vitro.
( baculovirus and Mammalian expression system are
often significant differences.)
● Chromatographic purifications +antibody precipitations
----the modifications of a single protein.
● 2D +different staining.
The ‘pearls-on-string’pattern is a telltale indication of protein
phosphorylation, although the introduction of charge
heterogeneity by deamidation of Asn or Gln residues to form
carboxylic acids can also generate such a pattern.
PTM mapping of a purified protein:
A variety of techniques can be used to determine the modified amino
acids.
Don’t forget the traditional method: Amino acid sequencing for aminoor carboxy-terminal processing!
Proteomic analysis of PTMs
Mann and
Jensen,
Nature
Biotech. 21,
255 (2003)
●
A Proteomics Approach to Understanding
Protein Ubiquitination
1)Nature Biotechnology,21,921-926(2003)
S.cerevisiae Strain SUB592(in which all ubiquitin genes were removed and a 6xHismyc-ubiquitin-coding plasmid was introduced) and the control strain SUB280(similar
to SUB592 except for the introduction of a wild-type ubiquitin plasmid)
Summary:
2) Genome Research,13:1389-1394(2003)
---The Comparative Proteomics of Ubiquitination in Mouse
It was a bioinformatics work.
Mus musculus proteome (based upon proteins from SWISSPROT and TrEMBL and additional peptides predicted by
Ensembl) was obtained from the EBI Proteome Analysis
Database(http://www.ebi.ac.uk/proteome/)
The Representative Transcript and Protein Set(RTPS)
UA InterPro domains----UA proteins
●
Proteomic Analysis of glycoprotein
Isotope-coded glycosylation-site-specific tagging(IGOT)
What ahout O-linked glycoproteins?
● There is no enzyme comparable to PNGase F for removing
intact O-linked sugars.
● Sequentially remove monosaccharides by using a panel
of exoglycosidase until only the Galbeta1,3 GalNAc core
remains attached to the serine or threonine residue.
● The core can then be released by O-glycosidase.
●The chemical method, such as beta-elimination, may be
more generally useful and effective.
Glycoprotein Gel Stain
Detection of glycoproteins and total protein on an SDS-polyacrylamide gel
using the Pro-Q Fuchsia Glycoprotein Gel Stain Kit.
CandyCane glycoprotein molecular weight standards
containing alternating glycosylated and nonglycosylated
proteins were electrophoresed through a 13% polyacrylamide
gel. After separation, the gel was stained with SYPRO Ruby
protein gel stain to detect all eight marker proteins (left).
Subsequently, the gel was stained by the standard periodic
acid–Schiff base (PAS) method in the Pro-Q Fuchsia
Glycoprotein Gel Stain Kit to detect the glycoproteins alpha2macroglobulin, glucose oxidase, alpha1-glycoprotein and
avidin.
Pro-Q™ Glycoprotein Stain (DDAO phosphate)
Molecular Formula: C15H18Cl2N3O5P (MW 422.20)
Glycome-Glycomics
Glycoproteome-Glycoprotomics
Glycan array
●
Proteomics and Protein phosphorylation
Phosphorylation
• Analysis of the entire complement of phosphorylated proteins in
cells: “phosphoproteome”
• Qualitative and quantitative information regarding protein
phosphorylation important
• Important in many cellular processes
– signal transduction, gene regulation, cell cycle, apoptosis
• Most common sites of phosphorylation: Ser, Thr, Tyr
• MS can be used to detect and map
locations for phosphorylation
– MW increase from addition of
phosphate group
– treatment with phosphatase allows
determination of number of phosphate
groups
– digestion and tandem MS allows for
determination of phosphorylation sites
Enrichment strategies to analyze
phosphoproteins/peptides
• Phosphospecific antibodies
– Anti-pY quite successful
– Anti-pS and anti-pT not as successful, but may be used (M.
Grønborg, T. Z. Kristiansen, A. Stensballe, J. S. Andersen, O.
Ohara, M. Mann, O. N. Jensen, and A. Pandey, “Approach for
Identification of Serine/Threonine-phosphorylated Proteins by
Enrichment with Phospho-specific Antibodies.” Mol. Cell.
Proteomics 2002, 1:517–527.
• Immobilized metal affinity chromatography (IMAC)
– Negatively charged phosphate groups bind to postively charged
metal ions (e.g., Fe3+, Ga3+) immobilized to a chromatographic
support
– Limitation: non-specific binding to acidic side chains (D, E)
• Derivatize all peptides by methyl esterification to reduce nonspecific binding by carboxylate groups.
• Ficarro et al., Nature Biotech. (2002), 20, 301.
