laboratori de proteòmica vall d’hebron institut d’oncologia francesc canals madrid 28/08/2012...
TRANSCRIPT
![Page 1: Laboratori de Proteòmica Vall d’Hebron Institut d’Oncologia Francesc Canals Madrid 28/08/2012 Human Proteome Project – CHR16 SRM Analysis of selected proteins](https://reader035.vdocuments.us/reader035/viewer/2022062719/56649ee45503460f94bf3b88/html5/thumbnails/1.jpg)
Laboratori de ProteòmicaVall d’Hebron Institut d’Oncologia
Francesc Canals
Madrid 28/08/2012
Human Proteome Project – CHR16
SRM Analysis of selected proteins (ABSciex 4000 QTRAP)
Vall d’Hebron Institute of Oncology
Vall d’Hebron University Hospital, Barcelona
![Page 2: Laboratori de Proteòmica Vall d’Hebron Institut d’Oncologia Francesc Canals Madrid 28/08/2012 Human Proteome Project – CHR16 SRM Analysis of selected proteins](https://reader035.vdocuments.us/reader035/viewer/2022062719/56649ee45503460f94bf3b88/html5/thumbnails/2.jpg)
1D- SDS-PAGE 12% - 100 mg each sample
Trypsin digestion
In solution – 100 mg
Ramos TCA/Acetone 5.93 mg/mLMCF7 4.92 mg/mLCCD18 7.30 mg/mL
SAMPLE PREPARATION
In gel – 15 bands :
~ 6.7 mg / band
A: RamosMW B: MCF7 C: CCD18
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
75
100
50
37
![Page 3: Laboratori de Proteòmica Vall d’Hebron Institut d’Oncologia Francesc Canals Madrid 28/08/2012 Human Proteome Project – CHR16 SRM Analysis of selected proteins](https://reader035.vdocuments.us/reader035/viewer/2022062719/56649ee45503460f94bf3b88/html5/thumbnails/3.jpg)
SHOTGUN LC-MSMS Analysis MCF7 sample
Esquire HCT Ion trap
• 15 bands *2uL (~400ng)
1093 proteins identified
• 200ng In solution digestion
152 proteins identified
4 of “our” 20 known proteins to analyze observed in Gel-LCMS of MCF7
29 of 160 total known proteins to analyze observed in Gel-LCMS of MCF7
![Page 4: Laboratori de Proteòmica Vall d’Hebron Institut d’Oncologia Francesc Canals Madrid 28/08/2012 Human Proteome Project – CHR16 SRM Analysis of selected proteins](https://reader035.vdocuments.us/reader035/viewer/2022062719/56649ee45503460f94bf3b88/html5/thumbnails/4.jpg)
Protein Name/ Accession ensembl gene id
external gene id description
P69905 ENSG00000188536 HBA2 hemoglobin, alpha 2 [Source:HGNC Symbol;Acc:4824]P07911 ENSG00000169344 UMOD uromodulin [Source:HGNC Symbol;Acc:12559]P23141 ENSG00000198848 CES1 carboxylesterase 1 [Source:HGNC Symbol;Acc:1863]O43303 ENSG00000103540 CCP110 centriolar coiled coil protein 110kDa [Source:HGNC Symbol;Acc:24342]P35858 ENSG00000099769 IGFALS insulin-like growth factor binding protein, acid labile subunit [Source:HGNC Symbol;Acc:5468]Q92793 ENSG00000005339 CREBBP CREB binding protein [Source:HGNC Symbol;Acc:2348]P15559 ENSG00000181019 NQO1 NAD(P)H dehydrogenase, quinone 1 [Source:HGNC Symbol;Acc:2874]P69849 ENSG00000103226 NOMO3 NODAL modulator 3 [Source:HGNC Symbol;Acc:25242]O43294 ENSG00000140682 TGFB1I1 transforming growth factor beta 1 induced transcript 1 [Source:HGNC Symbol;Acc:11767]Q6P996 ENSG00000179889 PDXDC1 pyridoxal-dependent decarboxylase domain containing 1 [Source:HGNC Symbol;Acc:28995]Q93009 ENSG00000187555 USP7 ubiquitin specific peptidase 7 (herpes virus-associated) [Source:HGNC