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Studies of the yeast pheromone pathway using quantitative proteomics Episode IV: Phosphoproteomes. Biological Sciences Division Pacific Northwest National Laboratory. Initial goals of PNNL efforts.

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slide1

Studies of the yeast pheromone pathway using quantitative proteomics

Episode IV: Phosphoproteomes

Biological Sciences Division

Pacific Northwest National Laboratory

initial goals of pnnl efforts
Initial goals of PNNL efforts
  • Identify phosphorylated species and quantify their changes in response to alpha factor treatment
  • Provide kinetic data for pathway modeling (e.g. time course studies)
slide4

Identification of phosphorylated proteins by

nanoLC-MS/MS of tryptic peptides

[M+2H]2+=1031.6

[M+2H-98]2+= 98

=49

y8

y6

y9-H2O

y10-H2O

y7

y11

y5

b10

y13

y12

y4

400

600

800

1000

1200

1400

1600

1800

2000

m/z

(K.FQS*EEQQQTEDELQDK.I)

challenges
Challenges
  • Low abundance of pathway proteins

- Ste5 ~500 molecules/cell and Fus3 ~20,000, molecules/cell vs. millions/cell for abundant proteins

  • Unknown number of pathway player modifications
  • Often low phosphorylation stoichiometry
  • Desire to make many quantitative measurements
focused analysis of affinity selected alpha pathway players and their complexes
Focused analysis of affinity selected alpha pathway players and their complexes

Tandem affinity purification (TAP) to expand results obtained from global view (identify other binding partners, verify and identify additional low level sites of modification, etc.)

LC-MS

imac i mmobilized m etal ion a ffinity c hromatography
IMAC(Immobilized Metal ion Affinity Chromatography)

Esterification

of carboxylic groups

O

To reduce interaction between COO

and metal ion in IMAC enrichment

-

process

aspartic acid (D)

glutamic

acid (E)

C

-

terminus

O

H

H

O

OH

H

H

O

N

O

N

C

C

O

N

C

C

C

O

C

H

OR

Fe3+

C

H

2

P

2

C

H

O

O

C

2

O

O

C

O

O

O

OH2

+

O

O

H

+

+

C

X

H

O

H

O

C

C

3

2

O

H

C

X

O

3

X : H or D

direct vs imac enriched nanolc ms analyses
Direct vs. IMAC enriched nanoLC-MS analyses

K.FQS*EEQQQTEDELQDK.I

50.50

Before IMAC enrichment

28.83

22.35

33.70

62.87

42.56

57.13

20.17

41.08

Relative Abundance

29.16

K.FQS*EEQQQTEDELQDK.I

After IMAC enrichment

61.97

39.27

50.60

74.59

36.65

27.90

0

10

20

30

40

50

60

70

80

90

100

Time (min)

yeast strain sub592
Yeast Strain: SUB592
  • Obtained from Dan Finley’s lab (Harvard)

(Peng et al., Nature Biotech vol. 21, 2003)

  • Endogenous ubiquitin genes knocked out
  • NH2-His-tagged ubiquitin gene supplied by plasmid
  • Cells grow and respond to alpha factor normally

(in plate assay)

Initial studies: Treat Yeast strain 592 with alpha factor, recover His-tagged proteins, greatly enrich phosphopeptides using IMAC, and identify peptides/proteins using nanoLC-MS/MS

proteins identified after dual his affinity purification and imac enrichment
Proteins identified after dual His affinity purification and IMAC enrichment

Untreated

(757 total)

Alpha treated

(703 total)

384

373

330

slide14

Use of Accurate Mass and Time (AMT) tags

Shotgun peptide identification and generation of AMT tag look-up table

High throughput analyses

Proteins

Proteins

Digestion

Digestion

SCX LC fractionation

Nano LC-FTICR MS

ID using AMT tags

nanoLC-MS/MS

Peptides (or features) identified by their accurate masses and LC separation times,

abundances determined

Peptide identification

Set of AMT tags providing a

“look-up” table of peptides identified by their accurate masses and LC separation times

slide15

The application of peptide AMT tags

Single LC-FTICR analysis

Locations of peptides identified from multiple “shotgun” LC-MS/MS analyses

Peptides identified using AMT tags

quantitation using stable isotope labeling with nanolc fticr amt tag approach

Treated

Non-Treated

IMAC

IMAC

1:1

Label heavy

Label light

Monoisotopic mass

Monoisotopic mass

4.05 Da

4.05 Da

AR ~1 (Light ÷

Heavy)

AR ~8

(Light ÷

Heavy)

Scan number

Scan number

Quantitation using stable-isotope labeling with nanoLC-FTICR AMT tag approach
  • Ability to quantify modified peptides independent of unmodified species
  • Ability to accurately detect and quantify at low stoichiometric ratios

IMAC selected phosphopeptides

a path forward
A path forward
  • Technology improvements for the masses:
  • - New metal free high resolution nanoLC system optimized for phosphoproteomics
  • - Improved characterization of modifications (using ECD/ETD, intact protein “top-down” approaches)
  • - Much higher throughput e.g. for time course studies and “fishing with an adjustable net”
  • Deliniation of phosphorylation sites, and abundances, for all known alpha pathway players (in progress)
  • Characterization of other modifications and other possible (fringe?) players
  • Studies of selected perturbations, time courses, etc……
slide18

Acknowledgements

Robert Maxwell Orna Resnekov

Kirsten Benjamin David Pincus

Roger Brent

PNNL Proteomics Team

David Camp Mary Lipton Joshua Adkins

Sample processing and automation

Eric Livesay Kim Hixson

Heather Mottaz Carrie Goddard

Marina Gritsenko Therese Clauss

Dave Prior

Data processing, software development and statistics

Gordon Anderson Matt Monroe

Mary Powers Dave Clark

Angela Norbeck Nikola Tolic

Gary Kiebel Eric Strittmater

Ken Auberry Sam Purvine

Kerry Steele Steve Callister

Deep Jaitly Niksa Blonder

Separations

Yufeng Shen Kostas Petritis

Rui Zhao David Simpson

Alex Shvartsburg Quanzhou Luo

Mass spectrometry

Ljiljana Pasa-Tolic Keqi Tang

Harold Udseth Anil Shukla

Tom Metz Tao Liu

Ron Moore David Anderson

Aleksey Tolmachev Rui Zhang

Fumin Li Jon Jacobs

Charley Langley Feng Yang

Jason Page Weijun Qian

Hyak Kang