PROFILING THE CHROMOSOME 16 BY HIGH-RESOLUTION DATA DEPENDENT MS: EXTRACTION/FRACTIONATION METHOD EVALUATION IN JURKAT CELLS. Spanish Human Proteome Project Consortium Chromosome 16 III Workshop of the Chromosome 16 Consortium December 2012, La Cristalera, Miraflores de la Sierra, Madrid
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Spanish Human Proteome Project Consortium Chromosome 16
PROFILING THE CHROMOSOME 16 BY HIGH-RESOLUTION DATA DEPENDENT MS: EXTRACTION/FRACTIONATION METHOD EVALUATION IN JURKAT CELLS. . Spanish Human Proteome Project Consortium Chromosome 16. III Workshop of the Chromosome 16 Consortium - PowerPoint PPT Presentation
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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
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
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.