Quantitative analysis of the mitochondrial proteome and phosphoproteome in the yeast Saccharomyces...

Quantitative analysis of the mitochondrial proteome and

phosphoproteome in the yeast Saccharomyces cerevisiae

Claire LEMAIRE

CNRS-UMR8221, CEA-IBITECS, Université Paris-Sud, I2BC, F-91191 Gif-sur-Yvette, FRANCE.

[email protected]

July 14th 2015 International Summit on Current Trends in Mass Spectrometry 2015

Mitochondrial respiratory chain complexes

2

3

Mitochondrion: an essential organelle

http://conceptcours.fr/www/term_s/spe/energie

External membrane Cristae

Internal membrane Matrix

Essential function: energy production

Mitochondrion

4

OXPHOS

The mitochondrial respiratory chain complexes (OXPHOS)

http://conceptcours.fr/www/term_s/spe/energie

External membrane Cristae

Internal membrane (IM) Matrix

Variations among the metabolism of the cell

I II III IV V

IM

Matrix

5

The mitochondrial respiratory chain complexes (OXPHOS)

Dysregulation

Pathologies

I II III IV V

Metabolic demand/various stresses, adjustement of the activity

IM

Matrix

Levels of regulation

6

Change in the expression level of proteins

Reversible interaction with effectors

Post-translational modifications

Post-TranslationalModifications

7

Post-TranslationalModifications

Glycosylation Ubiquitination

Phosphorylation

S-Nitrosylation

Methylation

N-Acetylation

Lipidation

SUMOylation

8

Importance of protein regulation by phosphorylation

9

Phosphorylation

Functionalproperties

Cellular localization

Interaction with partners

10

Phosphorylation

Pathologies

Link between phosphorylation and pathologies

11

Phosphorylation

Pathologies

Link between phosphorylation and pathologies

Abnormal phosphorylation events

Cancers and neurodegenerative diseases (Alzheimer’s, Parkinson’s or Huntington’s disease)

12

Pathologies

Mitochondria

In human, dysregulation of mitochondrial functions, particularly of the respiratory chain are associated with different pathologies (neurodegenerative, neuromuscular, cardiovascular diseases for example)

Link between mitochondria and pathologies

13

Phosphorylation

Pathologies

Mitochondria

?- Few data

(cellular extracts) - Qualitative

Link between mitochondria and phosphorylation?

14Data on phosphorylation in mitochondria: Reproducible Quantitative Obtained in different physiological conditions variations

Phosphorylation

Pathologies

Mitochondria

?

Link between mitochondria and phosphorylation?

