HPP ch16CIC bioGUNE RESULTS
MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP
La Cristalera, 28th and 29th August 2012
Introduction
ensembl_gene_id external_gene_id Uniprot description
ENSG00000103248 MTHFSD B7ZLC2_HUMAN/MTHSD_HUMAN methenyltetrahydrofolate synthetase domain containing [Source:HGNC Symbol;Acc:25778]
ENSG00000103429 BFAR BFAR_HUMAN bifunctional apoptosis regulator [Source:HGNC Symbol;Acc:17613]
ENSG00000183971 NPW NPW_HUMAN neuropeptide W [Source:HGNC Symbol;Acc:30509]
ENSG00000149926 FAM57B FA57B_HUMAN family with sequence similarity 57, member B [Source:HGNC Symbol;Acc:25295]
ENSG00000205890 AC108134.1 No prot product?
ENSG00000122390 NAA60 NAA60_HUMAN N(alpha)-acetyltransferase 60, NatF catalytic subunit [Source:HGNC Symbol;Acc:25875]
CIC bioGUNE 6 unknown proteins
MCF7 cells(breast cancer)
Ramos cells(lymphoma)
CCD18 cells(colon fibroblast)
·500 ug/ 50ul (10ug/ul) ·500 ug/ 700ul (0.7ug/ul) ·700ug/25ul (28ug/ul aprox.)
MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP La Cristalera, 28th and 29th August 2012
MarkerMarker
CCD18 10ug
CCD18 25ug
CCD18 50ug Ramos
10ug Ramos 50ug
Ramos 25ug
11
23456789
10
101112131415
16
17181920212223 23
22
21
20191817161514131211
89
7
65
432
MCF-7 RAMOS & CCD18
Gels
25ug 50ug
1
23456789
1011
1312
14
15
1617
MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP La Cristalera, 28th and 29th August 2012
Workflow25ug 50ug
1
23456789
1011
13 12
14
15
1617
TrypsinResuspension in FA0.1%
LC MS
Thermo LTQ-Orbitrap XL ETD
Waters nanoAcquitySymmetry 300 C18 UPLC trap
(180 µm x 20 mm, 5 µm)BEH130 C18 column
(75 μm x 200 mm, 1.7 μm)60 min. linear gradient 3%-40% B (ACN FA0.1%)
ANALYSIS by LC-MS
ch16HUMAN MASCOT SEQUEST
cic
HPP
DATABASES SEARCH ENGINES
MarkerMarker
CCD18 10ug
CCD18 25ug
CCD18 50ug Ramos
10ug Ramos 50ug
Ramos 25ug
11
23456789
10
101112131415
16
17181920212223 23
22
21
20191817161514131211
89
7
65
432
Ramos(10ug)
MCF7(50ug)
MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP La Cristalera, 28th and 29th August 2012
1st: DDAMCF7 1-17
Decoy (FDR<1%)
Exclusion lists 2nd: Excl listsexcMCF7 1-17
ch16
cic
HPP
MASCOT
HUMAN
Results MCF7: MASCOT searches
MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP La Cristalera, 28th and 29th August 2012
1031
58 0
2742
118 0
vs.HUMAN vs. Ch16 vs. HPP7
MCF7 cellsProts Pepts
HPP
Results MCF7: SEQUEST searches
1st: DDAMCF7 1-17
