HPP ch16 CIC bioGUNE RESULTS MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP

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


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)


1st: DDAMCF7 1-17

Decoy (FDR<1%)

Exclusion lists 2nd: Excl listsexcMCF7 1-17

ch16

cic

HPP

MASCOT

HUMAN

Results MCF7: MASCOT searches


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













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Results MCF7: NPW_HUMANAAGLLMGLR

DLTSPEPAAR

Vs. ch16

Vs. ch16

SISGPISK (Lanosterol synthase)

DKFSFDLGK (Peptidyl-prolyl cis-trans isomerase)

Inclusion lists – NO HPP HIT


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


Ramos cellsProts Pepts

1204

61 0

2979

141 0


Ramos cellsProts Pepts


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


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???...


NPW_HUMAN

Swissprot

TrEMBL

Results (7): MTHSFD_HUMANMEPRAGVSKQDIR

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)


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.


3rd ACQUISITION: Inclusion lists

Survey (30000 FWHM) Inclusion list


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













HPP ch16 CIC bioGUNE RESULTS MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP

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