15

I – General strategy

16

-

400 450 500 550 600 650 700 750 800 850 900 950 1000m/z0

100

%

1:580.24

779.27

773.34

729.33

556.91

417.93

523.73

511.25418.19

474.74

463.18

418.44461.21

495.21

556.57

528.22

557.23

580.75

694.26

674.30

612.74581.24

597.74654.27

640.27639.94

629.23

694.77

703.81

704.34

728.77

756.29

779.81

959.40916.91780.30

916.33801.38 837.37

836.89

820.32

872.42 902.43872.93

948.48917.36

947.42

923.32

959.95

960.39

967.34

968.36

989.39

m/z

-

400 450 500 550 600 650 700 750 800 850 900 950 1000m/z0

100

%

1:580.24

779.27

773.34

729.33

556.91

417.93

523.73

511.25418.19

474.74

463.18

418.44461.21

495.21

556.57

528.22

557.23

580.75

694.26

674.30

612.74581.24

597.74654.27

640.27639.94

629.23

694.77

703.81

704.34

728.77

756.29

779.81

959.40916.91780.30

916.33801.38 837.37

836.89

820.32

872.42 902.43872.93

948.48917.36

947.42

923.32

959.95

960.39

967.34

968.36

989.39

-

400 450 500 550 600 650 700 750 800 850 900 950 1000m/z0

100

%

1:-

400 450 500 550 600 650 700 750 800 850 900 950 1000m/z0

100

%

1:580.24

779.27

773.34

729.33

556.91

417.93

523.73

511.25418.19

474.74

463.18

418.44461.21

495.21

556.57

528.22

557.23

580.75

694.26

674.30

612.74581.24

597.74654.27

640.27639.94

629.23

694.77

703.81

704.34

728.77

756.29

779.81

959.40916.91780.30

916.33801.38 837.37

836.89

820.32

872.42 902.43872.93

948.48917.36

947.42

923.32

959.95

960.39

967.34

968.36

989.39

m/z

Identification and quantification of proteins accumulation and

phosphorylation level in well-defined physiological states

Mass spectrometrySTRATEGY

I1

17

Ser, Thr , Tyr

STRATEGY

I

-

400 450 500 550 600 650 700 750 800 850 900 950 1000m/z0

100

%

1:580.24

779.27

773.34

729.33

556.91

417.93

523.73

511.25418.19

474.74

463.18

418.44461.21

495.21

556.57

528.22

557.23

580.75

694.26

674.30

612.74581.24

597.74654.27

640.27639.94

629.23

694.77

703.81

704.34

728.77

756.29

779.81

959.40916.91780.30

916.33801.38 837.37

836.89

820.32

872.42 902.43872.93

948.48917.36

947.42

923.32

959.95

960.39

967.34

968.36

989.39

m/z

-

400 450 500 550 600 650 700 750 800 850 900 950 1000m/z0

100

%

1:580.24

779.27

773.34

729.33

556.91

417.93

523.73

511.25418.19

474.74

463.18

418.44461.21

495.21

556.57

528.22

557.23

580.75

694.26

674.30

612.74581.24

597.74654.27

640.27639.94

629.23

694.77

703.81

704.34

728.77

756.29

779.81

959.40916.91780.30

916.33801.38 837.37

836.89

820.32

872.42 902.43872.93

948.48917.36

947.42

923.32

959.95

960.39

967.34

968.36

989.39

-

400 450 500 550 600 650 700 750 800 850 900 950 1000m/z0

100

%

1:-

400 450 500 550 600 650 700 750 800 850 900 950 1000m/z0

100

%

1:580.24

779.27

773.34

729.33

556.91

417.93

523.73

511.25418.19

474.74

463.18

418.44461.21

495.21

556.57

528.22

557.23

580.75

694.26

674.30

612.74581.24

597.74654.27

640.27639.94

629.23

694.77

703.81

704.34

728.77

756.29

779.81

959.40916.91780.30

916.33801.38 837.37

836.89

820.32

872.42 902.43872.93

948.48917.36

947.42

923.32

959.95

960.39

967.34

968.36

989.39

m/z



Mass spectrometry

18

Ser, Thr , Tyr Glu, Asp

Site-directed mutagenesis

Permanent phosphorylated state

STRATEGY

I

-

400 450 500 550 600 650 700 750 800 850 900 950 1000m/z0

100

%

1:580.24

779.27

773.34

729.33

556.91

417.93

523.73

511.25418.19

474.74

463.18

418.44461.21

495.21

556.57

528.22

557.23

580.75

694.26

674.30

612.74581.24

597.74654.27

640.27639.94

629.23

694.77

703.81

704.34

728.77

756.29

779.81

959.40916.91780.30

916.33801.38 837.37

836.89

820.32

