- DATA DEPENDENT ANALYSIS -Proteomics Unit. Centro Nacional de Biotecnología

sHPPMiguel Marcilla Goldaracena

MCF7(Epithelial)

RAMOS(Lymphoid)

CCD18(Fibroblast)

CELL LINES AND FRACTIONATION TECHNIQUES

• SDS-PAGE

• Basic rpHPLC

• SDS-PAGE

Jurkat(Lymphoid)

• SDS-PAGE

• Basic rpHPLC

Systematic Comparison of Fractionation Methods for In-depth Analysis of Plasma Proteomes

Cao et al. Journal of Proteome Research. 2012.

“High pH reverse-phase HPLC exhibited the highest peptide resolutionand yielded the best depth of analysis with detection of the largest number of known lowabundance proteins (…)”

15+1 slices

SDS-PAGE

Protein Precipitation

Trypsin Digestion

Trypsin Digestion

15 fractions

Basic rpHPLC

LC-MS Analysis5600 triple TOF (ABSciex)

MCF7Protein Extract

rpHPLC SDS-PAGE

FDR (Peptide level) 1 % 1 %

# PSM 13582 35186

# Peptides 12379 25772

# Proteins 3894 5892

Mean Coverage (peptides/protein) 3.2 4.4

634 26323260

Basic rpHPLC(3894)

SDS-PAGE(5892)

SDS-PAGE vs BASIC rpHPLC (MCF7)

6526 unique proteins

0 2 4 6 8 10 12 140

1000

2000

3000

4000

5000

6000

7000

proteins peptides

Slice number

0 2 4 6 8 10 12 140

200

400

600

800

1000

1200

1400

proteins peptides

Fraction number

SDS-PAGE vs BASIC rpHPLC (FDR <1%, peptide level)

SDS-PAGE (15+1 slices) Basic rpHPLC (15 fractions)

238(3.6%)

6288(96.4 %)

Chormosome 16Other Chromosomes

Proteins Identified in MCF7 cells

837 (4%)

20859(96%)

Chormosome 16

Other Chromosomes

Protein Coding Genes

C. Gil

E. Oliv

eira

F. Canals

F. Elortz

a

F. Viva

nco

F.J. B

lanco

F. J. C

orrales

I. Casa

l

J. Abian

J. M. A

rizmendi

J. P. A

lbar

M. S

. del P

ino

S. Barce

ló0

2

4

6

8

10

12

14

8

13

10

11

13

12

13

10

1

2

1

Iden

tified

Pro

tein

s

TARGET PROTEINS IDENTIFIED IN MCF7 CELLS

“Known” proteins “Unknown” proteins

SDS-PAGE vs BASIC rpHPLC (MCF7)

Jurkat

RIPA

SDS 4%

Urea CHAPS

SDS-PAGE

Basic rpHPLC

SDS-PAGE

Basic rpHPLC

SDS-PAGE

Basic rpHPLC

Cell Lysis Fractionation LC-MS Analysis

5600 triple TOF (ABSciex)

Basic rpHPLC(8893)

rpHPLC SDS-PAGE

FDR (Peptide level) 1 % 1 %

# PSM 62248 58100

# Peptides 54513 41382

# Proteins 8893 7326

Mean Coverage (peptides/protein) 6.0 5.6

11662733 6160

SDS-PAGE(7326)

SDS-PAGE vs BASIC rpHPLC (Jurkat)

10059 unique proteins

305(3.0%)

10059(97.0%)

Chormosome 16Other Chromosomes

Proteins Identified from Jurkat cells

Protein Coding Genes

837 (4%)

20859(96%)

Chormosome 16

Other Chromosomes

TARGET PROTEINS IDENTIFIED IN MCF7 CELLS

C. Gil

E. Oliv

eira

F. Canals

F. Elortz

a

F. Viva

nco

F.J. B

lanco

F. J. C

orrales

I. Casa

l

J. Abian

J. M. A

rizmendi

J. P. A

lbar

M. S

. del P

ino

S. Barce

ló0

2

4

6

8

10

12

14

16

18

20

9

14

16

15

18

15

14

16

1 1

2

1

Iden

tified

Pro

tein

s

“Known” proteins “Unknown” proteins

CONCLUSIONS

• Data dependent LC-MS analysis allows the characterization of a significant number of proteins coded in chromosome 16 (More than 50% of “known” proteins are readily detected)

• Peptide fractionation by reversed phase chromatography at basic pH is a valuable alternative to SDS-PAGE and allows the identification of a higher number of proteins

Carmen González Adán AlpizarSeverine Gharbi Rosana Navajas

Alberto Paradela

Salvador MartínezAlberto Medina