Symbol;Acc:12630]P15170 ENSG00000103342 GSPT1 G1 to S phase transition 1 [Source:HGNC Symbol;Acc:4621]Q9NPB6 ENSG00000102981 PARD6A par-6 partitioning defective 6 homolog alpha (C. elegans) [Source:HGNC Symbol;Acc:15943]Q15911 ENSG00000140836 ZFHX3 zinc finger homeobox 3 [Source:HGNC Symbol;Acc:777]Q15554 ENSG00000132604 TERF2 telomeric repeat binding factor 2 [Source:HGNC Symbol;Acc:11729]P63279 ENSG00000103275 UBE2I ubiquitin-conjugating enzyme E2I [Source:HGNC Symbol;Acc:12485]Q9NUU7 ENSG00000168872 DDX19A DEAD (Asp-Glu-Ala-As) box polypeptide 19A [Source:HGNC Symbol;Acc:25628]Q16775 ENSG00000063854 HAGH hydroxyacylglutathione hydrolase [Source:HGNC Symbol;Acc:4805]Q08AM6 ENSG00000103043 VAC14 Vac14 homolog (S. cerevisiae) [Source:HGNC Symbol;Acc:25507]Q86W42 ENSG00000131652 THOC6 THO complex 6 homolog (Drosophila) [Source:HGNC Symbol;Acc:28369]
Protein Name/Accessionensembl_gene_id external_gene_iddescriptionP51170 ENSG00000166828 SCNN1G sodium channel, nonvoltage-gated 1, gamma [Source:HGNC Symbol;Acc:10602]Q7L3S4 ENSG00000179965 ZNF771 zinc finger protein 771 [Source:HGNC Symbol;Acc:29653]Q96S06 ENSG00000103227 LMF1 lipase maturation factor 1 [Source:HGNC Symbol;Acc:14154]O00219 ENSG00000103044 HAS3 hyaluronan synthase 3 [Source:HGNC Symbol;Acc:4820]Q2QL34 ENSG00000156968 MPV17L MPV17 mitochondrial membrane protein-like [Source:HGNC Symbol;Acc:26827]Q9NVC3 ENSG00000103042 SLC38A7 solute carrier family 38, member 7 [Source:HGNC Symbol;Acc:25582]
Proteins to be assayed by SRM in our lab
20 Known Proteins
6 Unknown Proteins
![Page 5: Laboratori de Proteòmica Vall d’Hebron Institut d’Oncologia Francesc Canals Madrid 28/08/2012 Human Proteome Project – CHR16 SRM Analysis of selected proteins](https://reader035.vdocuments.us/reader035/viewer/2022062719/56649ee45503460f94bf3b88/html5/thumbnails/5.jpg)
Phase 1: Selection of 5 known + 1 unknown proteins
Q92793 ENSG00000005339 CREBBP CREB binding protein [Source:HGNC Symbol;Acc:2348]Q93009 ENSG00000187555 USP7 ubiquitin specific peptidase 7 (herpes virus-associated) [Source:HGNC Symbol;Acc:12630]Q08AM6 ENSG00000103043 VAC14 Vac14 homolog (S. cerevisiae) [Source:HGNC Symbol;Acc:25507]Q86W42 ENSG00000131652 THOC6 THO complex 6 homolog (Drosophila) [Source:HGNC Symbol;Acc:28369]Q9NUU7 ENSG00000168872 DDX19A DEAD (Asp-Glu-Ala-As) box polypeptide 19A [Source:HGNC Symbol;Acc:25628]Q96S06 ENSG00000103227 LMF1 lipase maturation factor 1 [Source:HGNC Symbol;Acc:14154]
All observed in MCF7 in MaxQuant database (11 cell lines, M.Mann)
In Silico
MRM Pilot SoftwareRepositories dataSRM Atlas (Peptide Atlas)MaxQuant database
Shotgun data1D GelIn solution digestion
Previous Experimental data
+
SRM Method Generation
![Page 6: Laboratori de Proteòmica Vall d’Hebron Institut d’Oncologia Francesc Canals Madrid 28/08/2012 Human Proteome Project – CHR16 SRM Analysis of selected proteins](https://reader035.vdocuments.us/reader035/viewer/2022062719/56649ee45503460f94bf3b88/html5/thumbnails/6.jpg)
6
Excel Macro_MRM_GenerateMethodList © Adelina Acosta
Macro that generates a transition table with the specific format of Analyst software (ABSciex 4000 QTRAP).