872.42 902.43872.93

948.48917.36

947.42

923.32

959.95

960.39

967.34

968.36

989.39

m/z

-

400 450 500 550 600 650 700 750 800 850 900 950 1000m/z0

100

%

1:580.24

779.27

773.34

729.33

556.91

417.93

523.73

511.25418.19

474.74

463.18

418.44461.21

495.21

556.57

528.22

557.23

580.75

694.26

674.30

612.74581.24

597.74654.27

640.27639.94

629.23

694.77

703.81

704.34

728.77

756.29

779.81

959.40916.91780.30

916.33801.38 837.37

836.89

820.32

872.42 902.43872.93

948.48917.36

947.42

923.32

959.95

960.39

967.34

968.36

989.39

-

400 450 500 550 600 650 700 750 800 850 900 950 1000m/z0

100

%

1:-

400 450 500 550 600 650 700 750 800 850 900 950 1000m/z0

100

%

1:580.24

779.27

773.34

729.33

556.91

417.93

523.73

511.25418.19

474.74

463.18

418.44461.21

495.21

556.57

528.22

557.23

580.75

694.26

674.30

612.74581.24

597.74654.27

640.27639.94

629.23

694.77

703.81

704.34

728.77

756.29

779.81

959.40916.91780.30

916.33801.38 837.37

836.89

820.32

872.42 902.43872.93

948.48917.36

947.42

923.32

959.95

960.39

967.34

968.36

989.39

m/z



Mass spectrometry

2

19

Ser , Thr Ala Tyr Phe

Permanent dephosphorylated state

STRATEGY

I



Permanent phosphorylated state-

400 450 500 550 600 650 700 750 800 850 900 950 1000m/z0

100

%

1:580.24

779.27

773.34

729.33

556.91

417.93

523.73

511.25418.19

474.74

463.18

418.44461.21

495.21

556.57

528.22

557.23

580.75

694.26

674.30

612.74581.24

597.74654.27

640.27639.94

629.23

694.77

703.81

704.34

728.77

756.29

779.81

959.40916.91780.30

916.33801.38 837.37

836.89

820.32

872.42 902.43872.93

948.48917.36

947.42

923.32

959.95

960.39

967.34

968.36

989.39

m/z

-

400 450 500 550 600 650 700 750 800 850 900 950 1000m/z0

100

%

1:580.24

779.27

773.34

729.33

556.91

417.93

523.73

511.25418.19

474.74

463.18

418.44461.21

495.21

556.57

528.22

557.23

580.75

694.26

674.30

612.74581.24

597.74654.27

640.27639.94

629.23

694.77

703.81

704.34

728.77

756.29

779.81

959.40916.91780.30

916.33801.38 837.37

836.89

820.32

872.42 902.43872.93

948.48917.36

947.42

923.32

959.95

960.39

967.34

968.36

989.39

-

400 450 500 550 600 650 700 750 800 850 900 950 1000m/z0

100

%

1:-

400 450 500 550 600 650 700 750 800 850 900 950 1000m/z0

100

%

1:580.24

779.27

773.34

729.33

556.91

417.93

523.73

511.25418.19

474.74

463.18

418.44461.21

495.21

556.57

528.22

557.23

580.75

694.26

674.30

612.74581.24

597.74654.27

640.27639.94

629.23

694.77

703.81

704.34

728.77

756.29

779.81

959.40916.91780.30

916.33801.38 837.37

836.89

820.32

872.42 902.43872.93

948.48917.36

947.42

923.32

959.95

960.39

967.34

968.36

989.39

m/z



Mass spectrometry

2

20Structural and functional analysis of the respiratory complexes





Permanent phosphorylated state-

400 450 500 550 600 650 700 750 800 850 900 950 1000m/z0

100

%

1:580.24

779.27

773.34

729.33

556.91

417.93

523.73

511.25418.19

474.74

463.18

418.44461.21

495.21

556.57

528.22

557.23

580.75

694.26

674.30

612.74581.24

597.74654.27

640.27639.94

629.23

694.77

703.81

704.34

728.77

756.29

779.81

959.40916.91780.30

916.33801.38 837.37

836.89

820.32

872.42 902.43872.93

948.48917.36

947.42

923.32

959.95

960.39

967.34

968.36

989.39

m/z

-

400 450 500 550 600 650 700 750 800 850 900 950 1000m/z0

100

%

1:580.24

779.27

773.34

729.33

556.91

417.93

523.73

511.25418.19

474.74

463.18

418.44461.21

495.21

556.57

528.22

557.23

580.75

694.26

674.30

612.74581.24

597.74654.27

640.27639.94

629.23

694.77

703.81

704.34

728.77

756.29

779.81

959.40916.91780.30

916.33801.38 837.37

