A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues

Saravanan, Ramu S.; Rose, Jocelyn K. C.

doi:10.1002/pmic.200300789

Cited by 367 publications

(391 citation statements)

References 26 publications

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“…18 Efficient extraction of proteins is one of the most critical issues for proteome analysis. 19 Since bone is almost solid, which is physiologically mineralized, the classical protein extraction methods for soft tissues and cells may not be efficient for bone tissue. Therefore, it is necessary to develop efficient methods for protein extraction from bone tissue.…”

Section: Introductionmentioning

confidence: 99%

Method Development of Efficient Protein Extraction in Bone Tissue for Proteome Analysis

et al. 2007

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Exploring bone proteome is an important and challenging task for understanding the mechanisms of physiological/pathological process of bone tissue. However, classical methods of protein extraction for soft tissues and cells are not applicable for bone tissue. Therefore, method development of efficient protein extraction is critical for bone proteome analysis. We found in this study that the protein extraction efficiency was improved significantly when bone tissue was demineralized by hydrochloric acid (HCl). A sequential protein extraction method was developed for large-scale proteome analysis of bone tissue. The bone tissue was first demineralized by HCl solution and then extracted using three different lysis buffers. As large amounts of acid soluble proteins also presented in the HCl solution, besides collection of proteins in the extracted lysis buffers, the proteins in the demineralized HCl solution were also collected for proteome analysis. Automated 2D-LC-MS/MS analysis of the collected protein fractions resulted in the identification of 6202 unique peptides which matched 2479 unique proteins. The identified proteins revealed a broad diversity in the protein identity and function. More than 40 bone-specific proteins and 15 potential protein biomarkers previously reported were observed in this study. It was demonstrated that the developed extraction method of proteins in bone tissue, which was also the first large-scale proteomic study of bone, was very efficient for comprehensive analysis of bone proteome and might be helpful for clarifying the mechanisms of bone diseases.

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Section: Introductionmentioning

confidence: 99%

Method Development of Efficient Protein Extraction in Bone Tissue for Proteome Analysis

et al. 2007

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“…Although fewer research groups adopt this protocol, the procedure was recently judged more efficient in terms of the number of proteins detected in tomato root tissue, when compared to the number extracted with the standard trichloroacetic acid/acetone method [25]. Despite this result, the type of protocol employed is in fact greatly dependent upon the type of tissue and the subsequent purification strategies.…”

Section: Sample Preparationmentioning

confidence: 92%

Rooteomics: The Challenge of Discovering Plant Defense-Related Proteins in Roots

Mehta¹,

Magalhães²,

Souza³

et al. 2008

CPPS

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Abstract:In recent years, a strong emphasis has been given in deciphering the function of genes unraveled by the completion of several genome sequencing projects. In plants, functional genomics has been massively used in order to search for gene products of agronomic relevance. As far as root-pathogen interactions are concerned, several genes are recognized to provide tolerance/resistance against potential invaders. However, very few proteins have been identified by using current proteomic approaches. One of the major drawbacks for the successful analysis of root proteomes is the inherent characteristics of this tissue, which include low volume content and high concentration of interfering substances such as pigments and phenolic compounds. The proteome analysis of plant-pathogen interactions provides important information about the global proteins expressed in roots in response to biotic stresses. Moreover, several pathogenic proteins superimpose the plant proteome and can be identified and used as targets for the control of viruses, bacteria, fungi and nematode pathogens. The present review focuses on advances in different proteomic strategies dedicated to the challenging analysis of plant defense proteins expressed during bacteria-, fungi-and nematode-root interactions. Recent developments, limitations of the current techniques, and technological perspectives for root proteomics aiming at the identification of resistance-related proteins are discussed.

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“…An equal volume of phenol was then added and the samples were rehomogenized and centrifuged at 10 0006g for 30 min. The upper phenol phase was re-extracted twice with the above extraction buffer as reported previously [25]. Proteins were precipitated from the final phenol phase with five volumes of acetone at 2207C overnight and washed with cold methanol and acetone.…”

Section: Preparation Of Cauliflower Protein Extractsmentioning

confidence: 99%

Development of an integrated approach for evaluation of 2‐D gel image analysis: Impact of multiple proteins in single spots on comparative proteomics in conventional 2‐D gel/MALDI workflow

Yang¹,

Thannhauser²,

Li³

et al. 2007

Electrophoresis

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With 2-D gel mapping, it is often observed that essentially identical proteins migrate to different positions in the gel, while some seemingly well-resolved protein spots consist of multiple proteins. These observations can undermine the validity of gel-based comparative proteomic studies. Through a comparison of protein identifications using direct MALDI-TOF/ TOF and LC-ESI-MS/MS analyses of 2-D gel separated proteins from cauliflower florets, we have developed an integrated approach to improve the accuracy and reliability of comparative 2-D electrophoresis. From 46 spots of interest, we identified 51 proteins by MALDI-TOF/TOF analysis and 108 proteins by LC-ESI-MS/MS. The results indicate that 75% of the analyzed spots contained multiple proteins. A comparison of hit rank for protein identifications showed that 37 out of 43 spots identified by MALDI matched the top-ranked hit from the ESI-MS/MS. By using the exponentially modified protein abundance index (emPAI) to determine the abundance of the individual component proteins for the spots containing multiple proteins, we found that the top-hit proteins from 40 out of 43 spots identified by MALDI matched the most abundant proteins determined by LC-MS/MS. Furthermore, our 2-D-GeLC-MS/MS results show that the top-hit proteins in 44 identified spots contributed on average 81% of the spots' staining intensity. This is the first quantitative measurement of the average rate of false assignment for direct MALDI analysis of 2-D gel spots using a new integrated workflow (2-D gel imaging, "2-D GeLC-MS/MS", and emPAI analysis). Here, the new approach is proposed as an alternative to traditional gel-based quantitative proteomics studies.

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A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues

Cited by 367 publications

References 26 publications

Method Development of Efficient Protein Extraction in Bone Tissue for Proteome Analysis

Method Development of Efficient Protein Extraction in Bone Tissue for Proteome Analysis

Rooteomics: The Challenge of Discovering Plant Defense-Related Proteins in Roots

Development of an integrated approach for evaluation of 2‐D gel image analysis: Impact of multiple proteins in single spots on comparative proteomics in conventional 2‐D gel/MALDI workflow

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