A High-throughput Approach for Subcellular Proteome

Jiang, Xueying; Zhou, Hu; Zhang, Lei; Sheng, Quanhu; Li, Su-Jun; Li, Long; Hao, Pei; Li, Yixue; Xia, Qi-Chang; Wu, Jiarui; Zeng, Rong

doi:10.1074/mcp.m300117-mcp200

Cited by 74 publications

(34 citation statements)

References 54 publications

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“…Orthogonal 2-D LC-MS/MS was performed using a 2-D LCNano-LTQ workstation (Thermo Fisher Scientific, San Jose, CA, USA) [12,13]. The system comprised an SCX column (320 mm id6100 mm, Column Technology), two C18 RP trap columns (320 mm620 mm, C18, 5 mm, Column Technology), and one C18 RP capillary column (75 mm6150 mm, C18, 5 mm, Column Technology).…”

Section: -D Lc-ms/msmentioning

confidence: 99%

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

et al. 2007

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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.

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Section: -D Lc-ms/msmentioning

confidence: 99%

“…Tryptic peptides were first loaded onto the SCX and directly eluted onto the C18 column by salt step gradient. The two C18 columns were used alternatively either for loading or for separation [12][13][14].…”

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

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“…PSORT shows 59.5% accuracy with 50/84 proteins rightly predicted, while SubLoc shows 57.1% accuracy with 48/84 proteins rightly predicted. When proteins annotated as cytoplasmic, nuclear, and so on are considered as other location, TargetP shows an accuracy up to 81.0% with 68/84 proteins rightly predicted.We have shown that combinational usage of different bioinformatics tools can increase specificity for subcellular location prediction [24,25]. For the 34 proteins with no subcellular location annotation, about 50% (17/ 34) of them are predicted as cytoplasmic (10/34) or nuclear (7/34) by at least two bioinformatics tools at the same time.…”

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confidence: 99%

Two-dimensional gel electrophoresis maps of the proteome and phosphoproteome of primitively cultured rat mesangial cells

et al. 2005

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Mesangial cells (MC) play an important role in maintaining the structure and function of the glomerulus. The proliferation of MC is a prominent feature of many kinds of glomerular disease. The first reference 2-DE maps of rat mesangial cells (RMC), stained with silver staining or Pro-Q Diamond dye, have been established here to describe the proteome and phosphoproteome of RMC, respectively. A total of 157 selected protein spots, corresponding to 118 unique proteins, have been identified by MALDI-TOF-MS or LC-ESI-IT-MS/MS, in which 37 protein spots representing 28 unique proteins have also been stained with Pro-Q Diamond, indicating that they are in phosphorylated forms. All the identified proteins were bioinformatically annotated in detail according to their physiochemical characteristics, subcellular location, and function. Most of the separated or identified protein spots are distributed in the area of mass 10-70 kDa and pI 5.0-8.0. The identified proteins include mainly cytoplasmic and nuclear proteins and some mitochondrial, endoplasmic reticulum, and membrane proteins. These proteins are classified into different functional groups such as structure and mobility proteins (21.2%), metabolic enzymes (16.9%), protein folding and metabolism proteins (13.6%), signaling proteins (14.4%), heat-shock proteins (7.6%), and other functional proteins (12.7%). While structure and mobility proteins are mostly represented by protein spots with high abundance, signaling proteins are mostly represented by protein spots with relatively low abundance. Such a 2-DE database for RMC, especially with many signaling proteins and phosphoproteins characterized, will provide a valuable resource for comparative proteomics analysis of normal and pathologic conditions affecting MC function or pathologic progress.

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“…Distribution of proteins identified with GGE strategy in fractionation and sealing gel layers. Electrophoresis 2012, 33,[316][317][318][319][320][321][322][323][324] in the TSGE and TGD strategies, respectively, fell outside the typical limits of protein resolution by conventional IEF/ 2D-gel electrophoresis (r100 kDa; pI 4.5-8.5) [34,35] (Fig. 6).…”

Section: Comparative Characterization Of Membrane Proteins Identifiedmentioning

confidence: 99%

Electrophoretically driven SDS removal and protein fractionation in the shotgun analysis of membrane proteomes

et al. 2012

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SDS is mostly used to enhance the solubilization and extraction of membrane proteins due to its strong detergency and low cost. Nevertheless, SDS interferes with the subsequent procedures and needs to be removed from the samples. In this work, a special gradient gel electrophoresis (GGE) system was developed to remove SDS from the SDS-solubilized protein samples. As a proof-of-principle experiment, the GGE system was designed to be composed of an agarose loading layer, six polyacrylamide fractionation layers with different concentrations and a high-concentration polyacrylamide sealing layer. The advantages of the GGE system are that it not only can electrophoretically remove SDS efficiently so that the protein loss resulted from the repeated gel washing after electrophoresis was avoided, but also can reduce the complexity of the sample, prevent the precipitation of proteins after loading and avoid the loss of proteins with low molecular weight during the electrophoresis. Using GGE system, about 85% of SDS in the sample and gel was electrophoretically removed and the proteins were fractionated. Compared with the two representative gel-based sample cleanup methods reported in literature, GGE-based strategy significantly improved the identification efficiency of proteins in terms of the number and coverage of the identified proteins.

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A High-throughput Approach for Subcellular Proteome

Cited by 74 publications

References 54 publications

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

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

Two-dimensional gel electrophoresis maps of the proteome and phosphoproteome of primitively cultured rat mesangial cells

Electrophoretically driven SDS removal and protein fractionation in the shotgun analysis of membrane proteomes

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