836.89

820.32

872.42 902.43872.93

948.48917.36

947.42

923.32

959.95

960.39

967.34

968.36

989.39

-

400 450 500 550 600 650 700 750 800 850 900 950 1000m/z0

100

%

1:-

400 450 500 550 600 650 700 750 800 850 900 950 1000m/z0

100

%

1:580.24

779.27

773.34

729.33

556.91

417.93

523.73

511.25418.19

474.74

463.18

418.44461.21

495.21

556.57

528.22

557.23

580.75

694.26

674.30

612.74581.24

597.74654.27

640.27639.94

629.23

694.77

703.81

704.34

728.77

756.29

779.81

959.40916.91780.30

916.33801.38 837.37

836.89

820.32

872.42 902.43872.93

948.48917.36

947.42

923.32

959.95

960.39

967.34

968.36

989.39

m/z



Mass spectrometrySTRATEGY

I

3

II – Biological study model : the yeast Saccharomyces cerevisiae

21

22

Facultative aerobe study of various physiological states, dependent on culture conditions

WT Respiratory mutant

Fermentescible medium(glucose, galactose)

Respiratory medium(Lactate)

Molecular genetics techniques

Complete sequenced genome

Numerous homologies with the human respiratory chain

STUDY

MODEL

IISaccharomyces cerevisiae

III – First aim

23

Quantitative variations of the mitochondrial proteome and

phosphoproteome during fermentative and respiratory

growth in Saccharomyces cerevisiae

Renvoisé et al. , J Proteomics (2014) 106, 140-150 24

IV -Methodology

25

METHODOLOGY

IV

26

A B C

Growth in three conditions

Mitochondria purification

Protein quantification by LC-MS/MS

Respiration(Lactate)

Fermentation(Glucose)

Fermentation(Galactose)

A B C

Metabolism

(Substrate)

4 independant biological replicates for each of the 3 growth conditions

Trypsin digestion

1

2

3

METHODOLOGY

IV

software developed by the PAPPSO Platform/ Gif-sur-Yvette/ France

27

Mix the 3 samples in 1:1:1ratio

CH2O CD2O 13CD2O

Multiplex stable isotope dimethyl labeling (Boersema et al., Nat Protoc., 2009)

Strong Cation Exchange Chromatography (SCX)

Affinity Chromatography (IMAC)

Phosphopeptides quantification by LC-MS/MS

Enrichment in

Phosphopeptides

Labeling Efficiency:93-97,7%

4

5

Trypsic peptides

6

7

METHODOLOGY

IV

Primary amines Formaldehyde Cyanoborohydride

V – Results

28

V-1- Proteomic results

29

Variations of mitochondrial protein abundances

30

176(32%)

368(68%)

significative variationno variation

544 mitochondrial proteins quantified(only peptides quantified in at least 3 replicates/condition were keptOnly proteins quantified with at least two peptides were kept)

PROTEOMICS

V-1

Significant changes of protein abundances according to the substrate

31

Cluster168 proteins

Cluster233 proteins

Cluster349 proteins

Cluster426 proteins

1 2 3

3: Lactate

1: Glucose

2: Galactose

PROTEOMICS

V-1

1st groupClassification:2 major groups

32

Cluster168 proteins

Cluster233 proteins

Cluster349 proteins

Cluster426 proteins

1 2 3

1 2 3

1 2 3


3: Lactate

1: Glucose

2: Galactose

PROTEOMICS

V-1

2nd group

33

Cluster168 proteins

Cluster233 proteins

Cluster349 proteins

Cluster426 proteins

1 2 3

1 2 3

1 2 3


3: Lactate

1: Glucose

2: Galactose

PROTEOMICS

V-1

1 2 3

Differences glucose vs galactose

Conclusions

34

This study allows ,for the first time, an overall comparison of mitochondrial protein abundances in fermentation and respiration

Differences between the two fermentative substrates (glucose and galactose) are highlighted

Galactose could be considered as a substrate displaying an intermediate metabolism between fermentation and respiration

respiro-fermentative substrate

PROTEOMICS

V-1

V-2 Phosphoproteomic results

35

36

670 phosphosites

Phosphosites selection

LC-MS/MS identification

PHOSPHOROTEOMICS

V-2

37

670 phosphosites

650 (97%)

