Download y-ion series=A, AA, LAA, SLAA

Proteomic Based Approaches In
Developmental Biology
Why Proteomics?
• Alterations between cells, tissues, and embryos often are not
associated with changes in RNA levels, i.e. you cannot answer
everything by RNA-seq; protein stability, protein localization,
changes in PTMs, etc.
• Only approximately 10% of all RNAs with changes of 1.5X or greater
between two samples, from yeast to human, lead to a change of 1.5X
in protein levels.
• Establish in vivo interactomes as function of stage and tissue.
• Define in vivo PTMs as function of stage and tissue.
Proteomic Based Approaches
Proteomics
Generally refers to a survey of all the proteins in a
given tissue or cell type.
Directed Proteomic
The identification of a set of proteins that are in
complex with a defined protein of interest.
Quantitative Proteomics
Characterization of the relative amount of a set of
proteins between 2-16 samples.
Mass Spectrometry – A Brief Definition
1394.49
Mass Spectrometry is a technique for the production of charged
molecular species in vacuo, and their separation by magnetic and/or
electric fields based on the mass to charge ratio (m/z).
2000
2846.95
1742.46
2522.78
3000
1787.43
4000
989.30
861.24
5000
1584.48
6000
1252.42
1099.34
7000
750.24
Abundance (Intensity)
8000
1000
500
1000
1500
(m/z)
2000
2500
3000
Mass Spectrometry Data
From Andrew Link, Vanderbilt University
Basic Mass Spectrometer
From “Mass Spectrometry in Biotechnology by
Gary Siuzdak
Fundamental Parts of a Mass
Spectrometer
Ion source
1. MALDI
2. ESI
Mass analyzer
1. TOF
2. Ion trap
3. Quadrupole
4. Orbitrap
5. Magnetic sector
6. FTICR
Detector
Ionization Techniques
From “Mass Spectrometry in Biotechnology by
Gary Siuzdak
Properties of Different
Mass Analyzers
Mass
Separation
Mass
Accuracy
Resolution
FWHH
Scan
Rate
m/z
Range
TOF
Very good
Medium
Very fast
Very wide
10-100 ppm
20,000
(s)
Good
Medium
Fast
100-1000 ppm
20,000
(m s)
Ion trap
Quadrupol
e
Good
Low
Normal
100-1000 ppm
2,000
(s)
FTICR
Excellent
High
Normal
0.1-1 ppm
>100,000
(s)
0 – 4000
0 – 4000
Very wide
Ionization
Steen and Mann,
2004
m/z Not Enough
Verification of all proteins must be done by second phase MS
to obtain sequence data of at least 3 peptides per protein
b-ion
y-ion
=
=
=
=
CH2-OH
CH2-CH-(CH3)2
CH3
CH3
O
O
O
O
C-NH-CH-C-NH-CH-C-NH-CH-C-NH-CH
Peptide: SLAA
m/z Not Enough
Verification of all proteins done by second phase MS to
obtain sequence data of at least 3 peptides per protein.
y-ion
y-ion
y-ion
=
=
CH2-CH-(CH3)2
CH3
CH3
CH2-OH
=
=
O
O
O
O
C-NH-CH-C-NH-CH-C-NH-CH-C-NH-CH
Peptide: SLAA
e.g. y-ion series=A, AA, LAA, SLAA
m/z Not Enough
Verification of all proteins done by second phase MS to
obtain sequence data of at least 3 peptides per protein.
b-ion
b-ion
b-ion
b-ion
=
CH3
CH2-CH-(CH3)2
CH3
=
CH2-OH
=
=
O
O
O
O
C-NH-CH-C-NH-CH-C-NH-CH-C-NH-CH
Peptide: SLAA
y-ion series= A, AA, LAA, SLAA
b-ion series=S, SL, SLA, SLAA
TBX20 MS/MS Raw Data
~115D + H20 (16D) =
133D; i.e. Aspartic Acid
~115D
How do you what
proteins you have?
Protein Databases
• Database must contain what you need and little more:
• “Additional” proteins crashes specificity and discovery
rate, increase shared tryptic peptides.
• Additional” proteins greatly(!) increase time of
analysis.
• Need all available protein sequences from species of
interest derived from trEMBL and SWISSPROT (Everything
that is in NCBI, Sanger and EMBL).
Where Do Protein Sequences Come From?
And How Do They Translate Gene Sequences?
All protein sequences in NCBI, etc. come from the
UniProtDatabase which is divided into:
•
TrEMBL (automated annotation). Contains more
redundancy, includes sequence fragments and isoforms.
Especially useful for organisms that are not well annotated.
•
Swiss-Prot sequences (manually annotated). Sufficient for
well-annotated organisms, e.g. human, mouse, yeast,
E.coli, HSV-1 and Xenopus. This is not derived from the
sequencing of proteins.
How do you get the protein sequence?
How do you know where your protein sequence came from?
How do you know where your protein came from?
