hpp preliminary results la cristalera , 28-29 august 2012
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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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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
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
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)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
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
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