2D-LC/MS techniques for the identification of proteins in highly complex mixtures

Nägele, Edgar; Vollmer, Martin; Hörth, Patric; Vad, Cornelia

doi:10.1586/14789450.1.1.37

Cited by 96 publications

(73 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Each fraction was further separated by nanoflow reverse phase HPLC-MS/MS. Compared with online 2D LC-MS, the off-line 2D LC-MS work flow has been shown to display superior overall outcome in protein identification and sequence coverage (23,24). After merging the data obtained from different experiments, a total of 1116 unique proteins were identified to be present in both guard cells and mesophyll cells.…”

Section: Isolation Of Guard Cells and Mesophyll Cells From B Napusmentioning

confidence: 99%

Functional Differentiation of Brassica napus Guard Cells and Mesophyll Cells Revealed by Comparative Proteomics

Zhu

Dai

McClung

et al. 2009

Molecular & Cellular Proteomics

111

115

View full text Add to dashboard Cite

Guard cells (GC)1 are highly specialized cells that form tiny pores called stomata on the leaf surface. When environmental conditions change, guard cells can rapidly change shape so that the pores open or close to control leaf gas exchange and water transpiration. Mesophyll cells (MC) are mainly parenchyma cells between the upper and lower epidermis specialized for photosynthesis. Previous studies that focused on guard cell metabolism and response to environmental signals have revealed important features of functional differentiation of GC (1, 2). Compared with MC, GC contain few chloroplasts with very limited structures and thus possess very low photosynthetic capability. The Calvin cycle in GC only assimilates 2-4% of CO 2 fixed in MC (3). In contrast, GC contain abundant mitochondria and display a high respiratory rate, suggesting that oxidative phosphorylation is an important source of ATP to fuel the guard cell machinery (4). Using microarrays covering just one-third of the Arabidopsis genome, Leonhardt et al. (5) observed a differential abscisic acid (ABA) modulation of many guard cell ABA signaling components as well as key enzymes involved in carbon metabolism in GC and MC. This only available large scale genomics study identified 1309 guard cell expressed genes of which 64 transcripts mainly involved in transcription, signaling, and cytoskeleton were preferentially expressed in GC compared with MC. However, functional grouping of the genes revealed only a 1.9% higher representation of photosynthesis genes in MC than in GC. The percentages of genes in all other categories such as protein turnover, defense, signaling, channels and transporters, and metabolism are similar between the two distinct cell types (5). These proteins are known to play specific roles in guard cell functions (6). This highlights the necessity of studying guard cell functions at the protein level.To date, there have been very few analyses of single celltype proteomes in plants. Proteome analyses of trichomes from Arabidopsis (7) and tobacco (8) and root hairs from soybean (9) exist but have identified fewer than 100 proteins per proteome. The proteomes of pollen from different species have been relatively well studied, but the pollen grains are not single cells because they contain two/three-cell gametophytes (10). A critical factor for large scale proteomics analFrom the ‡Department

show abstract

Section: Isolation Of Guard Cells and Mesophyll Cells From B Napusmentioning

confidence: 99%

Functional Differentiation of Brassica napus Guard Cells and Mesophyll Cells Revealed by Comparative Proteomics

Zhu

Dai

McClung

et al. 2009

Molecular & Cellular Proteomics

111

115

View full text Add to dashboard Cite

show abstract

“…On the other hand, on-line 2-D LC-MS/MS required extensive column washing after each salt step in order to avoid ion suppression during the ESI step making the continuous online setup somewhat impractical. Another approach used online semicontinuous gradient [19,20], in which the salt gradient was splitted in order to give sufficient time to wash out the salt from the RP column. Although most reports claimed that the use of an organic modifier in offline mode or continuous gradient improved the 2-D LC separation and downstream MS/MS identifications, actually very few comparisons were made to elucidate the advantages and disadvantages of these different methods.…”

Section: Introductionmentioning

confidence: 99%

A fully automated 2‐D LC‐MS method utilizing online continuous pH and RP gradients for global proteome analysis

et al. 2007

View full text Add to dashboard Cite

The conventional 2-D LC-MS/MS setup for global proteome analysis was based on online and offline salt gradients (step and continuous) using strong-cation-exchange chromatography in conjunction with RP chromatography and MS. The use of the online system with step salt elution had the possibility of resulting in peptide overlapping across fractions. The offline mode had the option to operate with continuous salt gradient to decrease peak overlap, but exhibited decreased robustness, lower reproducibility, and sample loss during the process. Due to the extensive washing requirement between the chromatography steps, online continuous gradient was not an option for salt elution. In this report, a fully automated, online, and continuous gradient (pH continuous online gradient, pCOG) 2-D LC-MS/MS system is introduced that provided excellent separation and identification power. The pH gradient-based elution provided more basic peptides than that of salt-based elution. Fraction overlap was significantly minimized by combining pH and continuous gradient elutions. This latter approach also increased sequence coverage and the concomitant confidence level in protein identification. The salt and pH elution-based 2-D LC-MS/MS approaches were compared by analyzing the mouse liver proteome.

show abstract

“…For this purpose, the most usually used techniques are 2-dimensional electrophoresis (2-DE), off-gel fractionation, and 2-dimensional liquid chromatography (LC) [21][22][23] (Figure 3). …”

Section: Protein Separationmentioning

confidence: 99%

Proteomics and Metabolomics in Aortic Stenosis: Studying Healthy Valves for a Better Understanding of the Disease

Mouriño-Álvarez¹,

Laborde²,

Barderas³

2013

Calcific Aortic Valve Disease

View full text Add to dashboard Cite

2D-LC/MS techniques for the identification of proteins in highly complex mixtures

Cited by 96 publications

References 32 publications

Functional Differentiation of Brassica napus Guard Cells and Mesophyll Cells Revealed by Comparative Proteomics

Functional Differentiation of Brassica napus Guard Cells and Mesophyll Cells Revealed by Comparative Proteomics

A fully automated 2‐D LC‐MS method utilizing online continuous pH and RP gradients for global proteome analysis

Proteomics and Metabolomics in Aortic Stenosis: Studying Healthy Valves for a Better Understanding of the Disease

Contact Info

Product

Resources

About