Protein Entry Headers
>SOURCE(sp/tr)|ACCESSION|UNIPROT_ID DESCRIPTION ORGANISM GENE
>sp|Q04917|1433F_HUMAN 14-3-3 protein eta OS=Homo sapiens GN=YWHAH PE=1 SV=4
MGDREQLLQRARLAEQAERYDDMASAMKAVTELNEPLSNEDRNLLSVAYKNVVGARRSSWRVISSIEQKTMADGNEKKLEKVKA
YREKIEKELETVCNDVLSLLDKFLIKNCNDFQYESKVFYLKMKGDYYRYLAEVASGEKKNSVVEASEAAYKEAFEISKEQMQPT
HPIRLGLALNFSVFYYEIQNAPEQACLLAKQAFDDAIAELDTLNEDSYKDSTLIMQLLRDNLTLWTSDQQDE
EAGEGN
Analysis with xProteo of Tbx20-GFP (MALDI)
Source: 293 cells (Human Kidney Cell Line)
False
Positive
Junk
Further Look Into TBX20 Data
Proteomic Based Approaches
Proteomics
Generally refers to a survey of all the proteins in a
given tissue or cell type.
Directed Proteomic
The identification of a set of proteins that are in
complex with a defined protein of interest.
Quantitative Proteomics
Characterization of the relative amount of a set of
proteins between 2-16 samples.
Overview of Approach
Work Flow
Incubation with
Ab, 1hr at 4C.
Takes another
~8hrs to wash,
elute and
concentrate
(20ul)
Can go
from
isolating
tissue to
trypsin in 1
day
(a very long
day)
How Much Tissue is Enough?
Tissue Isolation
Need Large/Unlimited Source of Protein for Initial Optimization
e.g. TBX20: 293 Cells transfected with pcDNA 3.1-TBX20
CMV promoter driving epitope tagged Tbx20
Starting Material: N=15-20 X 150mm Dishes
After Optimization: Current record from mouse of an endogenous
protein 4fM (50 adult brains) Selimi et al. 2009
More likely: 500-1500 embryos
Alternative: ES cell differentiation
Why so much? Each step needs to be
optimized for an individual protein.
Initial Lysis Buffers Routinely Used
Worked
With
TBX20
Affinity Purification
Which Epitopes or Tags
1.
Ideal: High affinity, high specificity antibody against endogenous
protein.
1.
V5, Myc and Flg (1,3, 9X)
Poor. Commercial Abs of too low affinity and/or low specificity. (V5
worked with Tbx20 but pulled down some non-specific proteins).
2. Anti-HIS (3, 6, 9X); i.e. Nickel or Cobalt
Horrific. Very, very high non-specific interactions even in presence
of DNAse. If it does work will probably strip off any interacting
proteins.
4. HA (3X). Good. e.g. Bienvenu F. et al. 2010,
5. GFP. Better/Best but not an epitope tag and may interfere with protein
function, e.g. Selimi et al. 2009 (worked with Tbx20).
6. AVI-Tag (In Progress)
Avi-tag Tagged Tbx20
Avi-tag epitope knocked into Tbx20 locus
Biotinylation by BirA
Biotin
Biotin
Streptavidin
Applications of BirA-mediated biotinylation
Isolation and purification of small poplulations of
cells from living embryos
Purification of protein complexes
Identification of Targets Genes, i.e.Chrommatin IP
AP Tagged Tbx20
Construct knocked into Tbx5 containing mouse for Tg
Proteomic Based Approaches
Proteomics
Generally refers to a survey of all the proteins in a
given tissue or cell type.
Directed Proteomic
The identification of a set of proteins that are in
complex with a defined protein of interest.
Quantitative Proteomics
Characterization of the relative amount of a set of
proteins between 2-16 samples.
Overcomes the differential gel
running problem. You can use
software to quantitate
Best for
separating whole
cell lysatelooking at in tact
proteins!
You will lose Membrane proteins,
histones, highly acidic proteins
MS-based Quantitative Methods
• Precursor: Quantitation based on the relative intensities of
extracted ion chromatograms (XICs) for precursors within a
single data set. This is a widely used approach, which can be
used with any chemistry that creates a precursor mass shift.
For example, 18O, AQUA, ICAT, ICPL, Metabolic, SILAC,
etc., etc.
• Reporter: Quantitation based on the relative intensities of
fragment peaks at fixed m/z values within an MS/MS
spectrum. For example, iTRAQ and Tandem Mass Tags
• Replicate: Label free quantitation based on the relative
intensities of extracted ion chromatograms (XICs) for
precursors in multiple data sets aligned using mass and elution
time.
Stable Isotope Labeling Methods
• ‘Mass difference’ approaches
– Metabolic (Stable Isotope Labeling with Amino
acids in Cell culture SILAC)- introduces heavy
isotopes into sample
– Chemical labels (Isotope Coding with Affinity tags
ICAT)
– Enzymatic (O16/O18 labeling)
• Isobaric (equal mass) peptide tags
– iTRAQ &TMT
SILAC Labeling Reagents
•
•
13C- and 12C-Lysine (heavy – light = 6 D)
13C- and 12C-Arginine (heavy – light = 6 D)
C12->C13 adds one
neutron
C13 is chemically
indistinguishable
SILAC
(98%)
Prostate cancer
cell line PC3
PC3M
(low metastatic potential)
PC3M-LN4
(high metastatic potential)
Everley et al., MCP 2004
Labeling Efficiency of both isotopes
Ratio = 0.98
Doublet is
separated
You need to passage cells 3-4
times in heavy media,
measure incorporation
iTRAQ: Isotope Coding
8-plex isobaric tagging reagents:
iTRAQ
Great Free On-line Resource:
“The Expanding Role of Mass
Spectrometry in Biotechnology
by Gary Siuzdak