Protein characterization posttranslational modifications and proteinprotein interactions Week 10 Top down bottom up Top down Bottom up masscharge intensity Top down Bottom up ID: 600978
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Slide1
Proteomics Informatics –
Protein characterization: post-translational modifications and protein-protein interactions (Week 10)Slide2
Top down / bottom up
Top down
Bottom up
mass/charge
intensitySlide3
Top down Bottom up
Charge distribution
mass/charge
intensity
mass/charge
intensity
1+
2+
3+
4+
27+
31+Slide4
Top down Bottom up
Isotope distribution
mass/charge
intensity
mass/charge
intensitySlide5
Fragmentation
Top down
Bottom up
FragmentationSlide6
Correlations between modifications
Top down
Bottom upSlide7
Alternative Splicing
Top down
Bottom up
Exon
1
2
3Slide8
Top down
Kellie et al., Molecular BioSystems 2010
Protein
mass spectra
Fragment
mass spectraSlide9
Protein Complexes
A
B
A
C
D
Digestion
Mass spectrometrySlide10
Sowa et al., Cell 2009
Protein Complexes – specific/non-specific bindingSlide11
Protein Complexes – specific/non-specific binding
Choi et al., Nature Methods 2010Slide12
Tackett et al. JPR 2005
Protein Complexes – specific/non-specific bindingSlide13
Analysis of Non-Covalent Protein Complexes
Taverner et al., Acc Chem Res 2008Slide14
Non-Covalent Protein Complexes
Schreiber et al., Nature 2011Slide15
More / better quality interactions
Affinity Capture Optimization Screen
+
Cell extraction
Lysate
clearance/
Batch Binding
Binding/Washing/Eluting
SDS-PAGE
Filtration
LaCava
,
Hakhverdyan
,
Domanski
,
RoutSlide16
Over 20 different extraction and washing
conditions ~ 10 years or art.(41 pullouts are shown)
Molecular Architecture of the NPC
Actual model
Alber
F. et al. Nature (450) 683-694.
2007
Alber
F. et al. Nature (450) 695-700.
2007Slide17
Cloning nanobodies for GFP pullouts
Atypical heavy chain-only IgG antibody produced in
camelid
family – retain high affinity for antigen without light chain
Aimed to clone individual single-domain VHH antibodies against GFP – only ~15
kDa
, can be
recombinantly
expressed, used as bait for pullouts, etc.To identify full repertoire, will identify GFP binders through combination of high-throughput DNA sequencing and mass spectrometry
VHH clone for recombinant expressionSlide18
Cloning
llamabodies for GFP pullouts
Llama GFP immunization
Lymphocyte
total RNA
Crude serum
VHH
amplicon
454 DNA sequencing
RT / Nested
PCR
IgG
fractionation &
GFP affinity
purification
VHH DNA sequence library
GFP-specific
VHH fraction
LC-MS/MS
GFP-specific
VHH clones
Bone marrow aspiration
Serum bleed
500
400
300
1000
bp
No. of Reads
Read length (
bp
)
VH
VHH
Fridy
, Li
,
Keegan,
Chait
, RoutSlide19
CDR3: 100.0% (14/14); combined CDR: 100.0% (33/33); DNA count: 10
MAQVQLVESGGGLVQAGGSLR
LSCVASGRTFSGYAMGWFR
QTPGRER
EAVAAITWSAHSTYYSDSVK
DR
FTISIDNTRNTGYLQMNSLKPEDTAVYYCTVRHGTWFTTSRYWTDWGQGTQVTVS
CDR3: 100.0% (14/14); combined CDR: 72.7% (24/33;
DNA count: 1
MADVQLVESGGGLVQSGGSRTLSCA
ASGRVLATYHLGWF
RQSPGRER
EAVAAITWSAHSTYYSDSVK
GR
FTISIDNARNTGYLQMNSLKPEDTAVYYCTVRHGTWFTVSRYWTDWGQGTQVTVS
CDR3: 100.0% (14/14); combined CDR: 72.7% (24/33); DNA count: 1
MAQVQLVESGGALVQAGASLSVS
CAASGGTISKYNMAWFRR
APGRER
EAVAAITWSAHSTYYSDSVK
DR
FTISIDNTRNTGYLQMNSLKPEDTAVYYCTVRHGTWFTTSRYWTDWGQGTQVTVS
CDR3: 100.0% (14/14); combined CDR: 42.4% (14/33); DNA count: 1
