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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.

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Number of predicted (A) post-translation modifications (PTMs) and (B) sites for individual buffer systems and the two-step solubilization process. During protein identification in the MASCOT and Scaffold, acetylation, oxidation, phosphorylation and palmitoylation were set as variable modifications. Bars represent the (A) number of proteins and (B) PTM sites that contain the PTM of interest.
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ijms-16-00857-f004: Number of predicted (A) post-translation modifications (PTMs) and (B) sites for individual buffer systems and the two-step solubilization process. During protein identification in the MASCOT and Scaffold, acetylation, oxidation, phosphorylation and palmitoylation were set as variable modifications. Bars represent the (A) number of proteins and (B) PTM sites that contain the PTM of interest.

Mentions: Looking at the entire set of identified proteins from each buffer system tested after OFFGEL fractionation, acetylated, oxidized, phosphorylated and palmitoylated sites were detected by MASCOT and Scaffold (p ≥ 95%; Figure 4A,B). Greater numbers of these four PTMs were predicted after the two-step solubilization process and NDSB-containing buffer, with 856 and 746 acetylated sites in 628 and 582 proteins, 1992 and 1163 oxidized sites in 993 and 653 proteins, 335 and 304 phosphosites in 232 and 202 phosphorylated proteins and 135 and 121 palmitoylated sites located in 87 and 82 proteins, respectively. Detection of phosphorylated and palmitoylated protein sites was limited in all samples, but was higher with the two-step solubilization process and NDSB-containing buffer (Figure 4B). Predictions of other PTMs were carried out using the Big-PI plant predictor for glycosylphosphatidylinositol (GPI) site detection, and this suggested that most proteins containing GPI modifications also possessed palmitoylation sites (Table S3). Furthermore, myristoylated sites were predicted with the myristoylation (MYR) predictor tool locating 100 MYR signals of which, 16 contained GPI sites. Interestingly, 14 of these 16 predicted myristoylated proteins also contained palmitoylation sites (Table S4). Taken together and based on these selected PTMs, the data implies that the two-step solubilization process allowed for the detection of the highest number of predicted PTMs, suggesting that this combination of buffers may be better suited for PTM-based proteomic studies. Interestingly, for all four PTMs considered, sites of modification outnumbered the number of proteins, indicating that the recovery of multiple modified proteins was achieved.


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)

Number of predicted (A) post-translation modifications (PTMs) and (B) sites for individual buffer systems and the two-step solubilization process. During protein identification in the MASCOT and Scaffold, acetylation, oxidation, phosphorylation and palmitoylation were set as variable modifications. Bars represent the (A) number of proteins and (B) PTM sites that contain the PTM of interest.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-00857-f004: Number of predicted (A) post-translation modifications (PTMs) and (B) sites for individual buffer systems and the two-step solubilization process. During protein identification in the MASCOT and Scaffold, acetylation, oxidation, phosphorylation and palmitoylation were set as variable modifications. Bars represent the (A) number of proteins and (B) PTM sites that contain the PTM of interest.
Mentions: Looking at the entire set of identified proteins from each buffer system tested after OFFGEL fractionation, acetylated, oxidized, phosphorylated and palmitoylated sites were detected by MASCOT and Scaffold (p ≥ 95%; Figure 4A,B). Greater numbers of these four PTMs were predicted after the two-step solubilization process and NDSB-containing buffer, with 856 and 746 acetylated sites in 628 and 582 proteins, 1992 and 1163 oxidized sites in 993 and 653 proteins, 335 and 304 phosphosites in 232 and 202 phosphorylated proteins and 135 and 121 palmitoylated sites located in 87 and 82 proteins, respectively. Detection of phosphorylated and palmitoylated protein sites was limited in all samples, but was higher with the two-step solubilization process and NDSB-containing buffer (Figure 4B). Predictions of other PTMs were carried out using the Big-PI plant predictor for glycosylphosphatidylinositol (GPI) site detection, and this suggested that most proteins containing GPI modifications also possessed palmitoylation sites (Table S3). Furthermore, myristoylated sites were predicted with the myristoylation (MYR) predictor tool locating 100 MYR signals of which, 16 contained GPI sites. Interestingly, 14 of these 16 predicted myristoylated proteins also contained palmitoylation sites (Table S4). Taken together and based on these selected PTMs, the data implies that the two-step solubilization process allowed for the detection of the highest number of predicted PTMs, suggesting that this combination of buffers may be better suited for PTM-based proteomic studies. Interestingly, for all four PTMs considered, sites of modification outnumbered the number of proteins, indicating that the recovery of multiple modified proteins was achieved.

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