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20100428 presentation

weekely presentation

Sooa Lim

on 4 May 2010

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Transcript of 20100428 presentation

Phosphoproteomics Meeting 2010.04.28. Commencing Date: 7 September 2009
Expiry date: 6 September 2012 Attainment Of Milestone When do you expect the candidate to complete your next milestone? 22 June 2011 (?) Timeline Phosphoproteomics Hypothesis PhD Scholarship 2008 June 2009 June 2010 June 2011 June 1 year 1 year 1year 1year Comfirmation: Jan 2010 Mid-Candidature Review Thesis Review KAIST Project Experiment Plan 1. Result
Phosphoproteome of E.coli UQ and K12(MG1655)
It is identifed phospopeptide number from number of E. coli UQ and K12(MG1655)
-Identification of 105 phosphopeptides from 79 E. coli proteins with very high confidence.
It is determined with a prbability higher than 75% for 81 phosphorylation sites.
Identify phosphopeptides/ phosphorylation ratio:
Serine 55/67.9%, threonines 19/23.5%, tyrosine 7/8.6%

Classes of phosphorylated proteins as assigned by Blast 2GO
Biological process: 60 information
Cellular localization: 26 information
Among enzymes involved in the main pathways of carbohydate metaolism.
For example,
A. phosphorylation sites is detected on almost all glycolytic enzymes such as
enolase(eno), L-lactate dehydrogenase(IctE), triose-phosphate isomerase(tpi), malate dehydrogenase(citH),
glyceraldehyde-3-phosphate dehydrogenase(gap), pyruvate kinase(pykA), isomerase(pgi),
fructose-1,6-bisphosphate aldolase(fbaA),malate dehydrogenase(citH), phosphoglycerate mutase(pgm),
glucose-6-phosphate isomerase(pgi), fructose-1, 6-bisphosphate aldolase(fbaA), pyruvate dehydrogenase(pdhB),
phosphoglycerate knase(pgk), ybbT

Comparison of E.coli UQ and K12 Phosphoproteomes
Protein conservation analysis between these two phosphoproteomes revealed number of orthologous proteins.
Such as (14 proteins) fus(A), glmM, ptsl, tsf, ndk, deoB, mdh, pykF, pgi, eno, gpmM, pgk, gatY, pta
Some orthologous phosphoproteins contained conserved phosphorylation sites that were detected in both organisms.
Some orthologous phosphoproteins contained conserved specific phosphorylation sites that were detected in each organisms.
Direct compariosn of the general features of the Ser/Thr/Tyr phosphoproteome
Genome siz(ORFs)
Number of phosphoproteins
Number of detected phosphorylation events
Distribution of Ser/Thr/Tyr phosphorylation
Protein essentiality

Quantitative phosphoproteomic analysis reveals pathways of carbohydrate metabolism.
Can help identify, and possibly quantify the phosphorylation status of E. coli during developmental and physiological processes. Workflow


10 phosphopeptide
5 nonphosphopeptide

MRMpilot create method

Control Samples


C1= 0.5 mg/mL Dataset 10 (Standard proteins) a-casein
phosvitin C2=0.5 mg/mL
Weight = 0.05mg
Vol. = 100uL a-casein
phosvitin 1. Mixture (15uL)
Weight = 0.0075mg 2. 1:2 Dilution Mixture (15uL)
C4 = 0.25mg/mL
Weight = 0.0075mg 3. 1:10 dilution Mixture (15uL)
C5 = 0.05mg/mL
Weight = 0.0075mg Pho_a casein Depho_a casein 100% 100% C1= 0.5mg/mL Depho_a casein Pho_a casein C2= 0.5mg/mL
Weight = 0.05mg
Vol. = 100uL 10uL 10uL 4. Mixture
C3= 0.5mg/mL
Weight = 0.01mg
Vol. = 20uL Depho_a casein 100% 100% C2= 0.5mg/mL
Weight = 0.05mg
Vol. = 100uL Depho_a casein Pho_a casein Pho_a casein 5uL 5uL 5. 1:2 Dilution Mixture
C4= 0.25mg/mL
Weight = 0.01mg
Vol. = 20uL +10uL MillQ water Pho_a casein Depho_a casein 100% 100% Depho_a casein Pho_a casein C2= 0.5mg/mL
Weight = 0.05mg
Vol. = 100uL 6. Original
C5= 0.5mg/mL
Weight = 0.01mg
Vol. = 20uL 7. Original
C5= 0.5mg/mL
Weight = 0.01mg
Vol. = 20uL Enriched Samples (TiO2) Samples information
UQ_SCX_F1 1. Enriched Sample(O)
2. Enriched Sample(T) 3. Original _ unenriched Samples 4. Enriched Sample(O)
5. -20 Store GL Sciences Inc. GE healthcare Control Samples
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