La Cristalera, 5-6 November 2013.
SRM working plan.
Previous SRM analysis
• To deliver reliable SRM methods, we proposed a stepwise strategy that involves
data sharing and cross-validation across different laboratories and this process
must be optimized under standard conditions
• An initial set of 120 Chr-16 proteins was selected on the basis of their GPMDB
scores, log e in the range -175 to -6000, belonging to the group defined as
“known” Chr16 proteins.
• Each laboratory explored the detectability by SRM of the assigned proteins in
digests from at least three cell lines : MCF7, CCD18, and Ramos.
• For 106 out of the 120 proteins selected (88.3%) a minimum of three co-eluting
SRM transition signals were observed for a number of peptides ranging from 1-10
per protein, with an average of 4.16 peptides/protein.
• After the initial round of SRM method development at the six laboratories, a
second round of cross-validation was performed, assigning each of the final 51
proteins detected to two laboratories different from the laboratory that initially
developed the SRM method. After this second round of analysis validation, a total
of 149 peptides from 49 proteins.
-1186,1
-339,5
-392,6
-206,8
-173,1
-187,3
-564,2
-1400,2
-1076,2
-284,5
-384,3
-357,3
-361,5
-598,1
-365
-411,3
-543
-210,9
-369,4
-230,7
-187,1
-1039,8
-392,6
-901,2
-236,4
-177,7
-387,7
-702
-196,9
-271,3
-198,8
-1107,8
-435,1
-179,2
-285,4
-408,5
-312,2
-265
-335,9
-185
-203,5
-219,6
-1962,2
2
3
3
3
3
2
3
3
3
3
3
3
3
3
3
3
3
3
3
2
3
3
3
3
3
3
2
3
3
3
3
2
3
3
3
2
3
2
3
2
3
2
3
1
4
1
4
1
2
3
4
3
3
3
4
4
5
4
9
3
1
2
1
4
1
1
7
2
5
1
4
2
2
1
1
3
4
1
1
5
1
3
2
7
1
3
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
HUAECS
x
x
x
x
Jurkat
Peptides
Huh7
#
TC28
# Laboratories
CCD18
CNOT1
EIF3CL
ATP2A1
NUDT21
DNAJA2
RNF40
GOT2
HP
ALDOA
GSPT1
NQO1
RPS2
UQCRC2
CORO1A
FUS
TUFM
AARS
UBE2I
NOMO3
ABAT
VAC14
GTF3C1
TRAP1
TUBB3
COTL1
USP10
KIF22
SF3B3
HAGH
GLYR1
ACSM3
ARHGAP17
EDC4
THOC6
KLHDC4
CREBBP
USP7
ZG16B
VPS35
DECR2
DDX19A
DDX19B
SRRM2
loge
MCF7
A5YKK6
B5ME19
O14983
O43809
O60884
O75150
P00505
P00738
P04075
P15170
P15559
P15880
P22695
P31146
P35637
P49411
P49588
P63279
P69849
P80404
Q08AM6
Q12789
Q12931
Q13509
Q14019
Q14694
Q14807
Q15393
Q16775
Q49A26
Q53FZ2
Q68EM7
Q6P2E9
Q86W42
Q8TBB5
Q92793
Q93009
Q96DA0
Q96QK1
Q9NUI1
Q9NUU7
Q9UMR2
Q9UQ35
Gene_id
Ramos
Protein
Accession
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
Working plan for MRM WP 2013-14
•
•
•
•
All proteins identified by SG analyses will be distributed in packages of 48 protein across 8 labs.
Analysis will be also performed by pseudoSRM in additional 8 labs.
A minimum of 25 proteins must be analysed.
Development of absolute quantitation methods.
October 2013
Nov 2013
Dec 2013
Jan 2014
Feb2014
March 2014
April2014
May 2014
Design of SRM mehods. Peptides and transitions.
Assays in biological matrices (at least 1 cell line)
•
•
Consolidation of SRM methods (1-n cell lines)
Follow up meeting (SRM-psSRM)
•
•
•
•
Validation of assays in at least 2 labs
Chr16 SRM Database
MIAPE for SRM experiments
Follow up meeting (SRM-psSRM)
48 proteins/group
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Group 7
Group 8
MRM
IDIBELL-IAS
VHIO
UV
CNB
INIBIC
UCM
FJD-HNP
CIMA-NAVB
PMRM
PCB
CSIC IBB
VHIO
UV
CNB
UPV
CIB
CNB-CBM
CIC BIOGUNE
Excell with the proteins assigned to each group will be distributed
CRG
•
•
•
•
Preliminary assays based on experimental data
Matching with SG data.
Ranking by difficulty. Troubleshooting.
Follow up meeting (SRM-psSRM)
Topics for discussion
• MIDAS, Synthetic peptides
• Acceptance criteria.
• Reporting data.
• SRM database.
• Missing/difficult proteins.
• Gene expression data for cell line selection.
• Analysis of subcellular fractions.
• Analysis of plasma.
