Limits...
Exploring the Arabidopsis proteome: influence of protein solubilization buffers on proteome coverage.

Marondedze C, Wong A, Groen A, Serrano N, Jankovic B, Lilley K, Gehring C, Thomas L - Int J Mol Sci (2014)

Bottom Line: These include proteins with a role in signaling, transport, response to temperature stimuli and metabolism.This data may offer a functional bias on comparative analysis studies.In order to obtain a broader coverage, we propose a two-step solubilization protocol with first a detergent-free buffer and then a second step utilizing a combination of two detergents to solubilize proteins.

View Article: PubMed Central - PubMed

Affiliation: Biological and Environmental Sciences and Engineering Division, 4700 King Abdullah University of Science and Technology, Thuwal 23955-6900, Saudi Arabia. claudius.marondedze@kaust.edu.sa.

ABSTRACT
The study of proteomes provides new insights into stimulus-specific responses of protein synthesis and turnover, and the role of post-translational modifications at the systems level. Due to the diverse chemical nature of proteins and shortcomings in the analytical techniques used in their study, only a partial display of the proteome is achieved in any study, and this holds particularly true for plant proteomes. Here we show that different solubilization and separation methods have profound effects on the resulting proteome. In particular, we observed that the type of detergents employed in the solubilization buffer preferentially enriches proteins in different functional categories. These include proteins with a role in signaling, transport, response to temperature stimuli and metabolism. This data may offer a functional bias on comparative analysis studies. In order to obtain a broader coverage, we propose a two-step solubilization protocol with first a detergent-free buffer and then a second step utilizing a combination of two detergents to solubilize proteins.

Show MeSH
Enriched cellular compartments in (A) total unique and (B) membrane-associated proteins identified in the five buffer systems. Gene ontology (GO) analyses were performed using the entire dataset of identified proteins and then using the buffer-specific datasets separately. Cellular compartments that were detected as enriched are shown in the graph (p ≤ 0.05 and false discovery rate (FDR) ≤ 1%). Bars represent the ratio of enrichment against the total number of proteins for each category.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4307279&req=5

ijms-16-00857-f002: Enriched cellular compartments in (A) total unique and (B) membrane-associated proteins identified in the five buffer systems. Gene ontology (GO) analyses were performed using the entire dataset of identified proteins and then using the buffer-specific datasets separately. Cellular compartments that were detected as enriched are shown in the graph (p ≤ 0.05 and false discovery rate (FDR) ≤ 1%). Bars represent the ratio of enrichment against the total number of proteins for each category.

Mentions: Proteins identified after OFFGEL fractionation showed a bias towards some organelles and cellular compartments, particularly “coated vesicle”, which was enriched only in NDSB-containing buffer, followed by “respiratory chain” and “cytosol” (Figure 2A). Enrichment of the latter was expected as close to 25% of all experimentally localized proteins in the SUBcellular Arabidopsis database (SUBA) are cytosolic [25]. However, since the cytosol is an aqueous environment, a large portion of its proteins is hydrophilic, suggesting enrichment for hydrophilic proteins, particularly with ND- and NDSB-containing buffers. Enrichment for “Golgi apparatus” was only detected in ND-, NDSB-, and TRIT-containing buffers. Other categories showed similar levels of enrichment in the five buffer systems, with the exception of “mitochondrion” and “endoplasmic reticulum” (ER), which showed higher enrichment in NDSB-based buffer, “respiratory chain” in NDSB- and TRIT-containing buffers and “Golgi apparatus” that was not detected in CHAPS- and SDS-based buffers. In the two-step solubilization approach, all categories were represented and importantly, these categories showed greater enrichment than the respective ND-, NDSB-, and SDS-based buffers (Figure 2). Considering specifically the “small guanosine triphosphatase (GTPase)” (19 proteins), “cell surface receptor” (32 proteins) and “Golgi vesicle transport” (11 proteins) categories, unique isoforms were identified with different buffer systems (Table S2). The NDSB-containing buffer retrieved more proteins from each of the three categories while no protein from the “Golgi vesicle transport” category was detected with ND.


