PM-enrichment and maize root PM proteome
17
University of Missouri – Columbia Division of Biochemistry Bond Life Sciences Center Zhe Zhang (Jenny) Maize primary root zone-specific plasma membrane proteome
-
Upload
jennyjiji -
Category
Technology
-
view
510 -
download
1
Transcript of PM-enrichment and maize root PM proteome
University of Missouri – ColumbiaDivision of Biochemistry
Bond Life Sciences Center
Zhe Zhang (Jenny)
Maize primary root zone-specific plasma membrane proteome
Differential elongation during water stress
Spollen WG, et al., BMC Plant Biol. 2008; 8: 32.
What are plasma membrane-located proteins (e.g., transporters, aquaporins, receptors), that may function in water stress responses?
Cell division Elongation Differentiation
10,000xg 50,000xg
S1
P1
S2
P2 Brij-58
Crude Microsome (M)
Carbonate wash
Plasma Membrane(PM)
Crude Microsome (CM)
P2
1D SDS-PAGE gel
In-gel digestion
LC-MS/MS(LTQ-Orbi-trap)
Spectral Counting(Scaffold)
PM: simply “trapped” organelles?
Criteria for a successful PM prep ?
? Extraction preferred PM proteins
? PM protein are enriched
? Inner membrane domains recovered
PM-preferred protein preparation
Crude microsome prep(127)
Plasma membrane prep(130)
: Plasma membrane
: Endoplasmic reticulum
: Mitochondrion
: Cytosol
: Plastid
: Other
Crude microsome prep(127)
Plasma membrane prep(130)
: Plasma membrane
: Endoplasmic reticulum
: Mitochondrion
: Cytosol
: Plastid
: Other
PM-preferred protein preparation
-3
-2.5
-2
-1.5
-1
-0.5
0
0.5
1
1.5
2
PL MI ER CY NU PR PM
Log2
(P
M/C
M)
Cellular localizations
Average enrichment profile across 3 biological replica
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
Overall PM protein coverage
Brij-Carb (PM) Microsome (CM)
60 18No. of PM proteins:
3.33 fold more unique PM proteome
PM proteins enriched in CM87 26PM proteins enriched in PM
147 44
3.34-fold enriched PM proteome
Brij-58 inner membrane domain coverage
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]
1 2 3 4
AHA3104KDap=0.000332.5 fold enriched in PM
M6M4 M5
Zone-specific PM proteome?
Zone 1 (0-3 mm)
Zone 2 (3-7 mm)
Zone 3 (7-12 mm)
Zone 4 (12-20 mm)
Root tip
Cell division Elongation Differentiation
Two biological replica were analyzed, and results combined for data analysis.
Zone-specific proteins (79/1028)
13
0
2
4Zone 1
Zone 2
Zone 3
Zone 4
Root tip
9
Total: 28 zone-specifically presentproteins
1
4
0
24Zone 1
Zone 2
Zone 3
Zone 4
Root tip
Total: 29 zone-specifically absent proteins
10 4 8
Total: 22 zone-specifically gradient proteins
Gradient: Down
0
1
2
3
4
5
6
7
0-3mm 03-7mm 07-12mm 12-20mm
No
. of
un
iqu
e s
pe
ctra
Similar to XT1 (Xylosyltransferase 1)
Exp1
Exp2
0
2
4
6
8
10
12
14
16
18
0-3mm 03-7mm 07-12mm 12-20mm
No
. of
un
iqu
e s
pe
ctra
Similar to BGLU13 (Beta glucosidase 13)
Exp1
Exp2
0
2
4
6
8
10
12
14
0-3mm 03-7mm 07-12mm 12-20mm
No
. of
un
iqu
e s
pe
ctra
similar to AAA-type ATPase family protein
Exp1
Exp2
10
Gradient: UP
0
2
4
6
8
10
12
14
0-3mm 03-7mm 07-12mm 12-20mm
No
. of
un
iqu
e s
pe
ctra
Similar to Phenylalanine ammonia-lyase (PAL)
Exp1
Exp2
0
1
2
3
4
5
6
