HPPPreliminary ResultsLa Cristalera, 28-29 August 2012

Montserrat Carrascal, Joan Villanueva, Joaquín AbiánLP-CSIC/UAB

M. Carrascal 2011

J. AbiánENSG00000067365 METTL22 methyltransferase like 22 [Source:HGNC Symbol;Acc:28368]ENSG00000179583 CIITA class II, major histocompatibility complex, transactivator [Source:HGNC Symbol;Acc:7067]ENSG00000182601 HS3ST4 heparan sulfate (glucosamine) 3-O-sulfotransferase 4 [Source:HGNC Symbol;Acc:5200]ENSG00000140943 MBTPS1 membrane-bound transcription factor peptidase, site 1 [Source:HGNC Symbol;Acc:15456]ENSG00000178226 PRSS36 protease, serine, 36 [Source:HGNC Symbol;Acc:26906]ENSG00000197774 EME2 essential meiotic endonuclease 1 homolog 2 (S. pombe) [Source:HGNC Symbol;Acc:27289]

Protein share out : LP-CSIC/UAB

TARGET PEPTIDE SELECTION: Peptide Siever: list of potential peptides. Cys, Met and missed cleavage are eliminated. Other scores are calculated: STEPP and PeptideDetectabilityPredictor. Average of Peptide Siever, STEPP and PeptideDetectabilityPredictor. Filtering at 0.75 or 4 peptides/protein.

M.Carrascal 2012

M. Carrascal 2011

TARGET IONS

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SAMPLE PREPARATION:

Half of the MCF-7 and CDC-18 samples (aprox. 250 µg protein) were fractionated using the Offgel system (Agilent).12 fractions were obtained and digested with FASP.

INSTRUMENTAL ANALYSIS: The resulting extracts were injected in 2 different LC/MS systems

ANALYSIS VELOS:1/6 of each extract was injected.HPLC gradient 120 minMS programming: 1 MS followed by 30 Targeted MS/MS

ANALYSIS ORBITRAP:1/6 of each extract was injected.HPLC gradient 120 minMS programming: 1 MS followed by 10 Data Dependent MS/MS from inclusion listMS were obtained with FT at resolution 100000, MS/MS were obtained with IT

EXPERIMENTAL DESIGN

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TARGETED MS/MS : LTQ VELOS

Sample VH: MCF-7MS traces HPP_VallHebron_MRMvelos_Test1_120719_... 20-Jul-12 12:57:59

RT: 0.00 - 130.11

0 20 40 60 80 100 120Time (min)

0

10

20

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60

70

80

90

100

0

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Rel

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bund

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496041.23

426436.87

522142.86

664251.77

330730.84

699053.96

722255.423220

30.30762857.99

844063.13

16647115.252263

24.261061576.98

15545108.28

14559102.05

69714.40

443837.97

438037.60

377133.78 6555

51.22464139.23 7193

55.22319130.13

261126.472263

24.28820861.65

933968.85

989072.36

1139881.98

14240100.13

15139105.87

16647115.49

NL: 1.75E7TIC F: ITMS + p NSI E Full ms [400.00-1800.00] MS HPP_VallHebron_MRMvelos_Test1_120719_Fr07_07

NL: 3.66E7TIC F: ITMS + p NSI E Full ms [400.00-1800.00] MS HPP_VallHebron_MRMvelos_Test1_120719_Fr08_08

Offgel Fr6

Offgel Fr7

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DATA DEPENDENT WITH INCLUSION LIST: LTQ-ORBITRAP

RT: 0.00 - 140.02

0 20 40 60 80 100 120 140Time (min)

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Rel

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bund

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185641.90

256753.80

269255.50 7533

136.19

345868.15

241251.34

414980.00

439283.91

530198.67

173439.81

6742122.97

6210114.05

90525.72

40717.09

7635136.17

277055.79

261653.80 3374

64.60 358367.932495

52.20186942.36 3766

71.01 541898.504522

83.95157137.08 6832

122.716285

113.47106528.39 4819

88.7330815.32

NL: 2.17E9TIC F: FTMS + p NSI Full ms [400.00-1800.00] MS HPP_VallHebron_DDPorbi_Test1_120724_Fr06_06

