Limits...
Identification and bioinformatic analysis of the membrane proteins of synechocystis sp. PCC 6803.

Wang Y, Xu W, Chitnis PR - Proteome Sci (2009)

Bottom Line: Bioinformatic analysis revealed that the identified proteins can be functionally classified into 14 distinct groups according to the cellular functions annotated by Cyanobase, including the two largest groups hypothetical and unknown, and photosynthesis and respiration.Homology analysis indicates that the identified membrane proteins are more conserved than the rest of the proteome.The proteins identified in this study combined with other published proteomic data provide the most comprehensive Synechocystis proteome catalog, which will serve as a useful reference for further detailed studies to address protein functions through both traditional gene-by-gene and systems biology approaches.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, 50011, USA. wychun@ucsd.edu

ABSTRACT

Background: The membranes of Synechocystis sp. PCC 6803 play a central role in photosynthesis, respiration and other important metabolic pathways. Comprehensive identification of the membrane proteins is of importance for a better understanding of the diverse functions of its unique membrane structures. Up to date, approximately 900 known or predicted membrane proteins, consisting 24.5% of Synechocystis sp. PCC 6803 proteome, have been indentified by large-scale proteomic studies.

Results: To resolve more membrane proteins on 2-D gels for mass spectrometry identification, we separated integral proteins from membrane associated proteins and collected them as the integral and peripheral fractions, respectively. In total, 95 proteins in the peripheral fraction and 29 proteins in the integral fraction were identified, including the 5 unique proteins that were not identified by any previous studies. Bioinformatic analysis revealed that the identified proteins can be functionally classified into 14 distinct groups according to the cellular functions annotated by Cyanobase, including the two largest groups hypothetical and unknown, and photosynthesis and respiration. Homology analysis indicates that the identified membrane proteins are more conserved than the rest of the proteome.

Conclusion: The proteins identified in this study combined with other published proteomic data provide the most comprehensive Synechocystis proteome catalog, which will serve as a useful reference for further detailed studies to address protein functions through both traditional gene-by-gene and systems biology approaches.

No MeSH data available.


Related in: MedlinePlus

Separation of integral and membrane associated proteins. The total Synechocystis membranes were treated with 8.0 M urea to release the membrane associated proteins. The supernatants containing the released membrane associated proteins were collected as the peripheral fraction after centrifugation, and the insoluble pellet was collected as the integral fraction. The proteins in the peripheral fraction were further precipitated with 10% TCA, washed with ice-cold acetone, and re-solubilized by the multi-surfactant solution. The integral proteins were directly washed with acetone and solubilized with the same volume of multi-surfactant solution. Equal volume of samples from the peripheral fraction (Lane P) and the integral fraction (Lane I) were separated by SDS-PAGE. Total membranes containing 10 μg of chlorophyll were used as the loading control (Lane T). All the samples were incubated with denaturing dye (2% SDS and 0.1 M DTT) at room temperature for 4 h before separated by SDS-PAGE.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2666656&req=5

Figure 1: Separation of integral and membrane associated proteins. The total Synechocystis membranes were treated with 8.0 M urea to release the membrane associated proteins. The supernatants containing the released membrane associated proteins were collected as the peripheral fraction after centrifugation, and the insoluble pellet was collected as the integral fraction. The proteins in the peripheral fraction were further precipitated with 10% TCA, washed with ice-cold acetone, and re-solubilized by the multi-surfactant solution. The integral proteins were directly washed with acetone and solubilized with the same volume of multi-surfactant solution. Equal volume of samples from the peripheral fraction (Lane P) and the integral fraction (Lane I) were separated by SDS-PAGE. Total membranes containing 10 μg of chlorophyll were used as the loading control (Lane T). All the samples were incubated with denaturing dye (2% SDS and 0.1 M DTT) at room temperature for 4 h before separated by SDS-PAGE.

Mentions: The low abundance of some membrane proteins is one of the major hindrances to their identification and functional characterization. To analyze the membrane proteome of Synechocystis, we first enriched low abundant proteins by separating membranes into the peripheral and integral fractions. The peripheral fraction mainly contains the membrane associated proteins that were released from the lipid-bilayer by 8.0 M urea extraction, whereas the integral fraction mainly contains the integral membrane proteins that are refractory to the urea extraction. The fractionated proteins were separated by SDS-PAGE and stained with CBB. The result revealed that the protein migration patterns of the two fractions are not only different from each other, but also different from those of the total membrane proteins (Figure 1). The difference of the protein patterns was exhibited by the different dominant protein bands on the SDS-PAGE gel that are specific to either fraction, but not both (Figure 1), indicating that the fractionation is an efficient way to separate membrane associated proteins from integral proteins, which is in agree with the literature [4,5]. The 68 kDa major bands representing high abundant integral proteins PsaA and PsaB were present only in the lane for the total membrane proteins but not in the other two lanes, indicating that these two 11-TM containing highly hydrophobic proteins were not solubilized by the extraction buffer. In fact, no report has shown that these proteins can be solubilized in a buffer compatible for 2-DE. Both the peripheral and integral fractions contain many high molecular weight proteins that were not detected in our previous study using TCA/acetone precipitation of total membranes (Figure 1) [6]. Although the multiple surfactant solution used here is stronger than the rehydration buffer used in our previous study [6], detection of more high molecular weight proteins is more likely due to enrichment of low abundant proteins by serial extraction rather than the difference of solubilizing solution. This is supported by the observation that the serial extraction resolved more high molecular weight proteins than TCA/acetone precipitation did even we used the same solution for protein solubilization (unpublished data). Therefore, the serial extraction is an effective way for enriching low abundant membrane proteins.