• PTMs by SRM.
Searching for missing proteins
• Definition of the missing protein group is already made (NextProt) but it may
be worth to consider low abundance proteins which detection will be
challenging. If so, define criterion.
• Identify which tissues or cell lines should be explored to identify chr16 missing
proteins based on transcriptomic profiles and HPA evidences (for 95 chr16
missing proteins). Primary cells should be considered, in particular PBL.
• Study of secretome and body fluids.
• In some cases, subcellular fractionation or any other enrichment procedure
should be considered. The case of plasma.
• Searching on cells upon stimulation. Cell lines, type of stimuli and conditions
should be defined.
• Revise the biological function of the missing proteins, if known, to infer
potential localizations.
626 HPA antibodies
for Chr16 proteins, 95
for missing
2360 genes
840 protein coding genes
143 missing proteins
Protein expression
vectors for 64 Chr16
missing proteins
Shotgun
Proteomic
analysis
Protein profile
neXtProt v2013-10-10
ENSEMBL v73
UniProtKB v2013_09
HPA v11
120 OMIM hits
Obesity
Neurodegenerative diseases
Cancer
Coverage of 73% gene products in
Lymphoid cells
Epitelial cells
Fibroblasts
Transcriptomic
analysis
Data integration
Gene expression profile
Global and cell type specific outcomes
Correlation proteome/transcriptome
Proteome coverage and chromosome distribution
Chr16 proteome coverage
Tissue specific expression pattern of genes coding
for missing proteins. HBM
Color Key
4
6
8
Value
Heart
Ovary
Liver
Adipose
Brain
Colon
Breast
Thyroid
Adrenal
Kidney
Prostate
Lung
Testes
LymphNode
NX_Q9NNZ6_PRM3
NX_A8MZG2_C16orf90
NX_P0CG20_C16orf11
NX_Q9NQW5_PRDM7
NX_Q96A99_PTX4
NX_Q71RH2_FAM57B
NX_Q8NCV1_ADAD2
NX_A6NI56_CCDC154
NX_Q9GZS9_CHST5
NX_A1A4V9_C16orf93
NX_A6NHN6_NPIPL2
NX_Q6PL45_BRICD5
NX_Q7RTW8_OTOA
NX_Q96KJ4_MSLNL
NX_A2IDD5_CCDC78
NX_Q9NRR2_TPSG1
NX_O95177_C16orf3
NX_P17538_CTRB1
NX_Q6UXF7_CLEC18B
NX_P0CJ71_MTRNR2L4
NX_A6NF02_−
NX_A6NDY0_PABPN1L
NX_A6NKX4_SLC22A31
NX_A6NNN8_SLC38A8
NX_A8MU76_−
NX_B3SHH9_TMEM114
NX_O00634_NTN3
NX_O43749_OR1F1
NX_O75200_NPIPL1
NX_O95371_OR2C1
NX_P0CG43_FAM157C
NX_P47944_MT4
NX_P69849_NOMO3
NX_Q05996_ZP2
NX_Q6DWJ6_GPR139
NX_Q6GPI1_CTRB2
NX_Q86VD5_−
NX_Q8N6K4_−
NX_Q8NAQ8_−
NX_Q8NCF0_CLEC18C
NX_Q9NX77_−
NX_Q9P2W3_GNG13
NX_Q496H8_NRN1L
NX_Q6PRD7_CEMP1
NX_Q3KNW1_SNAI3
NX_O60359_CACNG3
NX_Q96LL3_C16orf92
NX_Q8TDN1_KCNG4
NX_O75783_RHBDL1
NX_P07438_MT1B
NX_B2RD01_CENPBD1
NX_Q6ZP98_C16orf47
NX_Q6ZRN7_−
NX_Q9BXE9_VN1R3
NX_Q7Z443_PKD1L3
NX_P78415_IRX3
NX_P78412_IRX6
NX_Q2KHN1_RNF151
NX_P0C7X3_CCNYL3
NX_H3BN30_C16orf97
NX_Q6PEW0_PRSS54
NX_Q96M29_TEKT5
NX_Q8WTQ4_C16orf78
NX_P0CG21_NHLRC4
NX_A6NJ64_−
NX_Q96M66_−
NX_Q2L4Q9_PRSS53
NX_Q96L46_CAPNS2
NX_Q99819_ARHGDIG
NX_Q8WWX8_SLC5A11
NX_Q9ULZ0_TP53TG3C
NX_Q9ULZ0_TP53TG3
NX_Q6ZVL8_−
NX_A5D8T8_CLEC18A
NX_Q96S07_PRR25
NX_O95484_CLDN9
NX_Q92617_NPIPL3
NX_Q9Y661_HS3ST4
NX_Q6ZTK2_−
NX_O95947_TBX6
NX_Q8WV35_LRRC29
NX_Q6ZNL0_−
NX_Q8TBY8_PMFBP1
NX_B4DS77_SHISA9
NX_Q6UXU4_GSG1L
NX_Q96S16_JMJD8
NX_Q9HC57_WFDC1
NX_Q86VI1_EXOC3L1
NX_Q68EN5_KIAA0895L
NX_Q2TAA8_TSNAXIP1
NX_A8MT33_SYCE1L
NX_Q3B7T3_BEAN1
NX_Q8WTX9_ZDHHC1
NX_Q96KV7_WDR90
NX_Q9H693_C16orf95
NX_Q96LX8_ZNF597