NL: 9.66E8TIC F: FTMS + p NSI Full ms [400.00-1800.00] MS HPP_VallHebron_DDPorbi_Test1_120724_Fr07_07

Offgel Fr6

Offgel Fr7

Sample VH: MCF-7MS traces

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ANALYSIS VELOS:

1.- The resulting RAW files were searched using Proteome Discoverer v1.3 against a database including only the 6 targeted proteins. Precursor tolerance 2Da. Product tolerance 0.8Da

2.- Matches with a Xcorr < 1.8 were discarded. Matches with Xcorr>1.8 were checked manually.

DATA ANALYSIS

ANALYSIS ORBITRAP: the RAW data was processed twice:

METHOD ORBITRAP 1:The RAW data was searched against a database with 6 proteins (MS tolerance of 10ppm).RESULT METHOD ORBITRAP 1: No hits were returned by the Proteome Discoverer software.

METHOD ORBITRAP 2:The targeted MS data was revised manually :

1.- Fragmentograms for each proteotypic peptide were obtained at z=1, 2, 3 and 4.To ensure selectivity resolution was reduced to 10 ppm

2.- Intense LCMS peaks or small peaks observed in two of the 4 fragmentograms were manually checked:- have the expected z values

- Is not related to isotopic contamination of other peptides

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CHROMOSOME 16 PROTEIN DESCRIPTION

Target peptides undetected

OffGel(12 fractions)

FASP digestion

LC-MS/MS

Protein DiscoveryManual inspection

Low amount of target proteins

Proteins not expressed in these cells

ensembl_gene_id external_gene_id Entry Protein.existenceENSG00000179583 CIITA P33076 Evidence at protein level

ensembl_gene_id external_gene_id atlas_celltypeENSG00000179583 CIITA CD56+ NK cell

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CHROMOSOME 16 PROTEIN DESCRIPTION

A1A5D9RDSFLGGGPGPEEPEDLALQLQQK(0)

O14874S(0)TSAT(1)DTHHVEMAR

O15553KLS(1)LSEGKMRPRS(1)LEVTISTGEKS(0)ST(1)PPRQS(1)PSRT(0)RS(1)PALPGGRTRS(1)PALPGGR

O43169F(0)LNEHPGGEEVLLEQAGVDASESFEDVGHSSDARFLNEHPGGEEVLLEQAGVDASESFEDVGHSSDAR

O43237C(0)RS(1)PGMLEPLGSSRCRS(1)PGMLEPLGSSRM(0)ALPPQEDATAS(1)PPRQK(0)M(0)TSERAPS(1)PSSRQ(0)PLLLS(1)EDEEDTK(0)RR(0)RET(1)PS(1)PRPMRRPT(1)LTTFFGRRSPS(1)PPEACRS(0)GT(1)PPRQGSITSPQANEQSVTPQRYLLGDAPVS(1)PSSQK

O43809L(0)PGGELNPGEDEVEGLK(0)R

O75113KQFS(1)LENVQEGEILHDAKQGS(1)LAMEELKR(0)FS(1)DSEER

O76021LGVSVS(1)PSRQ(0)GS(1)LAMEELK(0)SPSPPT(1)QHTGQPPGQPSAPSQLSAPR

UNIPROT2012_07

Chrom16.txt

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CHROMOSOME 16 PROTEIN DESCRIPTION

Peptides and proteins from T-CELLS in chromosome 16

Non-redundant peptides 421Phosphorylated 172

Non-phosphorylated 249

Unique peptides 265Phosphorylated 122

Non-phosphorylated 163Total proteins 56

Phosphoproteins 25

Peptides and proteins from PLASMA in chromosome 16

Non-redundant peptides 46Phosphorylated 10

Non-phosphorylated 36

Unique peptides 35Phosphorylated 8

Non-phosphorylated 27Total proteins 16

Phosphoproteins 8

M.Carrascal 2012

Review of other studies (different cells or tissues).

Creation of a database of spectra for MRM experiments.

M. Carrascal 2011M.Carrascal 2012

SEProt 2013 AND HPP

M. Carrascal 2011M.Carrascal 2012