Overview of ICAT Proteomics :

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Overview of ICAT Proteomics Oxygen stress in
Desulfovibrio vulgaris Aindrila
Mukhopadhyay Keasling Lab U C Berkeley and
LBNL GTL Annual Retreat July 23rd 2004
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Desulfovibrio vulgaris O2 stress experiment
t 5 hours
Nitrogen
C1
T0
Air
t 0
V1
0.3
Growth (OD)
Time (hours)
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Desulfovibrio vulgaris O2 stress experiment
T0
V1
C1
V1
C1
T0
307.07g
306.477g
307.234g
Harvest, wash Store pellet at 80oC
Thaw. Resuspend in 3ml 50mM Tris.HCL pH7.0
1 ml
clarify
Sonicate
T0
V1
C1
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ICAT Proteomics Strategy
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One dimensional LC MS
LC Gradient of 0-50 ACN
Total Ion Chromatogram
One TOF MS
Product Ion MSMS
Product Ion MSMS
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Sulfate Reduction Pathway
Sulfate
Sat (Sulfate Adenylyltransferase)
Adenylylsulfate
ApsA (Adenylylsulphate Reductase)
Sulfite
(Sulfite Reductase)
DvsB
H2S
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Protein Coverage
Reproducibility
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1D to 2D
0-50 Organic phase
C18 RP
DESALTING
Sample
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Summary of method optimization
Using 1D LCMS All three comparisons were done.
Unique protein hits Hits with HL ratio in
every comparison 22
Using 2D LCMS Done only for V1 vs C1 Hits at
99 confidence- Total no. of unique hits
89 Based on 177 peptides at 99 confidence Hits
with HL ratio 70
Using 2D LCMS with 2XSample Done only for V1
vs C1 Hits at 99 confidence- Total no. of
unique Protein hits 123 Based on 307 peptides at
99 confidence Hits with HL ratio 93
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LpdA
Pta
Acka
Pyruvate Acetyl-CoA
AcetylP
Acetaldehyde
EC 1.6.1.4
EC 2.3.1.8
EC 2.7.2.1
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Data posted on Biofiles
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FLKPGDAPEAEFCIK
IFCSPMGLK
C1
C1
HL 0.0948 Confidence 99
HL 0.325 Confidence 90
V1
V1
D.vulgaris VIMSS208706 Rubredoxin oxidoreductase
(Roo)
D.vulgaris VIMSS208706 Desulfoferrodoxin
(superoxide reductase, Rbo)
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Procedures for cell harvestingFor Proteomics
(estimated time 45mins) - 1.    D. vulgaris
cells are cultured in LS4D medium. Collect cells
at T ? hours.2.    Weigh the cell collecting
tubes.     3.    Harvest the cells by
centrifugation at 5000Xg for 10 min at 4oC.4.  
 Wash cells twice with vacuum degassed ice-cold
PBS buffer.       Wash volume  same as culture
volume 5.    Spin down cells again by
centrifugation at 5000Xg for 10 min at 4oC. 6.  
 Decant supernatant. Weigh the tube again to
calculate weight of cell pellets.7.    Store
pellet at -80oC.For Transcriptomics (estimated
time 15mins) - 1.    Collect the cells in a
50 mL falcon tube. 2. Harvest as in Proteomic
protocol. No wash. 3. Discard supernatant
via decanting eliminate all residual
supernatant by tapping rim of
collection tube (Falcon) with kimwipe.
4.    Quick freeze pellet in liquid
nitrogen5.    Store them at -80oC and ship on
dry ice overnight. Note earlier time
points are preferred and shorter the harvest time
the better.
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Metabolomics Overview

• Classes of metabolites
• Organic acid CoAs
• Nucleotides
• Amino acids

Separation of organic acid CoAs (D. vulgaris cell
extract) Extracted using TCA and analyzed via
LCMS.
Separation of amino acids and bases (E. coli cell
extract).
Lianhong Sun
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Extraction methods

• TCA
• Methanol
• Perchloric acid

SIM mass electropherograms of amino acids from D.
vulgaris lysate via methanol extraction
Metabolite database
Edward Baidoo
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Gene deletion library of Histidine Sensor Kinases

• 64 HKs are present in the D vulgaris genome
  (using the IPR005467 designation)
• 28 HKs have no RR close by (within 1-2 kb)
• 27 HKs are hybrid HKs

cat
ori
Plac
lacZ
lacI
Gene in D. vulgaris chromosome
Conjugate using E.coli S17-1 and selection for
Antibiotic resistance
One step

Single Crossover gene deletion
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Histidine Kinase gene deletion library Progress
Cam 0 - 20
Cam 0 20 Gent 40
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Preliminary experiments
LacZ activity in D.vulgaris
With suicide vector
With Broad host range vector
Electroporation with pSC27 ans pBMC7 worked
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Acknowledgements
Vince Martin Alyssa Redding Lianhong Sun Edward
Baidoo Sandra Villa Neil Renninger Tung
Chao Adam Arkin Eric Alm Hazen lab Dominique
and Sharon Singh lab Swapnil and Sara GTL
Project
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H Box
X Region
N
G1
F
G2
G3
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Designed 28 pairs of internal primers with UP and
DOWN Barcodes as well as 20bp CONSTANT regions
for future use in Microrarray work
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Bar-coding gene disruptions
Common primers for ALL Disrupted genes
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