Chemical derivatization to
enrich for phosphoproteins
• Developed because other
methods based on
affinity/adsorption (e.g.,
IMAC) displayed some
non-specific binding
• Chemical derivatization
methods may be overly
complex to be used
routinely
• Sensitivity may not be
sufficient for some
experiments (low pmol)
Phosphoprotein Stain
Phospho
PeppermintStick phosphoprotein
molecular weight standards
separated on a 13% SDS
polyacrylamide gel. The markers
contain (from largest to smallest)
beta-galactosidase, bovine serum
albumin (BSA), ovalbumin, betacasein, avidin and lysozyme.
Ovalbumin and beta-casein are
phosphorylated. The gel was stained
with Pro-Q Diamond phosphoprotein
gel stain (blue) followed by SYPRO
Ruby protein gel stain (red). The
digital images were pseudocolored.
Phosphoprotein Stain
Visualization of total protein and
phosphoproteins in a 2-D gel
Proteins from a Jurkat T-cell
lymphoma line cell lysate were
separated by 2-D gel electrophoresis
and stained with Pro-Q Diamond
phosphoprotein gel stain (blue)
followed by SYPRO Ruby protein gel
stain (red). After each dye staining,
the gel was imaged and the resulting
composite image was digitally
pseudocolored and overlaid.
T.H. Steinberg et al., Global quantitative phosphoprotein analysis using
Multiplexed Proteomics technology, Proteomics 2003, 3, 1128-1144
Global Analysis of Protein Phosphorylation
RAW 264.7 exposed to DEP
Pro-Q Diamond
3.54.5 5.1 5.5
6.0
7.0
98
55
37
30
Sypro Ruby
IEF
8.4 9.5
3.54.5 5.1 5.5
98
TNF convertase
MAGUK p55
PDI
Protein phosphatase 2A
JNK-1
p38 MAPK alpha
ERK-1
ERK-2
ErbB-2
TNF
HSP 27
20
Xiao, Loo, and Nel - UCLA
3
5
55
6 7
37
13
30
20
1
2
8
9
10
4
12
11
14
6.0
7.0
8.4 9.5
Identification of Low Abundance
Proteins
• The identification of low abundance
proteins in the presence of high
abundance proteins is problematic
(e.g., “needle in a haystack”)
• Pre-fractionation of complex protein
mixtures can alleviate some
difficulties
– gel electrophoresis, chromatography, etc
• Removal of known high abundance
proteins allows less abundant
species to be visualized and
detected
Identification of Low Abundance Proteins
GenWay Biotech
Proteinchip for analysis of protein phosphorylation
Molecular Cell, Vol. 13, 649–663, March 12, 2004
Endostatin’s Antiangiogenic Signaling Network
Methods:
DNA+Antibody Chip, Real-time PCR &
Immunocytochemistry
◆ Human Unigene Chip II containing 74834 elements covering
90% of all
human genes. This represents one of the largest gene sets interrogated
in array-based gene expression studies to date.
ratio-voting criteria (more than 2.0 and less than 0.5)
◆ analyzed the alteration in gene expression
after a 4 hr treatment of
primary isolated human dermal microvascular of endothelial cells (HDMVEC)
with 200 ng/ml of human recombinant endostatin (Pichia Pastoris).
16 characteristic genes were chosen for real-time
quantitative RT-PCR analysis using Taq-man technology
◆ Differentially
phosphorylated proteins were examined using antibody array
technology. After 30 min incubation with 500 ng/ml human recombinant
endostatin, whole cell protein lysates from HDMVECs were hybridized on
an antibody array containing 400 immobilized antibodies against wellstudied signaling proteins. The phosphorylation status of the tyrosine
residues from the cellular proteins bound to the array were detected using
a horse peroxidase-conjugated anti-phosphotyrosine antibody.
◆Immunocytochemistry
Results:
●Genome-Wide Expression Profiling: 6635 Unigene clusters to
24 different functional groups
● Protein-Level Regulation:
● Endostatin Downregulates Id1 and Id3
● Endostatin Downregulates HIF1-alpha
● Ephrins and TNF in Endostatin Signaling
● Endostatin Modulates NF-B Signaling
● AP-1 Transcription Factors Are Downregulated By
Endostatin
● Coagulation Cascade and Adhesion Molecules in Endostatin
Signaling
● STATs in Endostatin Signaling
● ETS-1 in Endostatin Signaling
Bioinformatics and protein modifications
This database is freely accessible on the Internet
through resources provided by the European
Bioinformatics Institute
(http://www.ebi.ac.uk/RESID),
and by the National Cancer Institute – Frederick
Advanced BiomedicalComputing Center
(http://www.ncifcrf.gov/RESID).