Exploring the Arabidopsis proteome: influence of protein solubilization buffers on proteome coverage.

Marondedze C, Wong A, Groen A, Serrano N, Jankovic B, Lilley K, Gehring C, Thomas L - Int J Mol Sci (2014)

Enriched cellular compartments in (A) total unique and (B) membrane-associated proteins identified in the five buffer systems. Gene ontology (GO) analyses were performed using the entire dataset of identified proteins and then using the buffer-specific datasets separately. Cellular compartments that were detected as enriched are shown in the graph (p ≤ 0.05 and false discovery rate (FDR) ≤ 1%). Bars represent the ratio of enrichment against the total number of proteins for each category.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307279&req=5

ijms-16-00857-f002: Enriched cellular compartments in (A) total unique and (B) membrane-associated proteins identified in the five buffer systems. Gene ontology (GO) analyses were performed using the entire dataset of identified proteins and then using the buffer-specific datasets separately. Cellular compartments that were detected as enriched are shown in the graph (p ≤ 0.05 and false discovery rate (FDR) ≤ 1%). Bars represent the ratio of enrichment against the total number of proteins for each category.
Mentions: Proteins identified after OFFGEL fractionation showed a bias towards some organelles and cellular compartments, particularly “coated vesicle”, which was enriched only in NDSB-containing buffer, followed by “respiratory chain” and “cytosol” (Figure 2A). Enrichment of the latter was expected as close to 25% of all experimentally localized proteins in the SUBcellular Arabidopsis database (SUBA) are cytosolic [25]. However, since the cytosol is an aqueous environment, a large portion of its proteins is hydrophilic, suggesting enrichment for hydrophilic proteins, particularly with ND- and NDSB-containing buffers. Enrichment for “Golgi apparatus” was only detected in ND-, NDSB-, and TRIT-containing buffers. Other categories showed similar levels of enrichment in the five buffer systems, with the exception of “mitochondrion” and “endoplasmic reticulum” (ER), which showed higher enrichment in NDSB-based buffer, “respiratory chain” in NDSB- and TRIT-containing buffers and “Golgi apparatus” that was not detected in CHAPS- and SDS-based buffers. In the two-step solubilization approach, all categories were represented and importantly, these categories showed greater enrichment than the respective ND-, NDSB-, and SDS-based buffers (Figure 2). Considering specifically the “small guanosine triphosphatase (GTPase)” (19 proteins), “cell surface receptor” (32 proteins) and “Golgi vesicle transport” (11 proteins) categories, unique isoforms were identified with different buffer systems (Table S2). The NDSB-containing buffer retrieved more proteins from each of the three categories while no protein from the “Golgi vesicle transport” category was detected with ND.

Bottom Line: These include proteins with a role in signaling, transport, response to temperature stimuli and metabolism.This data may offer a functional bias on comparative analysis studies.In order to obtain a broader coverage, we propose a two-step solubilization protocol with first a detergent-free buffer and then a second step utilizing a combination of two detergents to solubilize proteins.

View Article: PubMed Central - PubMed

Affiliation: Biological and Environmental Sciences and Engineering Division, 4700 King Abdullah University of Science and Technology, Thuwal 23955-6900, Saudi Arabia. claudius.marondedze@kaust.edu.sa.

ABSTRACT
The study of proteomes provides new insights into stimulus-specific responses of protein synthesis and turnover, and the role of post-translational modifications at the systems level. Due to the diverse chemical nature of proteins and shortcomings in the analytical techniques used in their study, only a partial display of the proteome is achieved in any study, and this holds particularly true for plant proteomes. Here we show that different solubilization and separation methods have profound effects on the resulting proteome. In particular, we observed that the type of detergents employed in the solubilization buffer preferentially enriches proteins in different functional categories. These include proteins with a role in signaling, transport, response to temperature stimuli and metabolism. This data may offer a functional bias on comparative analysis studies. In order to obtain a broader coverage, we propose a two-step solubilization protocol with first a detergent-free buffer and then a second step utilizing a combination of two detergents to solubilize proteins.

Show MeSH