NX_O43304_SEC14L5
NX_Q8NA31_CCDC79
NX_Q8IXQ8_PDZD9
NX_Q6NT04_TIGD7
NX_Q8N446_ZNF843
NX_Q9UJH8_METRN
NX_Q6ZSR6_−
NX_A6NKP2_SDR42E2
NX_Q8N635_MEIOB
NX_Q8IZ96_CMTM1
NX_Q9UJX0_OSGIN1
NX_Q9UND3_NPIP
NX_Q8WUS8_SDR42E1
NX_Q6URK8_TEPP
NX_A6NH52_TVP23A
NX_Q8IYS4_C16orf71
NX_P17027_ZNF23
NX_Q8IZF4_GPR114
NX_Q6ZTR7_FAM92B
NX_Q17RG1_KCTD19
NX_A6NCI4_VWA3A
NX_Q6UXS0_CLEC19A
NX_Q8IY82_CCDC135
NX_Q1X8D7_LRRC36
NX_Q8TAZ6_CMTM2
NX_Q6ZN54_DEF8
NX_A8K8V0_ZNF785
NX_Q96N20_ZNF75A
NX_Q5XG92_CES4A
NX_Q6P387_C16orf46
NX_Q96MU6_ZNF778
NX_P17023_ZNF19
NX_Q9H0I3_CCDC113
NX_Q8N0W5_IQCK
NX_P61236_YPEL3
NX_Q8NHV5_C16orf52
NX_Q7Z2F6_ZNF720
NX_A8K979_ERI2
NX_Q8N339_MT1M
NX_P54851_EMP2
NX_Q8WYQ9_ZCCHC14
NX_O60304_ZNF500
NX_Q08AH1_ACSM1
NX_O75541_ZNF821
NX_O95424_DEXI
NX_Q9BV97_ZNF747
NX_P98182_ZNF200
NX_Q9NX65_ZSCAN32
WhiteBloodCells
2
SkeletalMuscle
0
B/D oriented strategy
• Targeting disease related proteins.
 Identify proteins of chr16 involved in B/D conditions within the
consortium.
 Configuration of disease related protein lists within the different B/D areas
of the SpHPP.
• Targeted monitoring of proteins of cellular pathways of particular relevance
(metabolic or signaling pathways, etc). PTMs?
• Proposals from B/D related groups
• Integration with transcriptomic, metabolomics, others.
B/D-SpHPP
Coordinator: FJ. Blanco
Biology
Biomarkers (D/P/P/T)
1ª Phase: Known Proteins
2ª Phase: Unknown Proteins
Biobanks-ISCIII
CAIBER-ISCIII
(clinical research)
Neurologic
Disorders
Cardiovascular
Diseases
Cancer
Infectious
Diseases
Obesity
Rheumatic
Diseases
Chair: A. López-Munain
Co-chair: JM. Arizmendi
Chair: L. Rguez.-Padial
Co-Chair: F. Vivanco
Chair: C. Belda
Co-Chair: I. Casal
Chair: J. Fortun
Co-Chair: C. Gil
Chair: J. Prieto
Co-Chair:F. Corrales
Chair: FJ. Blanco
Co-Chair: JP. Albar
Muscular dystrophy
Parkinson diseases
Artherosclerosis
Valvular diseases
Brain tumors
Breast cancer
Colon cancer
Candidiasis
Obesity
NAFLD
Osteoarthritis
Rheumatoid arthritis
SLE
Red
RECAVA
Data management
• Data reporting, MIAPE. A common
• SRM database
Other topics for discussion. Analytical procedures
• Standardization of LC conditions. Reference peptides for retention time
normalization.
• Acceptance criteria:
 number of peptides and transitions
 MIDAS
 Synthetic peptides for validation
• Validation across labs (2-3).
• SOPs.
Proposal for extended working plan
• SRM for SG proteins. All, according with the proposed pipeline. Results warranted.
Reduce the workload if we agree to tackle missing proteins and B/D.
• SRM for missing proteins
 Biological analysis of chr16 missing proteins (databases). Groups
 Biophysical features of chr16 missing proteins (Pino’s work).
 Identification of tissues and cell lines for missing proteins search. Transcriptomic,
HPA, others. CIMA.
 Development of SRM assays.
• SRM for B/D
 Specific proteins, drivers of disease, already described by B/D groups.
 Configuration of disease related lists of proteins (form datasets of B/D groups), any
of them from chr16?
 Analysis of all lists and identification of cellular pathways and corresponding
proteins of special interest (biological, belong to chr16, common across lists, etc.)
 Development of SRM assays.