7
8
9
10
0-3mm 03-7mm 07-12mm 12-20mm
No
. of
un
iqu
e s
pe
ctra
Similar to SEC14 family protein/phosphoglyceride transfer family protein
Exp1
Exp2
0
2
4
6
8
10
12
0-3mm 03-7mm 07-12mm 12-20mm
No
. of
un
iqu
e s
pe
ctra
Putative protein kinase
Exp1
Exp2
0
2
4
6
8
10
12
0-3mm 03-7mm 07-12mm 12-20mm
No
. of
un
iqu
e s
pe
ctra
Kinase-like protein
Exp1
Exp2
4
Region-2 tops
0
5
10
15
20
25
30
35
0-3mm 3-7mm 7-12mm 12-20mm
Similar to arabidopsis ankyrin-like protein/dehydration-responsive
protein-related
Exp1
Exp2
0
2
4
6
8
10
12
14
0-3mm 3-7mm 7-12mm 12-20mm
Similar to arabidopsis EIF4A1
Exp1
Exp2
0
2
4
6
8
10
12
14
0-3mm 3-7mm 7-12mm 12-20mm
Putative glycosyl group transferring transferase
Exp1
Exp2
0
0.5
1
1.5
2
2.5
3
3.5
0-3mm 3-7mm 7-12mm 12-20mm
Similar to arabidopsis hexose transporter
Exp1
Exp2
8
Zone-specific (present or absent)
1-1 1-2 2-1 2-2 3-1 3-2 4-1 4-2
membrane protein gi|195616684 |gb|ACG30172.1| (+2)41 kDa 0 0 0 0 0 0 3 3
flavonoid O-methyltransferase gi|148337327 |gb|ABQ58826.1| (+99)40 kDa 0 0 0 0 0 0 4 2
DREPP2 protein gi|195635963 |gb|ACG37450.1| (+2)25 kDa 0 0 0 0 0 0 2 1
plasma membrane integral protein ZmPIP1-5 gi|13447797 |gb|AAK26756.1| (+5)31 kDa 0 0 0 0 0 0 2 3
plasma membrane intrinsic protein gi|162460423 |ref|NP_001105616.1| (+4)30 kDa 0 0 0 0 0 2 5 3
plasma membrane intrinsic protein gi|162460423 |ref|NP_001105616.1| (+4)30 kDa 0 0 0 0 0 2 5 3
unknown gi|219886991 |gb|ACL53870.1| (+2)66 kDa 0 0 0 0 0 0 4 2
unknown gi|194703914 |gb|ACF86041.1| (+7)42 kDa 0 0 0 0 0 0 2 1
unnamed protein product gi|227285654 |emb|CAY04840.1|37 kDa 0 0 0 0 0 0 3 1
unnamed protein product gi|219897604 |emb|CAW95663.1| (+7)57 kDa 0 0 0 0 1 0 2 2
unknown gi|194704188 |gb|ACF86178.1| (+9)37 kDa 0 0 0 0 0 0 2 2
hypothetical protein gi|195639232 |gb|ACG39084.1| (+1)36 kDa 1 0 1 0 1 0 3 2
GDP-mannose 4,6 dehydratase 2 gi|195624368 |gb|ACG34014.1| (+2)42 kDa 3 2 0 0 0 0 0 0
similar to CSLA02; mannan synthase/
transferase, transferring glycosyl groups gi|194690694 |gb|ACF79431.1| (+2)59 kDa 2 9 0 0 0 0 0 0
similar to C2 domain-containing protein gi|238014338 |gb|ACR38204.1|33 kDa 2 2 0 0 0 0 0 0
similar to myosin heavy chain related gi|219970780 |emb|CAX00109.1| (+2)51 kDa 4 3 2 0 0 0 0 0
similar to guanylate-binding family protein gi|238014796 |gb|ACR38433.1|69 kDa 2 4 3 6 0 0 2 5
Similar to RHD4 (root hair defective 4);
phosphatidylinositol-4,5-bisphosphate 5-
phosphatase/ phosphatidylinositol-4-
phosphate phosphatase
gi|238011180 |gb|ACR36625.1|68 kDa 2 4 2 3 0 0 2 2
Hypothetical protein [Zea mays] gi|195620054 |gb|ACG31857.1| (+1)53 kDa 3 12 3 9 0 0 1 3
Calcium-binding EF hand family protein gi|55741047 |gb|AAV64190.1| (+1)38 kDa 3 2 4 2 1 0 0 2
0-3mm 3-7mm 7-12mm12-20mmIdentified Proteins
Accession
Number
Molecular
Weight
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
CollaboratorsBobE Sharp (MU)George E Davis (MU)Ian Kovalchuk (U. of Lethbridge, Canada)Manfred Heinlein (U. of Basel, Switzerland)
Peck LabJeff AndersonBationaShahollariYing WanJenny ZhangHolly Ziobro--------------------George CyriacJarrod Dye