No Decoy
Exclusion lists 2nd: Excl listsexcMCF7 1-17
SEQUEST
Description ΣCoverage Prots. Unique pepts. Pepts. PSMsNPW_HUMAN 42.42 1 2 8 26
Sequence PSM Ambiguity Modifications Activation Type XCorr Ion Inject Time [ms] Charge m/z [Da] MH+ [Da] ΔM [ppm] RT [min] First Scan Spectrum FileAAGLLMGLR Unambiguous CID 1.55 100 2 451.27 901.53 4.88 13.49 1035 14_HPP7.RAWAAGLLMGLR Unambiguous CID 1.27 100 2 451.27 901.53 4.61 12.49 910 14_HPP7.RAW
DTLSPEPAAR Unambiguous CID 1.14 100 2 528.77 1056.53 2.39 41.90 4351 14_HPP7.RAWAAGLLMGLR Unconsidered CID 0.87 100 2 451.27 901.53 4.34 16.65 1306 13_HPP6.RAWMAWRPGER Unconsidered CID 0.87 87 2 501.75 1002.49 -2.35 12.15 845 22_HPP15.RAWAAGLLMGLR Unconsidered CID 0.85 100 2 451.27 901.53 3.53 12.44 901 13_HPP6.RAW
RDVSRPAVDPAANR Unconsidered CID 0.84 100 2 762.40 1523.80 -2.43 51.67 5586 14_HPP7.RAWDTLSPEPAAR Unconsidered CID 0.80 24 2 528.77 1056.54 3.43 11.81 815 14_HPP7.RAWAAGLLmGLR Unconsidered M6(Oxidation) CID 0.74 100 2 459.27 917.53 4.28 27.84 2269 22_HPP15.RAW
LGLPcLAPGPF Unconsidered C5(Carbamidomethyl) CID 0.68 100 2 571.31 1141.61 1.90 39.02 3659 22_HPP15.RAWLGLPcLAPGPF Unconsidered C5(Carbamidomethyl) CID 0.68 100 2 571.31 1141.61 1.90 39.73 3542 21_HPP14.RAWAAGLLMGLR Unconsidered CID 0.67 100 2 451.27 901.53 4.54 19.20 1669 14_HPP7.RAWAAGLLMGLR Unconsidered CID 0.67 100 2 451.27 901.53 0.55 15.64 1291 14_HPP7.RAWAAGLLmGLR Unconsidered M6(Oxidation) CID 0.66 100 2 459.27 917.53 4.88 27.19 2196 22_HPP15.RAWAAGLLMGLR Unconsidered CID 0.66 100 2 451.27 901.53 4.61 34.78 2991 13_HPP6.RAWAAGLLMGLR Unconsidered CID 0.61 100 2 451.27 901.53 4.00 15.60 1219 13_HPP6.RAWRSPYLWR Unconsidered CID 0.60 100 2 489.27 977.53 -1.80 47.88 5056 24_HPP17.RAW
RRSSQAGIPVR Unconsidered CID 0.57 21 2 613.86 1226.71 2.09 11.65 755 11_HPP4.RAWAAGLLMGLR Unconsidered CID 0.55 100 2 451.27 901.53 2.37 20.30 1776 14_HPP7.RAW
AAGLLMGLRR Unconsidered CID 0.53 100 2 529.32 1057.63 -2.92 12.06 913 24_HPP17.RAWAAGLLMGLR Unconsidered CID 0.52 100 2 451.27 901.53 4.47 16.65 1408 14_HPP7.RAW
LGLPcLAPGPF Unconsidered C5(Carbamidomethyl) CID 0.50 100 2 571.31 1141.61 -0.45 42.82 4000 13_HPP6.RAWAAGLLMGLRR Unconsidered CID 0.50 100 2 529.32 1057.63 -3.50 30.56 2472 13_HPP6.RAWAAGLLmGLR Unconsidered M6(Oxidation) CID 0.47 100 2 459.26 917.52 -4.96 42.89 4008 8_HPP1.RAW
