PROFILING THE CHROMOSOME 16 BY HIGH-RESOLUTION DATA DEPENDENT MS:
EXTRACTION/FRACTIONATION METHOD EVALUATION IN JURKAT CELLS.
Spanish Human Proteome ProjectConsortium Chromosome 16
III Workshop of the Chromosome 16 ConsortiumDecember 2012, La Cristalera, Miraflores de la Sierra, Madrid
INDEX
METHODOLOGY
Methanol/Chloroform precipitationIn-solution digestionOff-line HPLC-RP Basic pH
5600-Ttof (CID)
Cut bandsAutomatic In-gel digestion
Digestor (Bruker)
30 fractions / pooled into 10
HPLC-RP Acid pH
15 bands
Jurkat T cells
1D-SDS PAGE
RIPA CHAPS/UREA 4% SDS
METHODOLOGY
6 Experimentsx
2 biological replicates
TOTAL PROTEOME COVERAGE
FDR 1% Protein level
92278 peptides12159 proteins
TOTAL PROTEINS / PEPTIDES IDENTIFIED
R.1 R.1 R.1 R.1 R.1 R.1R.2 R.2 R.2 R.2 R.2 R.2
CHAPS CHAPSRIPA RIPA4% SDS 4% SDS
1D SDS-PAGE HPLC-RP
TOTAL PROTEOME COVERAGE
PROTEIN OVERLAPPING BETWEEN REPLICATES
1415 3755 1456
CHAPS 1D SDS-PAGE
Replicate 1Replicate 2
67211001 2098
CHAPS HPLC-RP
Replicate 1Replicate 2
TOTAL PROTEOME COVERAGE
Good overlapping between replicate experiments for the Gel or off-line basicRP prefractionation methods.
COMPARING FRACTIONATION METHODS
4737
CHAPS – Replicate 2
474 4082
1D SDS-PAGEHPLC-RP
5350905 2449
RIPA – Replicate 2
1D SDS-PAGEHPLC-RP
PROTEIN OVERLAPPING BETWEEN FRACTIONATION METHODS
4% SDS – Replicate 2
4794871 2431
1D SDS-PAGEHPLC-RP
COMPARING FRACTIONATION METHODS
For all the cell-lysing conditions tested, the in-solution digestion-basicRP-LC/MS workflow was by far the most compatible (between 20-30% more proteins identified).
R.1 R.1 R.1 R.1 R.1 R.1R.2 R.2 R.2 R.2 R.2 R.2
CHAPS CHAPSRIPA RIPA4% SDS 4% SDS
1D SDS-PAGE HPLC-RP
More exclusive proteins
identified by HPLC-RP
COMPARING FRACTIONATION METHODS
NUMBER OF EXCLUSIVE PROTEINS PER EXPERIMENT
COMPARING CELL-LYSING CONDITIONS
COMPARING PROTEIN EXTRACTION METHODS
HPLC-RP1D SDS-PAGE
CHAPS/ UREA
RIPA
4% SDS
TOTAL PROTEINS / PEPTIDES
2 Replicates
Different cell-lysis conditions gave similar proteome coverage in the Gel-LC-MS workflow. CHAPS lysis enabled greater protein identification in the basic-RP-LC/MS workflow. The excess of detergents that would alter protein precipitation and digestion.
More exclusive proteins identified by CHAPS lysis combined with in-solution digestion/off-line HPLC separation.