MAQVQLEESGGGLVQAGDSLT
LSCSASGRTFTNYAMAWSRQA
PGKE
RELLAAIDAAGGATYYSD
SVKGR
FTISIDNTRNTGYLQMNSLKPEDTAVYYCTVRHGTWFTTSRYWTDWGQGTQVTVS
CDR3: 100.0% (14/14); combined CDR: 42.4% (14/33); DNA count: 1
MAQVQLVESGGGRVQAGGSLTL
SCVGSEGIFWNHVMGWFR
QSPGKDREFVA
RISKIGGTTN
YADSVKGR
FTISIDNTRNTGYLQMNSLKPEDTAVYYCTVRHGTWFTTSRYWTDWGQGTQVTVS
CDR1
CDR2
CDR3
Underlined regions are covered by MS
Rank sequences according to:
CDR3 coverage; Overall coverage;
Combined CDR coverage; DNA counts;
Identifying full-length sequences from peptidesSlide20
Sequence diversity of 26 verified
anti-GFP nanobodies
Of ~200 positive sequence hits, 44 high confidence clones were synthesized and tested for expression and GFP binding: 26 were confirmed GFP binders.
Sequences have characteristic conserved VHH residues, but significant diversity in CDR regions.
FR1
CDR1
FR2
CDR2
CDR3
FR3
FR4Slide21
HIV-1
gp120
Lipid Bilayer
gp41
MA
CA
NC
PR
IN
RT
RNA
Particle
Genome
env
rev
vpu
tat
nef
3
’
LTR
5
’
LTR
vif
gag
pol
vpr
CA
MA
NC
p6
PR
RT
IN
gp41
gp120
9,200 nucleotidesSlide22
Genetic-Proteomic Approach
Tagged Viral Protein
Tag
Protein Complex
SDS-PAGE
*
Mass SpectrometrySlide23
I-Dirt
for Specific Interaction
3xFLAG Tagged HIV-1
WT HIV-1
Infection
Light
Heavy
(
13
C labeled Lys, Arg)
1:1 Mix
Immunoisolation
MS
I-DIRT
=
I
sotopic
D
ifferentiation of
I
nteractions as
R
andom or
T
argeted
Lys
Arg
(+6 daltons)
(+6 daltons)
Modified from Tackett AJ
et al
., J Proteome Res. (2005) 4, 1752-6. Slide24
IDIRT and Reverse IDIRT
Env-3xFLAG
Vif-3xFLAG
Luo
, Jacobs, Greco, Cristae,
Muesing
,
Chait
, RoutSlide25
Protein Exchange
Vif-3F
Heavy labeled Vif-3F lysate
IP in heavy labeled Vif-3F lysate
Vif-3F
Light labeled
wt
lysate
Incubation with light labeled
wt
lysate
Vif-3F
15min
Vif-3F
5min
Stable
Interactor
Vif-3F
Interactor
with fast exchange
60min Slide26
Env
Time Course SILACDifferentially labeled infection harvested at early or late stage of infection
Distinguish proteins that interact with
Env
at early or late stage during infection
Early during infection
Late during infectio
n
Light
Heavy
(
13
C labeled Lys,
Arg
)
1:1 Mix
Immunoisolation
MS
Early
interactor
Late
interactorSlide27
M/Z
Peptides
Fragments
Fragmentation
Proteolytic
Peptides
Enzymatic Digestion
Protein
Complex
Chemical Cross-Linking
MS
MS/MS
Isolation
Cross-Linked
Protein Complex
Interaction Partners by
Chemical Cross-LinkingSlide28
M/Z
Peptides
Fragments
Fragmentation
Proteolytic
Peptides
Enzymatic Digestion
Protein
Complex
Chemical Cross-Linking
MS
MS/MS
Isolation
Cross-Linked
Protein Complex
Interaction Sites by
Chemical Cross-LinkingSlide29
Cross-linking
protein
n peptides with reactive groups
(n-1)n/2 potential
ways to cross-link peptides pairwise
+ many additional uninformative forms
Protein A +
IgG
heavy chain 990 possible
peptide pairs
Yeast NPC
˜
10
6
possible
peptide pairsSlide30
Protein
Crosslinking
by Formaldehyde
~1%
w/v
Fal
20 – 60 min
~0.3%
w/v
Fal
5 – 20 min
1/100 the volume
LaCavaSlide31
Protein
Crosslinking
by Formaldehyde
RED:
triplicate experiments,
FAl
treated
grindate
BLACK:
duplicated experiments, FAl treated cells (then ground)SCORE: Log Ion Current / Log protein abundance
Akgöl
, LaCava,
RoutSlide32
Cross-linking
Mass spectrometers have a limited dynamic range and it therefore important to limit the number of possible reactions not to dilute the cross-linked peptides.
