hpp preliminary results la cristalera, 28-29 august 2012 montserrat carrascal, joan villanueva,...
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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.
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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
M.Carrascal 2012
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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
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lativ
e A
bu
nd
an
ce
496041.23
426436.87
522142.86
664251.77
330730.84
699053.96
722255.423220
30.30762857.99
844063.13
16647115.25
226324.26
1061576.98
15545108.28
14559102.05
69714.40
443837.97
438037.60
377133.78 6555
51.22464139.23 7193
55.22319130.13
261126.47
226324.28
820861.65
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)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lativ
e A
bu
nd
an
ce
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 541898.50
452283.95
157137.08 6832
122.716285
113.47106528.39
481988.73308
15.32
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 Discovery
Manual 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
M.Carrascal 2012
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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
UNIPROT
2012_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
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