LGLPcLAPGPF Unconsidered C5(Carbamidomethyl) CID 0.47 100 2 571.31 1141.61 1.90 38.20 3873 14_HPP7.RAWAAGLLMGLRR Unconsidered CID 0.45 100 2 529.32 1057.63 -4.42 42.45 3681 9_HPP2.RAW
Description ΣCoverage Prots. Unique pepts. Pepts. PSMsMTHSD_HUMAN 6.28 1 1 2 2
Sequence PSM Ambiguity Modifications Activation Type XCorr Ion Inject Time [ms] Charge m/z [Da] MH+ [Da] ΔM [ppm] RT [min] First Scan Spectrum FileMEPRAGVSKQDIR Unambiguous CID 1.61 100 3 496.26 1486.78 -2.76 21.21 1914 4_HPP7_4_excl.RAW
NYSVPIGLDSR Unconsidered CID 0.43 100 3 407.55 1220.63 4.92 21.02 1891 4_HPP7_4_excl.RAW
Description ΣCoverage Prots. Unique pepts. Pepts. PSMsNPW_HUMAN 42.42 1 2 8 26
Sequence PSM Ambiguity Modifications Activation Type XCorr Ion Inject Time [ms] Charge m/z [Da] MH+ [Da] ΔM [ppm] RT [min] First Scan Spectrum FileAAGLLMGLR Unambiguous CID 1.55 100 2 451.27 901.53 4.88 13.49 1035 14_HPP7.RAWAAGLLMGLR Unambiguous CID 1.27 100 2 451.27 901.53 4.61 12.49 910 14_HPP7.RAW
DTLSPEPAAR Unambiguous CID 1.14 100 2 528.77 1056.53 2.39 41.90 4351 14_HPP7.RAWAAGLLMGLR Unconsidered CID 0.87 100 2 451.27 901.53 4.34 16.65 1306 13_HPP6.RAWMAWRPGER Unconsidered CID 0.87 87 2 501.75 1002.49 -2.35 12.15 845 22_HPP15.RAWAAGLLMGLR Unconsidered CID 0.85 100 2 451.27 901.53 3.53 12.44 901 13_HPP6.RAW
RDVSRPAVDPAANR Unconsidered CID 0.84 100 2 762.40 1523.80 -2.43 51.67 5586 14_HPP7.RAWDTLSPEPAAR Unconsidered CID 0.80 24 2 528.77 1056.54 3.43 11.81 815 14_HPP7.RAWAAGLLmGLR Unconsidered M6(Oxidation) CID 0.74 100 2 459.27 917.53 4.28 27.84 2269 22_HPP15.RAW
LGLPcLAPGPF Unconsidered C5(Carbamidomethyl) CID 0.68 100 2 571.31 1141.61 1.90 39.02 3659 22_HPP15.RAWLGLPcLAPGPF Unconsidered C5(Carbamidomethyl) CID 0.68 100 2 571.31 1141.61 1.90 39.73 3542 21_HPP14.RAWAAGLLMGLR Unconsidered CID 0.67 100 2 451.27 901.53 4.54 19.20 1669 14_HPP7.RAWAAGLLMGLR Unconsidered CID 0.67 100 2 451.27 901.53 0.55 15.64 1291 14_HPP7.RAWAAGLLmGLR Unconsidered M6(Oxidation) CID 0.66 100 2 459.27 917.53 4.88 27.19 2196 22_HPP15.RAWAAGLLMGLR Unconsidered CID 0.66 100 2 451.27 901.53 4.61 34.78 2991 13_HPP6.RAWAAGLLMGLR Unconsidered CID 0.61 100 2 451.27 901.53 4.00 15.60 1219 13_HPP6.RAWRSPYLWR Unconsidered CID 0.60 100 2 489.27 977.53 -1.80 47.88 5056 24_HPP17.RAW
RRSSQAGIPVR Unconsidered CID 0.57 21 2 613.86 1226.71 2.09 11.65 755 11_HPP4.RAWAAGLLMGLR Unconsidered CID 0.55 100 2 451.27 901.53 2.37 20.30 1776 14_HPP7.RAW
