Novel Application of Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) in Shotgun Proteomics: Comprehensive Profiling of Rat Kidney Proteome

Hao, Piliang; Guo, Tiannan; Li, Xin; Adav, Sunil S.; Yang, Jie; Wang, Meng

doi:10.1021/pr100037h

Cited by 84 publications

(91 citation statements)

References 39 publications

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“…Two hundred micrograms of peptides from each condition were individually labeled with respective isobaric tags (control sample, 114; iberin-treated sample, 115), followed by 2 h of incubation, quenching by water, desalting using C 18 solid-phase extraction cartridge, and then vacuum centrifugation to dryness. The iTRAQ-labeled peptides were reconstituted in buffer A (10 mM ammonium acetate, 85% acetonitrile, 0.1% formic acid) and fractionated using an electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) column (200 by 4.6 mm, 5-m particle size, 200-Å pore size) by a high-performance liquid chromatography (HPLC) system (Shimadzu, Japan) at flow rate of 1.0 ml/min, using our previously optimized protocol (20). The HP liquid chromatograms were recorded at 280 nm, and fractions were collected online using an automated fraction collector.…”

Section: Methodsmentioning

confidence: 99%

Comparative Systems Biology Analysis To Study the Mode of Action of the Isothiocyanate Compound Iberin on Pseudomonas aeruginosa

Tan

Liu

Chua

et al. 2014

Antimicrob Agents Chemother

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Food is now recognized as a natural resource of novel antimicrobial agents, including those that target the virulence mechanisms of bacterial pathogens. Iberin, an isothiocyanate compound from horseradish, was recently identified as a quorum-sensing inhibitor (QSI) of the bacterial pathogen Pseudomonas aeruginosa. In this study, we used a comparative systems biology approach to unravel the molecular mechanisms of the effects of iberin on QS and virulence factor expression of P. aeruginosa. Our study shows that the two systems biology methods used (i.e., RNA sequencing and proteomics) complement each other and provide a thorough overview of the impact of iberin on P. aeruginosa. RNA sequencing-based transcriptomics showed that iberin inhibits the expression of the GacA-dependent small regulatory RNAs RsmY and RsmZ; this was verified by using gfp-based transcriptional reporter fusions with the rsmY or rsmZ promoter regions. Isobaric tags for relative and absolute quantitation (iTRAQ) proteomics showed that iberin reduces the abundance of the LadS protein, an activator of GacS. Taken together, the findings suggest that the mode of QS inhibition in iberin is through downregulation of the Gac/Rsm QS network, which in turn leads to the repression of QS-regulated virulence factors, such as pyoverdine, chitinase, and protease IV. Lastly, as expected from the observed repression of small regulatory RNA synthesis, we also show that iberin effectively reduces biofilm formation. This suggests that small regulatory RNAs might serve as potential targets in the future development of therapies against pathogens that use QS for controlling virulence factor expression and assume the biofilm mode of growth in the process of causing disease. Q uorum sensing (QS) is a cell-to-cell communication system widely distributed among bacteria in which small diffusible signal molecules are employed to regulate gene expression in a dose-dependent manner (1). After reaching a threshold concentration, the QS signal molecules will bind to and activate their receptors, which results in a coordinated population expression of QS-regulated genes. These genes include those that upregulate the synthesis of QS signal molecules (autoinduction) but, more importantly, they also include genes that encode virulence factors required for bacterial infections (2). Thus, QS inhibitors (QSIs) have been proposed as antipathogenic agents and have been shown to attenuate the capability of pathogens to cause infections (3, 4). QSIs possess different modes of action, including interfering with the synthesis of quorum-sensing signaling molecules (5) or competitively binding to the QS signal receptors (6). The regulation of the bacterial QS systems is complex, and this further expands the targets for the design of novel QSIs (7,8).Isothiocyanates (ITCs) are biologically active compounds found in cruciferous vegetables and have gained research interest as cancer chemopreventive agents (9). Sulforaphane (SFN) (10), allyl isothiocyanate (AITC) (11), and phenethyl isothioc...

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Section: Methodsmentioning

confidence: 99%

Comparative Systems Biology Analysis To Study the Mode of Action of the Isothiocyanate Compound Iberin on Pseudomonas aeruginosa

Tan

Liu

Chua

et al. 2014

Antimicrob Agents Chemother

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Section: Introductionmentioning

confidence: 99%

“…To this effect, various techniques of peptide separation prior to reverse phase liquid chromatography (RPLC) hyphenated to mass spectrometry (MS) detection are employed: electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) [18,19], hydrophilic interaction chromatography (HILIC) [20], ion exchange chromatography (IEX) (e.g., strong cation exchange [SCX]) [21], and RPLC with alkaline and acidic mobile phases [22,23] three-dimensional sample separations were also envisaged and developed for different purposes: reverse phase (RP)-SCX-RP approach allowing higher detection of hydrophilic peptides and consequently enhancing the quality of protein identification and quantification [24,25], three-dimensional (3D)-LC/MS setup for enrichment and identification of biotinylated proteins [26], and combinatorial use of SCX (ERLIC-SCX) chromatographic separations for phosphoproteome studies [27].…”

Section: Introductionmentioning

confidence: 99%

Comparison of fractionation strategies for offline two-dimensional liquid chromatography tandem mass spectrometry analysis of proteins from mouse adipose tissue