For identification of a cross-linked
peptide pair,
both peptides have to be sufficiently long and required to give informative fragmentation.
High mass accuracy MS/MS is recommended because the spectrum will be a mixture of fragment ions from two peptides.
Because the cross-linked
peptides
are often large,
CAD is not ideal, but instead ETD is recommended.Slide33
Phosphopeptide identification
mprecursor = 2000 Da
D
m
precursor
= 1 Da
D
mfragment = 0.5 DaPhosphorylation
Localization of modificationsSlide34
Localization (d
min=3)
m
precursor
= 2000 Da
D
m
precursor
= 1 Da
D
m
fragment
= 0.5 Da
Phosphorylation
d
min
>=3 for 47% of human tryptic peptides
Localization of modificationsSlide35
Localization (d
min=2)
m
precursor
= 2000 Da
D
m
precursor
= 1 Da
D
m
fragment
= 0.5 Da
Phosphorylation
d
min
=2 for 33% of human tryptic peptides
Localization of modificationsSlide36
Localization (d
min=1)
m
precursor
= 2000 Da
D
m
precursor
= 1 Da
D
m
fragment
= 0.5 Da
Phosphorylation
d
min
=1 for 20% of human tryptic peptides
Localization of modificationsSlide37
Localization
(d=1*)
m
precursor
= 2000 Da
D
m
precursor
= 1 Da
D
m
fragment
= 0.5 Da
Phosphorylation
Localization of modificationsSlide38
Peptide with two possible modification sites
Localization of modificationsSlide39
Peptide with two possible modification sites
MS/MS spectrum
m/z
Intensity
Localization of modificationsSlide40
Peptide with two possible modification sites
MS/MS spectrum
m/z
Intensity
Matching
Localization of modificationsSlide41
Peptide with two possible modification sites
MS/MS spectrum
m/z
Intensity
Matching
Which
assignment
does
the data support?
1
,
1
or
2
, or
1
and
2
?
Localization of modificationsSlide42
AAYYQK
Visualization of evidence for localization
AAYYQKSlide43
Visualization of evidence for localizationSlide44
Visualization of evidence for localization
3
2
1
3
2
1Slide45
Estimation of global false
localization rate using decoy sites
By counting how many times the
phosphorylation
is localized to amino acids that can not be
phosphorylated
we can estimate the false localization rate as a function of amino acid frequency.
Amino acid frequency
False localization frequency
YSlide46
How much can we trust a
single localization assignment?
If we can generate the distribution of scores for assignment 1 when 2 is the correct assignment, it is possible to estimate the probability of obtaining a certain score by chance for a given peptide sequence and MS/MS spectrum assignment.
Slide47
Is it a mixture or not?
If we can generate the distribution of scores for assignment 2 when 1 is the correct assignment, it is possible to estimate the probability of obtaining a certain score by chance for a given peptide sequence and MS/MS spectrum assignment.
Slide48
1
and
2
1
1
or
2
Ø
Localization of modificationsSlide49
Proteomics Informatics –
Protein characterization: post-translational modifications and protein-protein interactions
(Week 10)