AAGLLMGLRR Unconsidered CID 0.53 100 2 529.32 1057.63 -2.92 12.06 913 24_HPP17.RAWAAGLLMGLR Unconsidered CID 0.52 100 2 451.27 901.53 4.47 16.65 1408 14_HPP7.RAW
LGLPcLAPGPF Unconsidered C5(Carbamidomethyl) CID 0.50 100 2 571.31 1141.61 -0.45 42.82 4000 13_HPP6.RAWAAGLLMGLRR Unconsidered CID 0.50 100 2 529.32 1057.63 -3.50 30.56 2472 13_HPP6.RAWAAGLLmGLR Unconsidered M6(Oxidation) CID 0.47 100 2 459.26 917.52 -4.96 42.89 4008 8_HPP1.RAW
LGLPcLAPGPF Unconsidered C5(Carbamidomethyl) CID 0.47 100 2 571.31 1141.61 1.90 38.20 3873 14_HPP7.RAWAAGLLMGLRR Unconsidered CID 0.45 100 2 529.32 1057.63 -4.42 42.45 3681 9_HPP2.RAW
Description ΣCoverage Prots. Unique pepts. Pepts. PSMsMTHSD_HUMAN 6.28 1 1 2 2
Sequence PSM Ambiguity Modifications Activation Type XCorr Ion Inject Time [ms] Charge m/z [Da] MH+ [Da] ΔM [ppm] RT [min] First Scan Spectrum FileMEPRAGVSKQDIR Unambiguous CID 1.61 100 3 496.26 1486.78 -2.76 21.21 1914 4_HPP7_4_excl.RAW
NYSVPIGLDSR Unconsidered CID 0.43 100 3 407.55 1220.63 4.92 21.02 1891 4_HPP7_4_excl.RAW
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MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP La Cristalera, 28th and 29th August 2012
Results MCF7: NPW_HUMANAAGLLMGLR
DLTSPEPAAR
Vs. ch16
Vs. ch16
SISGPISK (Lanosterol synthase)
DKFSFDLGK (Peptidyl-prolyl cis-trans isomerase)
Inclusion lists – NO HPP HIT
MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP La Cristalera, 28th and 29th August 2012
Scan event 1035
Scan event 4351
Scan event 1035
Scan event 4351
1st: DDARAMOS 1-23
Decoy (FDR<1%)
ch16
cic
HPP
MASCOT
HUMAN
Results RAMOS: MASCOT searches
1204
61 0
2979
141 0
vs.HUMAN vs. Ch16 vs. HPP7
Ramos cellsProts Pepts
1204
61 0
2979
141 0
vs.HUMAN vs. Ch16 vs. HPP7
Ramos cellsProts Pepts
MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP La Cristalera, 28th and 29th August 2012
Results: HPP target+decoy searches
1276
85
1473
8159 4 50 9
vs.HUMAN vs. Ch16 vs.HUMAN vs. Ch16
MCF7 RAMOS
ProteinsProts FWD Prots Decoy
3650
214
4106
25259 4 52 9
vs.HUMAN vs. Ch16 vs.HUMAN vs. Ch16
MCF7 RAMOS
PeptidesPepts FWD Pepts Decoy
MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP La Cristalera, 28th and 29th August 2012
Mascot No decoy (p<0.05)
Summary
1-Protein quant issue
2-No HPP proteins identified:
*Only NPW_HUMAN was identified – false positive
*Different approaches?
Inclusion lists???...
MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP La Cristalera, 28th and 29th August 2012
1st: DDA
MCF7 1-17
vs. ch16 DB
vs. HUMAN
incMCF7
Workflow (2): MCF7 cells
3rd: Incl lists
Inclusion lists
Exclusion lists 2nd: Excl lists
excMCF7 1-17
vs. ch16 DB
vs. HUMAN
vs. ch16 DB vs. HUMAN
ch16 HUMAN
MASCOT SEQUEST
S
M
S
M
S M S M
Acquisition
Confirmation
HPP7
DATABASES SEARCH ENGINES
Results: Exclusion lists
DDA Exclist DDA Exclist
Vs. Chr16 Vs. HUMAN
NO HACER COMPARATIVA, NO MERECE LA PENA, SOLO CITAR NUMEROS TOTALES CH16 Y HUMAN
MCF7 cells
ensembl_gene_id external_gene_id Uniprot description
ENSG00000103248 MTHFSD B7ZLC2_HUMAN/MTHSD_HUMAN methenyltetrahydrofolate synthetase domain containing [Source:HGNC Symbol;Acc:25778]
ENSG00000103429 BFAR BFAR_HUMAN bifunctional apoptosis regulator [Source:HGNC Symbol;Acc:17613]
ENSG00000183971 NPW NPW_HUMAN neuropeptide W [Source:HGNC Symbol;Acc:30509]
ENSG00000149926 FAM57B FA57B_HUMAN family with sequence similarity 57, member B [Source:HGNC Symbol;Acc:25295]
ENSG00000205890 AC108134.1 No prot product?
ENSG00000122390 NAA60 NAA60_HUMAN N(alpha)-acetyltransferase 60, NatF catalytic subunit [Source:HGNC Symbol;Acc:25875]
CIC bioGUNE 6 unknown proteins
MCF7 cells
·500 ug/ 50ul (10ug/ul)
MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP La Cristalera, 28th and 29th August 2012
Results (1): MASCOT searches
1031
2742
58 1180 0
Proteins Peptides
MCF7 cellsvs HUMAN vs ch16 vs HPP7
High confidence hits (FDR<1%)Mascot vs ch16 & HUMAN DB.
HPP hits = 0
MCF7 1 - 17
1st ACQUISITION
Survey (30000 FWHM) DDA CID 6 most intense
MS/MS trigger 1000 counts
Vs. HPP DB
Vs. Ch16 DB
Vs. Human DB
2nd ACQUISITION: Exclusion lists
Survey (30000 FWHM) DDA CID 6 most intense.
MS/MS trigger 400 counts
3rd ACQUISITION: Inclusion lists
Survey (30000 FWHM) Inclusion list
MS/MS trigger 400 counts
Workflow (2)Incl lists
Excl lists
Searches & Peptide listsSequest Relaxed searches
·No decoy
·High & Medium confidence hits
·Define inclusion lists
Mascot stringent searches
·Decoy
·High confidence hits (FDR<1%)
·Define protein Ids & exclusion lists
1st: DDA
MCF7 1-17
vs. HPP7 DB
vs. HUMAN
incMCF7
Workflow (3): HPP7 DB
3rd: Incl lists
Inclusion lists
Exclusion lists 2nd: Excl lists
excMCF7 1-17
vs. HPP7 DB
vs. HUMAN
vs. ch16 DB vs. HUMAN
ch16 HUMAN
SEQUESTMASCOT
S
M
S
M
S M S M
Acquisition
Confirmation
HPP7
Results (2): SEQUEST searchesHigh, med and low confidence hits (no decoy) vs. HPP7 DB
Description ΣCoverage Prots. Unique pepts. Pepts. PSMsNPW_HUMAN 42.42 1 2 8 26