Phosphosites selection LC-MS/MS

identification

LC-MS/MS quantification

PHOSPHOROTEOMICS

V-2

38

670 phosphosites

650

330 (49%)

LC-MS/MS identification


Present in at least3 replicates


PHOSPHOROTEOMICS

V-2

39

LC-MS/MS identification670

phosphosites

650

330

289(43%)


Normalization according to protein abundance


PHOSPHOROTEOMICS

V-2

Present in at least3 replicates

Variations of mitochondrial protein phosphorylation

40

289 mitochondrial phosphosites quantified, of which 214 were new

90(31%)

199(69%)

significative variationno variation

PHOSPHOROTEOMICS

V-2

Variations of mitochondrial protein phosphorylation

41

90 mitochondrial phosphosites quantified with significative variation

1 condition

2 conditions

3 conditions9

42

39

PHOSPHOROTEOMICS

V-2

Significant changes of phosphorylation according to the substrate

42

Cluster P316 sites

Cluster P28 sites

Cluster P45 sites

Cluster P17 sites

Cluster P5

3 sites

1 2 3

39 varying phosphosites quantified in the 3 conditions

PHOSPHOROTEOMICS

V-2

1 2 3

1 2 3

1 2 3

Clusters P1 to P4 : same pattern as clusters 1 to 4

Classification

Significant changes of phosphorylation according to the substrate

43

Cluster P316 sites

Cluster P28 sites

Cluster P45 sites

Cluster P17 sites

Cluster P5

3 sites

PHOSPHOROTEOMICS

V-2

Additional cluster P5: specific regulation in galactose

1 2 3

44

300 mitochondrial proteins were identified as phosphorylated ≈ 30% mitochondrial proteins

289 phosphorylation sites were quantified of which 214 were new significant enlargement of the yeast mitochondrial phosphosites map

1/3 of the phosphorylation sites varies from the 3 culture conditions tested (glucose, galactose, lactate)

Specific regulation seems to occur in galactose (respiro-fermentative medium)

Conclusions PHOSPHOROTEOMICS

V-2

45

Variations of mitochondrial protein abundances and protein phosphorylation

according to their metabolic pathways

Proteins associated with metabolic pathways according to the MIPS functional classification ( http:// mips.helmholtz-muenchen.de)

Conclusion: The most regulated metabolic pathway at both proteomic and phosphoproteomic levels is energy metabolism

UnknownTransport

TranscriptionProtein synthesis

Protein fateNucleotide metabolism

Mitochondria biogenesisMetabolism of vitamins, cofactors and prosthetic groups

Lipid metabolismEnergy

Cell rescue, defense and virulenceCell cycle and DNA processing

C-compound and carbohydrate metabolismAmino acid metabolism

0 10 20 30 40 50 60 70

Proteins

Phosphosites

Number

OXPHOS proteins

46

OXPHOS

31 OXPHOS proteins were reproducibly quantified in the 3 growth conditions and showed different abundances according to the condition

19 of these proteins were phosphorylated, displaying 37 phosphosites

important role of phosphorylation in the regulation of the respiratory chain

For most of them, proteins were more abundant and more phosphorylated in lactate medium (respiratory condition) except for 2 proteins

C II (CIII)

2

(CIV)2 CV

matrix

Intermembranary space

NDI1

NDE1Innermembrane

In progress

47



Permanent phosphorylated state

2


Structural and functional analysis of the respiratory complexes


3

Acknowledgements

48

The team « Regulation of mitochondrial energy-transducing complexes »

Francis Erwan

Elodie

Mehdi Qian

Deborah

Margaux Claire

Tiona

Francis ErwannBoscoLaurélie

Acknowledgements

49

SBIGEM, Saclay

Gwenaëlle Le RouxChristophe Carles

Financial support and encouragements: Bruno Robert, head of department B3S

Thank you for your attention

Michel ZivyLudovic BonhommeMarlène Davanture

Benoit ValotThierry Bailleau

I.B.G.C. ,Bordeaux

Marie-France GiraudDaniel Brèthes

Isabelle Larrieu

Anne DevinMichel Rigoulet

Ohio State UniversityColumbus, U.S.A.

