PM-enrichment and maize root PM proteome

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jennyjiji

This is a short talk presentation I gave for Danforth Plant Research Center\'s Fall Symposium (2010).

Maize primary root zone-specific plasma membrane proteome Zhe Zhang (Jenny) University of Missouri – Columbia Division of Biochemistry Bond Life Sciences Center

Differential elongation during water stress Elongation Cell division Differentiation What are plasma membrane-located proteins (e.g., transporters, aquaporins, receptors), that may function in water stress responses? Spollen WG, et al., BMC Plant Biol. 2008; 8: 32.

1D SDS-PAGE gel In-gel digestion LC-MS/MS (LTQ-Orbi-trap) Spectral Counting (Scaffold) PM: simply “trapped” organelles? 50,000xg 10,000xg S1 S2 P1 P2 Brij-58 Carbonate wash P2 Crude Microsome (CM) Crude Microsome (M) Plasma Membrane (PM)

Criteria for a successful PM prep ? ,[object Object]

Inner membrane domains recovered,[object Object]

12 : Plasma membrane 21 12 20 47 : Endoplasmic reticulum : Mitochondrion 43 25 : Cytosol 31 10 : Plastid 19 45 : Other 22 Crude microsome prep (127) Plasma membrane prep (130) PM-preferred protein preparation

In Log2(PM/CM) euation: PM: Plasma Membrane Prep; CM: Crude Microsome prep; Subcellular locations: PL: plastid; MI: mitochondrion; ER: Endoplasmic reticulum; CY: cytosol; NU: nucleus; PR: peroxisome; PM: plasma membrane

No. of PM proteins: 60 18 3.33 fold more unique PM proteome 87 26 PM proteins enriched in CM PM proteins enriched in PM Overall PM protein coverage Brij-Carb (PM) Microsome (CM) 147 44 3.34-fold enriched PM proteome

Brij-58 inner membrane domain coverage 2 M4 3 4 M6 M5 1 AHA3 104KDa p=0.00033 2.5 fold enriched in PM 1: (K)GPGEEVFSGSTCK(Q) (192-204) [E1-E2 ATPase domain] 2: (R)VHSTIDKYAER(G) (444-454) 3: (K)HKDEAMAHIPVEDLIEK(A) (542-558) [Hydrolase domain] 4: (R)KHICGMTGDGVNDAPALK(K) (581-598) [Hydrolase domain]

Zone-specific PM proteome? Elongation Cell division Differentiation Zone 4 (12-20 mm) Zone 3 (7-12 mm) Zone 2 (3-7 mm) Zone 1 (0-3 mm) Root tip Two biological replica were analyzed, and results combined for data analysis.

Zone-specific proteins (79/1028) 13 1 10 4 8 Zone 4 Zone 4 0 4 Zone 3 Zone 3 2 0 Zone 2 Zone 2 9 4 Zone 1 24 Zone 1 Root tip Root tip Total: 28 zone-specifically presentproteins Total: 29 zone-specifically absent proteins Total: 22 zone-specifically gradient proteins

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PM-enrichment and maize root PM proteome

1. Maize primary root zone-specific plasma membrane proteome Zhe Zhang (Jenny) University of Missouri – Columbia Division of Biochemistry Bond Life Sciences Center

2. Differential elongation during water stress Elongation Cell division Differentiation What are plasma membrane-located proteins (e.g., transporters, aquaporins, receptors), that may function in water stress responses? Spollen WG, et al., BMC Plant Biol. 2008; 8: 32.

3. 1D SDS-PAGE gel In-gel digestion LC-MS/MS (LTQ-Orbi-trap) Spectral Counting (Scaffold) PM: simply “trapped” organelles? 50,000xg 10,000xg S1 S2 P1 P2 Brij-58 Carbonate wash P2 Crude Microsome (CM) Crude Microsome (M) Plasma Membrane (PM)

4. In gel v.s. in solution digestion

6. PM protein are enriched

8. 12 : Plasma membrane 21 12 20 47 : Endoplasmic reticulum : Mitochondrion 43 25 : Cytosol 31 10 : Plastid 19 45 : Other 22 Crude microsome prep (127) Plasma membrane prep (130) PM-preferred protein preparation

9. In Log2(PM/CM) euation: PM: Plasma Membrane Prep; CM: Crude Microsome prep; Subcellular locations: PL: plastid; MI: mitochondrion; ER: Endoplasmic reticulum; CY: cytosol; NU: nucleus; PR: peroxisome; PM: plasma membrane

10. No. of PM proteins: 60 18 3.33 fold more unique PM proteome 87 26 PM proteins enriched in CM PM proteins enriched in PM Overall PM protein coverage Brij-Carb (PM) Microsome (CM) 147 44 3.34-fold enriched PM proteome

11. Brij-58 inner membrane domain coverage 2 M4 3 4 M6 M5 1 AHA3 104KDa p=0.00033 2.5 fold enriched in PM 1: (K)GPGEEVFSGSTCK(Q) (192-204) [E1-E2 ATPase domain] 2: (R)VHSTIDKYAER(G) (444-454) 3: (K)HKDEAMAHIPVEDLIEK(A) (542-558) [Hydrolase domain] 4: (R)KHICGMTGDGVNDAPALK(K) (581-598) [Hydrolase domain]

12. Zone-specific PM proteome? Elongation Cell division Differentiation Zone 4 (12-20 mm) Zone 3 (7-12 mm) Zone 2 (3-7 mm) Zone 1 (0-3 mm) Root tip Two biological replica were analyzed, and results combined for data analysis.

13. Zone-specific proteins (79/1028) 13 1 10 4 8 Zone 4 Zone 4 0 4 Zone 3 Zone 3 2 0 Zone 2 Zone 2 9 4 Zone 1 24 Zone 1 Root tip Root tip Total: 28 zone-specifically presentproteins Total: 29 zone-specifically absent proteins Total: 22 zone-specifically gradient proteins

14. Gradient: Down 10

15. Gradient: UP 4

16. Region-2 tops 8

17. Zone-specific (present or absent)

18. Conclusion & perspectives Conclusion: PM-enrichment protocol worked well Background info on well-watered condition established Perspectives: WS vs WW comparison is underway Biological replica is planned

19. Peck Lab Jeff Anderson BationaShahollari Ying Wan Jenny Zhang Holly Ziobro -------------------- George Cyriac Jarrod Dye Collaborators BobE Sharp (MU) George E Davis (MU) Ian Kovalchuk (U. of Lethbridge, Canada) Manfred Heinlein (U. of Basel, Switzerland)

Editor's Notes

Our previous work on maize primary root adaptation to water deficit showed that cell elongation is maintained in the apical region of the growth zone but progressively inhibited further from the apex.
Criteria: minimum peptide per protein >=2; Protein identification probability>99.0%; Peptide identification probability>95.0%; Unique peptide probability>95.0%.
PAL is a key biosynthetic catalyst in phenylpropanoid assembly in plants and fungi, and is involved in the biosynthesis of a wide variety of secondary metabolites such as flavanoids, furanocoumarinphytoalexins and cell wall components. These compounds are important for normal growth and in responses to environmental stress.Kinase-like protein is actually found (traced to its original literature), a downstream kinase of Pto, called “Pto-interactor-1, found in tomato. SEC14 family protein: expressed in chloroplast and has transporter activity; shares a great deal of homology with Patellin 1/2, a transporter

PM-enrichment and maize root PM proteome

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