Sequence PSM Ambiguity Modifications Activation Type XCorr Ion Inject Time [ms] Charge m/z [Da] MH+ [Da] ΔM [ppm] RT [min] First Scan Spectrum FileAAGLLMGLR Unambiguous CID 1.55 100 2 451.27 901.53 4.88 13.49 1035 14_HPP7.RAWAAGLLMGLR Unambiguous CID 1.27 100 2 451.27 901.53 4.61 12.49 910 14_HPP7.RAW
DTLSPEPAAR Unambiguous CID 1.14 100 2 528.77 1056.53 2.39 41.90 4351 14_HPP7.RAWAAGLLMGLR Unconsidered CID 0.87 100 2 451.27 901.53 4.34 16.65 1306 13_HPP6.RAWMAWRPGER Unconsidered CID 0.87 87 2 501.75 1002.49 -2.35 12.15 845 22_HPP15.RAWAAGLLMGLR Unconsidered CID 0.85 100 2 451.27 901.53 3.53 12.44 901 13_HPP6.RAW
RDVSRPAVDPAANR Unconsidered CID 0.84 100 2 762.40 1523.80 -2.43 51.67 5586 14_HPP7.RAWDTLSPEPAAR Unconsidered CID 0.80 24 2 528.77 1056.54 3.43 11.81 815 14_HPP7.RAWAAGLLmGLR Unconsidered M6(Oxidation) CID 0.74 100 2 459.27 917.53 4.28 27.84 2269 22_HPP15.RAW
LGLPcLAPGPF Unconsidered C5(Carbamidomethyl) CID 0.68 100 2 571.31 1141.61 1.90 39.02 3659 22_HPP15.RAWLGLPcLAPGPF Unconsidered C5(Carbamidomethyl) CID 0.68 100 2 571.31 1141.61 1.90 39.73 3542 21_HPP14.RAWAAGLLMGLR Unconsidered CID 0.67 100 2 451.27 901.53 4.54 19.20 1669 14_HPP7.RAWAAGLLMGLR Unconsidered CID 0.67 100 2 451.27 901.53 0.55 15.64 1291 14_HPP7.RAWAAGLLmGLR Unconsidered M6(Oxidation) CID 0.66 100 2 459.27 917.53 4.88 27.19 2196 22_HPP15.RAWAAGLLMGLR Unconsidered CID 0.66 100 2 451.27 901.53 4.61 34.78 2991 13_HPP6.RAWAAGLLMGLR Unconsidered CID 0.61 100 2 451.27 901.53 4.00 15.60 1219 13_HPP6.RAWRSPYLWR Unconsidered CID 0.60 100 2 489.27 977.53 -1.80 47.88 5056 24_HPP17.RAW
RRSSQAGIPVR Unconsidered CID 0.57 21 2 613.86 1226.71 2.09 11.65 755 11_HPP4.RAWAAGLLMGLR Unconsidered CID 0.55 100 2 451.27 901.53 2.37 20.30 1776 14_HPP7.RAW
AAGLLMGLRR Unconsidered CID 0.53 100 2 529.32 1057.63 -2.92 12.06 913 24_HPP17.RAWAAGLLMGLR Unconsidered CID 0.52 100 2 451.27 901.53 4.47 16.65 1408 14_HPP7.RAW
LGLPcLAPGPF Unconsidered C5(Carbamidomethyl) CID 0.50 100 2 571.31 1141.61 -0.45 42.82 4000 13_HPP6.RAWAAGLLMGLRR Unconsidered CID 0.50 100 2 529.32 1057.63 -3.50 30.56 2472 13_HPP6.RAWAAGLLmGLR Unconsidered M6(Oxidation) CID 0.47 100 2 459.26 917.52 -4.96 42.89 4008 8_HPP1.RAW
LGLPcLAPGPF Unconsidered C5(Carbamidomethyl) CID 0.47 100 2 571.31 1141.61 1.90 38.20 3873 14_HPP7.RAWAAGLLMGLRR Unconsidered CID 0.45 100 2 529.32 1057.63 -4.42 42.45 3681 9_HPP2.RAW
Description ΣCoverage Prots. Unique pepts. Pepts. PSMsMTHSD_HUMAN 6.28 1 1 2 2
Sequence PSM Ambiguity Modifications Activation Type XCorr Ion Inject Time [ms] Charge m/z [Da] MH+ [Da] ΔM [ppm] RT [min] First Scan Spectrum FileMEPRAGVSKQDIR Unambiguous CID 1.61 100 3 496.26 1486.78 -2.76 21.21 1914 4_HPP7_4_excl.RAW
NYSVPIGLDSR Unconsidered CID 0.43 100 3 407.55 1220.63 4.92 21.02 1891 4_HPP7_4_excl.RAW
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