Patrice Hamel

Collaborations

52

75%

11%

8%2%

4%

mitochondriapolysomescytoplasmenucleusothers

Cellular localization of the 724 proteins identified

53

Median

Maximum

Minimum

1:50%

2:50%

1Q

2Q

3Q

4Q

Q : quartile

.

.

Middle of 1

Middle of 2

54

Q : quartile

Median

Maximum

Minimum

1:50%

2:50%

1Q

2Q

3Q

4Q ..

Middle of 1

Middle of 2

outlier

Variations of mitochondrial protein abundances and protein phosphorylation

according to their metabolic pathways

55

Proteins associated with metabolic pathways according to the MIPS functional classification ( http:// mips.helmholtz-muenchen.de)

Conclusion: The most regulated metabolic pathway at both proteomic and phosphoproteomice levels is energy metabolism

Localization of phosphorylation sites on yeast OXPHOS proteins

56

C II (CIII)2 (CIV)2 CV

matrix

Intermembranary space

NDI1

SDH2 RIP1

NDE1Innermembrane

SDH1

COR1

ATP2

INH1

OXPHOS

Globally, the level of phosphorylation varied in the same direction as protein amounts, except for 2 proteins (Rip1 and Atp2)

57

Stable Isotope Dimethyl labeling of trypsic peptides

RegularFormaldehyde/ cyanoborohydride

DeuteredFormaldehyde/cyanoborohydride

Deutered and C13-labeledFormaldehyde/ cyanoborodeuteride

Reaction of peptide primary amines with formaldehyde - base Schiff, reduced by addition of cyanoborohydride

58

- SILAC: stable isotope labeling by amino acids in cell culture cells are grown in culture media lacking essential (auxotrophic) amino acid(s). These amino acids are then supplied in either their natural form or in a stable isotope form to cause in vivo incorporation of the labeled amino acid(s). The differentially labeled samples are mixed and digested with a protease (most often trypsin) to obtain samples that can be readily analyzed by the mass spectrometer.

- iTRAQ : isobaric tagging for relative and absolute quantification

The ITRAQ method is based on the covalent labeling of the N-terminus and side chain amines of peptides from protein digestions with tags of varying mass. There are currently two mainly used reagents: 4-plex and 8-plex,

https://en.wikipedia.org/wiki/N-terminus

https://en.wikipedia.org/wiki/Side_chain

https://en.wikipedia.org/wiki/Amine

https://en.wikipedia.org/wiki/Peptide

61

- Labeled peptides were dried by vacuum centrifugation and re-suspended in ACN/H2O:30/70, 0.5% formic acid, pH2- Sample fractionation by strong cation exchange chromatography(Bonhomme et al. , Mol Cell Proteomics, 2012, 11,957-972) using a Zorbax BioSCX-Series II column on a Ultimate LC system combined with a Famos autosampler and a Switchos II microcolumn switch system (LC packings)- Samples automatically collected to form 12 fractions- Each fraction was dried by vacuum centrifugation and re-suspended in ACN/H2O:30/70, 250mM acetic acid- Incubation with Phos-select affinity gel during 1H- Washed in SigmaPrep spin column 2 fold with ACN/H2O:30/70, 250mM acetic acid- Elution of phosphopeptides in SigmaPrep spin column with ACN/H2O:30/70, 0.4M ammonium hydroxideEnrichment very effective :24% phosphorylated peptides (non-enriched samples: 4 phosphopeptides)88% of unphosphorylated peptides contained one ASP or GLU - limit of IMAC methodology (strong affinity for acidic peptides)

Phosphoproteomic analysis

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The phosphopeptides were identified in 299 proteins of which :

- 150 displayed 1 site of phosphorylation- 72 displayed 2 sites of phosphorylation- 27 displayed 3 sites of phosphorylation- 50 displayed 4 sites of phosphorylation or more

- 71% serine- 16% threonine- 0.6% tyrosine- 12.6% not precisely localized in the peptide sequence

Quantitative analysis of the mitochondrial proteome and phosphoproteome in the yeast Saccharomyces...

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