PTMs are ubiquitous and dynamic. Their presence
must be:
● Predicted by computational sequence analysis
● Determined by:
efficient and sensitive experimental proteomic techniques
genetic, biochemical and analytical techniques
Hepatitis C Virus and Proteomics
The Hepatitis C Virus (HCV)
 Genus of Hepacivirus, member of Flaviviridae, a family
comprising enveloped (+)strand RNA viruses.
 Complex secondary structure at the 5’NTR and part of
the core sequences serves as IRES.
 Hepatitis C virus infection represents a major problem of
public health with around 350 millions of chronically
infected individuals worldwide. The frequent evolution
towards severe liver disease and cancer are the main
features of HCV chronic infection.
Brechot C Cell Death and Differentiation(2003)10,S27-S38
Brechot C Cell Death and Differentiation(2003)10,S27-S38
Brechot C Cell Death and Differentiation(2003)10,S27-S38
Dubuisson J . Biochimie 85(2003)295-301
Human vesicle-associated membrane protein-associated protein A(hVAP-A)
Matthew J.Evans PNAS 2004 101:13038-43
DNA/RNA Affinity Chromatography
Column
Wash
Protein
Loading
Elution
Highly Purifed
Transcription Factor
Contaminant
proteins
Additional Readings
• R. Aebersold and M. Mann, Mass spectrometry-based
proteomics, Nature (2003), 422, 198-207.
• M. B. Goshe and R. D. Smith, “Stable isotope-coded proteomic
mass spectrometry.” Curr. Opin. Biotechnol. 2003; 14: 101-109.
• W. A. Tao and R. Aebersold, “Advances in quantitative
proteomics via stable isotope tagging and mass spectrometry.”
Curr. Opin. Biotechnol. 2003; 14: 110-118.
• S. D. Patterson and R. Aebersold, “Proteomics: the first decade
and beyond.” Nature Genetics 2003; 33 (suppl.): 311-323.
• M. Mann and O. N. Jensen, “Proteomic analysis of posttranslational modification.” Nature Biotech. 2003; 21: 255-261.
• D. T. McLachlin and B. T. Chait, “Analysis of phosphorylated
proteins and peptides by MS.” Curr. Opin. Chem. Biol. 2001; 5:
591-602.
• S. Gygi et al., “Quantitative analysis of complex protein mixtures
using isotope-coded affinity tags.” Nature Biotech. 1999; 17:
994-999.
Proteomics in Practice: A Laboratory Manual of
Proteome Analysis
Reiner Westermeier, Tom Naven
Wiley-VCH, 2002
PART I: PROTEOMICS
TECHNOLOGY
Introduction
Expression Proteomics
Two-dimensional Electrophoresis
Spot Handling
Mass Spectrometry
Protein Identification by Database
Searching
Methods of Proteomics
PART II: COURSE MANUAL
Step 1: Sample Preparation
Step 2: Isoelectric Focusing
Step 3: SDS Polyacrylamide Gel Electrophoresis
Step 4: Staining of the Gels
Step 5: Scanning of Gels and Image Analysis
Step 6: 2D DIGE
Step 7: Spot Excision
Step 8: Sample Destaining
Step 9: In-gel Digestion
Step 10: Microscale Purification
Step 11: Chemical Derivatisation of the Peptide Digest
Step 12: MS Analysis
Step 13: Calibration of the MALDI-ToF MS
Step 14: Preparing for a Database Search
Step 15: PMF Database Search Unsuccessful
Proteins and Proteomics: A Laboratory Manual
Richard J. Simpson
Cold Spring Harbor Laboratory (2002)
Chapter 1. Introduction to Proteomics
Chapter 2. One–dimensional Polyacrylamide Gel Electrophoresis
Chapter 3. Preparing Cellular and Subcellular Extracts
Chapter 4. Preparative Two–dimensional Gel Electrophoresis with
Immobilized pH Gradients
Chapter 5. Reversed–phase High–performance Liquid Chromatography
Chapter 6. Amino– and Carboxy– terminal Sequence Analysis
Chapter 7. Peptide Mapping and Sequence Analysis of Gel–resolved Proteins
Chapter 8. The Use of Mass Spectrometry in Proteomics
Chapter 9. Proteomic Methods for Phosphorylation Site Mapping
Chapter 10. Characterization of Protein Complexes
Chapter 11. Making Sense of Proteomics: Using Bioinformatics to Discover a
Protein’s Structure, Functions, and Interactions