andrey lisitsa - C-HPP

Russian Human Proteome Organization
Chr 18: Transcriptome vs. Proteome (transcriptoproteome) for
Liver tissue and HepG2 cells
ANDREY LISITSA
INSTITUTE OF BIOMEDICAL CHEMISTRY, MOSCOW
www.ibmc.msk.ru
www.c‐hpp.org
Victor Zgoda & Arthur Kupylov
Kate Poverennaya & Kate Ilgisonis
CHR18
Elena Ilyina, Dmitry Alexeev
Elena Arkhipova, Alexey Chernoborvkin
Nathaly Moskaleva & Olly Kiseleva
ALEX ARCHAKOV
RHUPO Founder (since 2002)
C‐HPP Principal Investigator (since 2011)
ELENA PONOMARENKO
C‐HPP Coordinator / Bioinformatics
TRANSCRIPTO-PROTEOME PROFILING
CHROMOSOME 18
LIVER
RNA‐Seq:
RT‐PCR
5
log10(transcript copies per cell)
SOLiD,Illumina
HepG2 CELL LINE
‐3
4
3
2
1
Selected Reaction Monitoring
0
‐1
‐1
1
‐2
‐3
log10(protein copies per cell)
3
TRANSCRIPTO-PROTEOME PROFILING
ARE WE SURE THE TRANSCRIPTOME
DATASETS ARE O’KEY?
SOLiD / Illumina / qRT‐PCR
WE USE TRANSCRIPTOME TO «VERIFY»
PROTEOME. SO…
WHY WE ARE SUSPICTIONS TO THE
PROTEOME DATA? Selected Reaction Monitoring:
Visual Perception*
DETECTED & QUANTIFIED
LIGHT (ENDOGENOUS)
MIGHT BE DETECTED, NOT QUANTIFIED
HEAVY (REFERENCE)
RT
* SkyLine (MacCoss Lab, Washington) SpectroDive (Biognosys AG, Switzerland)
NOT DETECTED (N/D)
PROTEOME PROFILING FOR CHR 18
(Selected Reaction Monitoring)
Blood plasma
(265 proteins)
0
10
6
249
0
Liver tissue
(262 proteins)
HepG2 cell line
3
0
Archakov et al., Exp.Rev. in Proteomics, 2014, submitted
(255 proteins)
«Ping» the ‐Оme by SRM
Take telephone books of different countries
‐ e.g. cells, tissue, plasma
Destroy books in the shredder
‐ sample prep
Glue some fragments
‐ bioinformatics
…AND CALL!
> 5000 calls // 249 responses // 3 types of biomaterial
CHR 18: PINGING THE HEPG2 CELL LINE
in progress
(12)
Distribution histogram of the number of protein species copies per HepG2 cell (CpC)
undetected (8)
Number of proteins
120
1 peptide
(35)
2 peptides
CV<100%
(85)
1 peptide
100
2 peptides: protein range>1 order
80
>2 orders
(24)
2 peptides: protein range ≤ 1 order
60
2 orders
(37)
40
1 order
(76)
20
0
1
2
3
4
5
6
7
Log10 (CpC)
8
9
10
CHR18: PINGING THE LIVER PROTEOME
in progress (8)
undetected
(8)
Distribution histogram of the number of protein species copies per liver cell (CpC)
Number of proteins
80
1 peptide (39)
1 peptide
70
2 peptides
CV<100%
(118)
>2 orders
(23)
2 peptides: protein range>1 order
60
50
2 peptides: protein range ≤ 1 order
40
2 orders
(27)
30
1 order
(54)
20
10
0
1
2
3
4
5
6
7
8
Log10 (CpC)
9
10 11
LIVER
Log10(portein copies per 1 liver cell)
12
y = 0,8115x + 0,5397
R² = 0,554
10
8
6
LIVER to PLASMA
4
R2 = 0.003
2
0
0
1
2
3
4
5
6
7
8
Log10(protein copies per single HepG2 cell)
9
10
HepG2
12
R² = 0.0186
SHOTGUN**
10
8
1
6
log 10 (iBAQ)
log 10 (proteins copy per HepG2 cell)
HepG2: shotgun vs. SRM
4
2
0.9
0.8
0.7
0
‐2
‐1
0
1
2
0.6
log 10 (transcripts per HepG2 cell, RPKM)
0.5
SRM*
0.4
y = 0,0514x + 0,6787
R² = 0,2383
0.3
*Zgoda et al., 2013, JPR
**Geiger et al. & Mann, 2012, MCP
‐2
‐1
0
1
2
3
log 10 (transcripts per HepG2 cell, RPKM)
Conclusions
Chromosome‐centric approach is opposed to the
fully automated data workflows applied in routine
in proteomics
We have to collect the SRM‐signals and revise them
manually to understand the destinations of our
“SRM‐calls”. These calls could be bound to:
‐ proteins
‐ proteins + PTMs
‐ proteins + drugs (low molecular weight cmpds)
‐ AS proteins
‐ lipids
… and may other things
www.c‐hpp.org
Victor Zgoda & Arthur Kupylov
Kate Poverennaya & Kate Ilgisonis
CHR18
Elena Ilyina, Dmitry Alexeev
Elena Arkhipova, Alexey Chernoborvkin
Nathaly Moskaleva & Olly Kiseleva
ALEX ARCHAKOV
RHUPO Founder (since 2002)
C‐HPP Principal Investigator (since 2011)
ELENA PONOMARENKO
C‐HPP Coordinator / Bioinformatics