From SRMAtlas Data:
![Page 7: Laboratori de Proteòmica Vall d’Hebron Institut d’Oncologia Francesc Canals Madrid 28/08/2012 Human Proteome Project – CHR16 SRM Analysis of selected proteins](https://reader035.vdocuments.us/reader035/viewer/2022062719/56649ee45503460f94bf3b88/html5/thumbnails/7.jpg)
1. Copy the TSV table in Worksheet “TSV” 2. Run Macro
![Page 8: Laboratori de Proteòmica Vall d’Hebron Institut d’Oncologia Francesc Canals Madrid 28/08/2012 Human Proteome Project – CHR16 SRM Analysis of selected proteins](https://reader035.vdocuments.us/reader035/viewer/2022062719/56649ee45503460f94bf3b88/html5/thumbnails/8.jpg)
In worksheet “MRM_List”: all transitions from TSV table in the right format for Analyst software
![Page 9: Laboratori de Proteòmica Vall d’Hebron Institut d’Oncologia Francesc Canals Madrid 28/08/2012 Human Proteome Project – CHR16 SRM Analysis of selected proteins](https://reader035.vdocuments.us/reader035/viewer/2022062719/56649ee45503460f94bf3b88/html5/thumbnails/9.jpg)
In worksheet “Filtered_List”: selected transitions according to specific criteria
from TSV table in the right format for Analyst software
![Page 10: Laboratori de Proteòmica Vall d’Hebron Institut d’Oncologia Francesc Canals Madrid 28/08/2012 Human Proteome Project – CHR16 SRM Analysis of selected proteins](https://reader035.vdocuments.us/reader035/viewer/2022062719/56649ee45503460f94bf3b88/html5/thumbnails/10.jpg)
SRM Methods
Exp. data derived Method In Silico derived Methods
MRM Pilot generated methods for each protein
4 transitions / peptide20-30 peptides/ protein
SRM Atlas Data for all 6 proteins
Filtered using Excel Macro
+ Observed MSMS data : shotgun + MaxQuant
5 transitions / peptide 5 peptides/ protein
LC-MS conditions: 15 cm x 75 mm column; 300 nl/min; 90 min gradient
![Page 11: Laboratori de Proteòmica Vall d’Hebron Institut d’Oncologia Francesc Canals Madrid 28/08/2012 Human Proteome Project – CHR16 SRM Analysis of selected proteins](https://reader035.vdocuments.us/reader035/viewer/2022062719/56649ee45503460f94bf3b88/html5/thumbnails/11.jpg)
A: RamosMW B: MCF7 C: CCD18
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
75
100
50
37
A: RamosMW B: MCF7 C: CCD18
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
75
100
50
37
RamosMCF7CCD18
Solution Digests
SRM - ANALYSIS
Exp. data derived Method
6 x In Silico derived Methods
Exp. data derived Method
CREBBP 265 kDa Band 1VAC14 87 kDa Band 3LMF1 65 kDa Band 4
In Silico derived Methods
Bands 1-7
![Page 12: Laboratori de Proteòmica Vall d’Hebron Institut d’Oncologia Francesc Canals Madrid 28/08/2012 Human Proteome Project – CHR16 SRM Analysis of selected proteins](https://reader035.vdocuments.us/reader035/viewer/2022062719/56649ee45503460f94bf3b88/html5/thumbnails/12.jpg)
Q92793 CREBBP SRM Atlas In Silico CCD1
8
MCF
7
Ram
os
SRM Atlas In SilicoN Dif.
TransitionsDAFLTLAR 3 3QAASTSGPTPAASQALNPQAQK 4 x x x 4
In Solution Digestion Ramos Band A1
SRM - RESULTS
“KNOWN” PROTEINS
Ramos Band A2-3
Q93009 USP7 SRM Atlas In Silico CCD1
8
MCF
7
Ram
os
SRM AtlasN Dif.
Transitions
FDDDVVSR 5 x x x 5IIGVHQEDELLEC[160]LSPATSR 4 4IQDYDVSLDK 4 x 4LLEIVSYK 5 x x x 5LSEVLQAVTDHDIPQQLVER 4 4
In Solution Digestion
![Page 13: Laboratori de Proteòmica Vall d’Hebron Institut d’Oncologia Francesc Canals Madrid 28/08/2012 Human Proteome Project – CHR16 SRM Analysis of selected proteins](https://reader035.vdocuments.us/reader035/viewer/2022062719/56649ee45503460f94bf3b88/html5/thumbnails/13.jpg)
Q08AM6 VAC14 SRM Atlas In Silico CCD1
8
MCF
7
Ram
os
SRM Atlas In SilicoN Dif.