PROTEIN OVERLAPPING BETWEEN EXTRACTION METHODS
5163
492
637586
1D SDS-PAGE HPLC-RP
1338
546
614
6386
2 Replicates
COMPARING PROTEIN EXTRACTION METHODS
CHROMOSOME 16 COVERAGE
CHROMOSOME 16 COVERAGE
CHROMOSOME 16 PROTEINS/PEPTIDES
4000 péptidos447 proteínas
351 genes
R.1 R.1 R.1 R.1 R.1 R.1R.2 R.2 R.2 R.2 R.2 R.2
CHAPS CHAPSRIPA RIPA4% SDS 4% SDS
1D SDS-PAGE HPLC-RP
Chr 16 PROTEINS: SUBCELLULAR LOCALIZATION
CHAPS lysis was better in recovering most sub-cellular compartments even for membrane proteins. However, our ability to identify membrane proteins is low and we should considerate using plasma membrane enrichment methods (subcellular fractionation, cell-surface biotinylation…)
CHROMOSOME 16 COVERAGE
PIKE
MAPPINGPOST-TRANSLATIONAL MODIFICATIONS OF THE CHR 16
MAPPING POST-TRANSLATIONAL MODIFICATIONS OF THE CHR 16
ACETYLATION PHOSPHORYLATION
2,26
3,33
2,35
2,85
2,00
4,01
2,23 2,38 2,53
3,002,58
3,01
0,00
0,50
1,00
1,50
2,00
2,50
3,00
3,50
4,00
4,50
1 2 3 4 5 6 7 8 9 10 11 12
% o
f ace
tyla
ted
pep
tid
es
Chr 16: % of acetylated peptides
R.1 R.1 R.1 R.1 R.1 R.1R.2 R.2 R.2 R.2 R.2 R.2
CHAPS CHAPSRIPA RIPA4% SDS 4% SDS
1D SDS-PAGE HPLC-RP
TOTAL:1317 P-Peptides / 865 P-ProteinsChr 16: 66 P-Peptides / 42 P-Proteins
Jurkat CHAPS HPLC-RP Replicate 2
Average of 3% of peptides found acetylated
Chr 16:56 peptides N-term acetylated
Can we use alternative strategies to complement this coverage of Chr16 PTMs?
ISSUE OF ISOFORMS• Test of Protein Grouping
FDR 1% Protein level
Replicate 2 Jurkat
- - - - - -PG PG PG PG PG PG
CHAPS CHAPSRIPA RIPA4% SDS 4% SDS
1D SDS-PAGE HPLC-RP
- - - - - -PG PG PG PG PG PG
CHAPS CHAPSRIPA RIPA4% SDS 4% SDS
1D SDS-PAGE HPLC-RP
TOTAL Chr 16
TOTAL/Chr 16 PROTEINS AND PEPTIDES
TEST OF PROTEIN GROUPING
IMPORTANTE: Exportar datos siempre de la misma forma
COMPARING MASS SPECTROMETERS
MASS SPECTROMETER: Triple ToF 5600 (CNB) vs. Q-Exactive (UPV)
CHAPS/UREA
10 Fractions
FDR 1% Protein level
Rep 1CNB
Rep 1UPV
Rep 2CNB
Both mass spectrometers are comparable
Rep 1CNB
Rep 1UPV
Rep 2CNB
TOTAL Chr 16
TOTAL/Chr 16 PROTEINS AND PEPTIDES Jurkat CHAPS HPLC-RP
MASS SPECTROMETER: Triple ToF 5600 (CNB) vs. Q-Exactive (UPV)
9723
1218
532
1315
Replicate 1 - CNBReplicate 1 - UPVReplicate 2 - CNB
PROTEIN OVERLAPPING Jurkat CHAPS HPLC-RP
MASS SPECTROMETER: Triple ToF 5600 (CNB) vs. Q-Exactive (UPV)
FDR 1% Protein level
4346
11
345
Replicate 1 - CNBReplicate 1 - UPVReplicate 2 - CNB
TOTAL Chr 16
Reproducibility
SUMMARY We have tested various workflows to increase our coverage of the Chr16.
We have used strong detergents to better solubilize and resolve membrane proteins and show that due to the low compatibility with in-solution digestion, proteins were not so efficiently recovered.
Our observation was that CHAPS cell-lysis coupled to basic-RP-LC/MS provided the best results.
We are combining various approaches to gain more insight and coverage of proteins of low solubility and their post-translational modification profiles:• Cell-surface biotinylation• Phosphopeptide enrichment
We are generating an increasing number of mass spectras and we will build an MS/MS library that will hopefully be used for MRM validations.
ACKNOWLEDGEMENTS
Adán AlpízarSilvia Juárez
Sergio CiordiaMarisol
Fernández
Rosana NavajasSeverine GharbiMiguel Marcilla
Alberto ParadelaCarmen González
Gonzalo Martínez
Alberto MedinaSalvador Martínez
Antonio RamosMiguel Ángel
López
Virginia Pavón
Lola Segura
Mª Carmen MenaFernando Roncal
Manuel Lombardía
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