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