Sajic

Varesio

Szántó

et al. 2015

Analytical Biochemistry

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a b s t r a c tIn the frame of protein identification from mouse adipose tissue, two strategies were compared for the offline elution of peptides from a strong cation exchange (SCX) column in two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) analyses. First, the salt gradient (using K + as displacing agent) was evaluated from 25 to 500 mM KCl. Then, a less investigated elution mode using a pH gradient (using citric acid and ammonium hydroxide) was carried out from pH 2.5 to 9.0. Equal amounts of peptide digest derived from mouse adipose tissue were loaded onto the SCX column and fractionated according to the two approaches. A total of 15 fractions were collected in two independent experiments for each SCX elution strategy. Then, each fraction was analyzed on a nanoLC-MS/MS platform equipped with a column-switching unit for desalting and enrichment. No substantial differences in peptide quality characteristics (molecular weight, isoelectric point, or GRAVY [grand average of hydropathicity] index distributions) were observed between the two datasets. The pH gradient approach was found to be superior, with 27.5% more unique peptide identifications and 10% more distinct protein identifications compared with the salt-based elution method. In conclusion, our data imply that the pH gradient SCX fractionation is more desirable for proteomics analysis of entire adipose tissue.Ó 2015 Elsevier Inc. All rights reserved.White adipose tissue (WAT) 3 is a complex metabolic and endocrine organ that secrets a variety of hormones, termed adipokines, into the circulation and thus regulates glucose, lipid, and energy homeostasis in mammalian organisms [1]. Pathological alterations in these various functions are well-established risk factors for metabolic disorders such as obesity, insulin resistance, and type 2 diabetes [2,3]. Investigations aimed at uncovering modifications in WAT protein expression are of important clinical interest in the quest to identify new therapeutic targets or candidate biomarkers for these prevalent pathologies [4,5]. Scientific prominence of adipose tissue proteomics along with an increasing number of adipose tissue proteomics studies per year [6][7][8][9][10][11][12][13][14] was recently observed by Peinado and coworkers [15]. The main hurdles in the isolation and identification of adipose tissue proteins are its high lipid content combined with low protein amount and a high proteome complexity related to significant tissue vascularization and the presence of a multitude of functionally important cell types located in different tissue fractions [4,16,17].Reducing protein sample complexity in two-dimensional liquid chromatography (2D-LC) investigations is a crucial issue. To this effect, various techniques of peptide separation prior to reverse phase liquid chromatography (RPLC) hyphenated to mass spectrometry (MS) detection are employed: electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) [18,19], hydrophilic interaction chromatography (HILIC) [20], ion exch...

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“…17 Hao et al extended it to the fractionation of complex peptide mixtures such as whole tryptic digests. 18,19 This was effected by starting with so high a level of ACN -initially, 90% -that hydrophilic interaction was considerably stronger than electrostatic repulsion. With 0.1% acetic acid present, permitting the ionization of whatever carboxyl groups are present in the peptides, then even basic peptides can be retained to some extent.…”

mentioning

confidence: 99%

“…With this combination there is a uniform distribution of peptides among the collected fractions, with peptides eluting in order of decreasing isoelectric point and increasing hydrophilicity. [18][19][20] This order of elution is comparable in some respects to that of isoelectric focusing but is accomplished without ampholines or a complex mixture of buffering salts. The separation of peptides based on isoelectric point has been exploited for the study of protein/ peptide deamidation 21,22 as the original residue, Asn, and its deamidated products n-Asp and isoAsp have different pKa values.…”

mentioning

confidence: 99%

The Use of Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) for Proteomics Research

Ng¹,

Hao²,

Sze³

2014

Mass Spectrometry Letters

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Characterization and studies of proteome are challenging because biological samples are complex, with a wide dynamic range of abundance. At present the proteins are identified by digestion into peptides, with subsequent identification of the peptides by mass spectrometry (MS). MS is a powerful technique for the purpose, but it cannot identify every peptide in such complex mixtures simultaneously. For accurate analysis and quantification it is important to separate the peptides first by chromatography into fractions of a size that MS can handle. With these less complex fractions, the probability is increased of identifying peptides of low abundance that would otherwise experience ion suppression effects due to the presence of peptides of high abundance. Enrichment for peptides with certain post-translational modifications helps to increase their detection rates as well. Electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) is a mixed-mode chromatographic technique which combines the use of electrostatic repulsion and hydrophilic interaction. This review provides an overview of ERLIC and its various proteomics applications. ERLIC has been demonstrated to have good orthogonality to reverse phase liquid chromatography (RPLC), making it useful as a first dimension in multidimensional liquid chromatography (MDLC) and fractionation of digests in general. Peptides elute in order of their isoelectric points and polarity. ERLIC has also been successfully utilized for the enrichment for phosphopeptides and glycopeptides, facilitating their identification. In addition, it is promising for the study of peptide deamidation. ERLIC performs comparably well or better than established methods for these various applications, and serves as a viable and efficient workflow alternative.

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Novel Application of Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) in Shotgun Proteomics: Comprehensive Profiling of Rat Kidney Proteome

Cited by 84 publications

References 39 publications

Comparative Systems Biology Analysis To Study the Mode of Action of the Isothiocyanate Compound Iberin on Pseudomonas aeruginosa

Comparative Systems Biology Analysis To Study the Mode of Action of the Isothiocyanate Compound Iberin on Pseudomonas aeruginosa

Comparison of fractionation strategies for offline two-dimensional liquid chromatography tandem mass spectrometry analysis of proteins from mouse adipose tissue

The Use of Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) for Proteomics Research

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