Identification and bioinformatic analysis of the membrane proteins of synechocystis sp. PCC 6803.

Wang Y, Xu W, Chitnis PR - Proteome Sci (2009)

Separation of integral and membrane associated proteins. The total Synechocystis membranes were treated with 8.0 M urea to release the membrane associated proteins. The supernatants containing the released membrane associated proteins were collected as the peripheral fraction after centrifugation, and the insoluble pellet was collected as the integral fraction. The proteins in the peripheral fraction were further precipitated with 10% TCA, washed with ice-cold acetone, and re-solubilized by the multi-surfactant solution. The integral proteins were directly washed with acetone and solubilized with the same volume of multi-surfactant solution. Equal volume of samples from the peripheral fraction (Lane P) and the integral fraction (Lane I) were separated by SDS-PAGE. Total membranes containing 10 μg of chlorophyll were used as the loading control (Lane T). All the samples were incubated with denaturing dye (2% SDS and 0.1 M DTT) at room temperature for 4 h before separated by SDS-PAGE.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2666656&req=5

Figure 1: Separation of integral and membrane associated proteins. The total Synechocystis membranes were treated with 8.0 M urea to release the membrane associated proteins. The supernatants containing the released membrane associated proteins were collected as the peripheral fraction after centrifugation, and the insoluble pellet was collected as the integral fraction. The proteins in the peripheral fraction were further precipitated with 10% TCA, washed with ice-cold acetone, and re-solubilized by the multi-surfactant solution. The integral proteins were directly washed with acetone and solubilized with the same volume of multi-surfactant solution. Equal volume of samples from the peripheral fraction (Lane P) and the integral fraction (Lane I) were separated by SDS-PAGE. Total membranes containing 10 μg of chlorophyll were used as the loading control (Lane T). All the samples were incubated with denaturing dye (2% SDS and 0.1 M DTT) at room temperature for 4 h before separated by SDS-PAGE.
Mentions: The low abundance of some membrane proteins is one of the major hindrances to their identification and functional characterization. To analyze the membrane proteome of Synechocystis, we first enriched low abundant proteins by separating membranes into the peripheral and integral fractions. The peripheral fraction mainly contains the membrane associated proteins that were released from the lipid-bilayer by 8.0 M urea extraction, whereas the integral fraction mainly contains the integral membrane proteins that are refractory to the urea extraction. The fractionated proteins were separated by SDS-PAGE and stained with CBB. The result revealed that the protein migration patterns of the two fractions are not only different from each other, but also different from those of the total membrane proteins (Figure 1). The difference of the protein patterns was exhibited by the different dominant protein bands on the SDS-PAGE gel that are specific to either fraction, but not both (Figure 1), indicating that the fractionation is an efficient way to separate membrane associated proteins from integral proteins, which is in agree with the literature [4,5]. The 68 kDa major bands representing high abundant integral proteins PsaA and PsaB were present only in the lane for the total membrane proteins but not in the other two lanes, indicating that these two 11-TM containing highly hydrophobic proteins were not solubilized by the extraction buffer. In fact, no report has shown that these proteins can be solubilized in a buffer compatible for 2-DE. Both the peripheral and integral fractions contain many high molecular weight proteins that were not detected in our previous study using TCA/acetone precipitation of total membranes (Figure 1) [6]. Although the multiple surfactant solution used here is stronger than the rehydration buffer used in our previous study [6], detection of more high molecular weight proteins is more likely due to enrichment of low abundant proteins by serial extraction rather than the difference of solubilizing solution. This is supported by the observation that the serial extraction resolved more high molecular weight proteins than TCA/acetone precipitation did even we used the same solution for protein solubilization (unpublished data). Therefore, the serial extraction is an effective way for enriching low abundant membrane proteins.

Bottom Line: Bioinformatic analysis revealed that the identified proteins can be functionally classified into 14 distinct groups according to the cellular functions annotated by Cyanobase, including the two largest groups hypothetical and unknown, and photosynthesis and respiration.Homology analysis indicates that the identified membrane proteins are more conserved than the rest of the proteome.The proteins identified in this study combined with other published proteomic data provide the most comprehensive Synechocystis proteome catalog, which will serve as a useful reference for further detailed studies to address protein functions through both traditional gene-by-gene and systems biology approaches.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, 50011, USA. wychun@ucsd.edu

ABSTRACT

Background: The membranes of Synechocystis sp. PCC 6803 play a central role in photosynthesis, respiration and other important metabolic pathways. Comprehensive identification of the membrane proteins is of importance for a better understanding of the diverse functions of its unique membrane structures. Up to date, approximately 900 known or predicted membrane proteins, consisting 24.5% of Synechocystis sp. PCC 6803 proteome, have been indentified by large-scale proteomic studies.

Results: To resolve more membrane proteins on 2-D gels for mass spectrometry identification, we separated integral proteins from membrane associated proteins and collected them as the integral and peripheral fractions, respectively. In total, 95 proteins in the peripheral fraction and 29 proteins in the integral fraction were identified, including the 5 unique proteins that were not identified by any previous studies. Bioinformatic analysis revealed that the identified proteins can be functionally classified into 14 distinct groups according to the cellular functions annotated by Cyanobase, including the two largest groups hypothetical and unknown, and photosynthesis and respiration. Homology analysis indicates that the identified membrane proteins are more conserved than the rest of the proteome.

Conclusion: The proteins identified in this study combined with other published proteomic data provide the most comprehensive Synechocystis proteome catalog, which will serve as a useful reference for further detailed studies to address protein functions through both traditional gene-by-gene and systems biology approaches.

No MeSH data available.


Related in: MedlinePlus