TransitionsDSGLYLK 3 3FDLVSFIPLLR 4 x x 4GPFIIR 3 3LAADPDPNVK 3 x 3 3QAEPTPDDALPK 4 4VAALEIEK 3 3
In Solution Digestion Ramos Band A3
Ramos Band A7
Q86W42 THOC6 SRM Atlas In Silico CCD1
8
MCF
7
Ram
os
SRM AtlasN Dif.
Transitions
AFSLSF 1 x 1AVPLAVPLGQTEVFQALQR 4 x 4EVQTIEVYK 4 x 4HLLSAGDGEVK 5 5SPEVLSGGEDGAVR 5 9 x x x 5 9SSTPTTIFPIR 6 x x 6
In Solution Digestion
SRM - RESULTS
“KNOWN” PROTEINS
![Page 14: Laboratori de Proteòmica Vall d’Hebron Institut d’Oncologia Francesc Canals Madrid 28/08/2012 Human Proteome Project – CHR16 SRM Analysis of selected proteins](https://reader035.vdocuments.us/reader035/viewer/2022062719/56649ee45503460f94bf3b88/html5/thumbnails/14.jpg)
Ramos Band A1/2/4
Q9NUU7 DDX19A SRM Atlas In Silico CCD1
8
MCF
7
Ram
os
SRM AtlasN Dif.
Transitions
DGNPDNETYLHR 5 x 5DPNSPLYSVK 5 7 x x 5 7EEETLDTIK 4 x 4FYPELK 4 x 4LDTDDLDEIEK 2 5 x 5 5SNLVDNTNQVEVLQR 5 7 x x 5 8VLVTTNVC[CAM]AR 4 x 4 5YPQC[CAM]LC[CAM]LSPTYELALQTGK 3 x x x 3
In Solution Digestion
Q96S06 LMF1 SRM Atlas In Silico CCD1
8
MCF
7
Ram
osSRM Atlas In Silico
N Dif. Transitions
LLASDAEALSLLAHNPFAGRPPPR 2 x 2
In Solution Digestion Ramos Band A4
SRM - RESULTS
“KNOWN” PROTEINS
“UNKNOWN” PROTEIN
![Page 15: Laboratori de Proteòmica Vall d’Hebron Institut d’Oncologia Francesc Canals Madrid 28/08/2012 Human Proteome Project – CHR16 SRM Analysis of selected proteins](https://reader035.vdocuments.us/reader035/viewer/2022062719/56649ee45503460f94bf3b88/html5/thumbnails/15.jpg)
SRM -CONCLUSIONS
• Better results by In Silico prediction than SRM Atlas
• Limited shotgun data utility
• Confidence by: MSMS spectra, different cell lines
• Known Proteins: Enough peptides /protein
• 3-5 transitions/peptide => optimize transitions
• Unknown Proteins => fractionate ?
![Page 16: Laboratori de Proteòmica Vall d’Hebron Institut d’Oncologia Francesc Canals Madrid 28/08/2012 Human Proteome Project – CHR16 SRM Analysis of selected proteins](https://reader035.vdocuments.us/reader035/viewer/2022062719/56649ee45503460f94bf3b88/html5/thumbnails/16.jpg)
Laboratori de ProteòmicaVall dHebron Institut d’Oncologia
Núria ColoméJoan Josep BechLuna MartínAdelina Acosta
ACKNOWLEDGMENTS
UCTS
Fátima Nuñez
Ricardo GonzaloM. Ángeles Artaza
Proteomics Laboratory
![Page 17: Laboratori de Proteòmica Vall d’Hebron Institut d’Oncologia Francesc Canals Madrid 28/08/2012 Human Proteome Project – CHR16 SRM Analysis of selected proteins](https://reader035.vdocuments.us/reader035/viewer/2022062719/56649ee45503460f94bf3b88/html5/thumbnails/17.jpg)
Thank You !
Laboratori de ProteòmicaVall dHebron Institut d’Oncologia