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Resin-assisted enrichment of N-terminal peptides for characterizing proteolytic processing.

ABSTRACT: A resin-assisted enrichment method has been developed for specific isolation of protein N-terminal peptides to facilitate LC-MS/MS characterization of proteolytic processing, a major form of posttranslational modifications. In this method, protein thiols are blocked by reduction and alkylation, and protein lysine residues are converted to homoarginines. Protein N-termini are selectively converted to reactive thiol groups, and the thiol-containing N-terminal peptides are then captured by a thiol-affinity resin with high specificity (>97%). The efficiencies of these sequential reactions were demonstrated to be nearly quantitative. The resin-assisted N-terminal peptide enrichment approach was initially applied to a cell lysate of the filamentous fungus Aspergillus niger. Subsequent C-MS/MS analyses resulted in the identification of 1672 unique protein N-termini or proteolytic cleavage sites from 690 unique proteins.

SUBMITTER: Kim JS

PROVIDER: S-EPMC3782086 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Resin-assisted enrichment of N-terminal peptides for characterizing proteolytic processing.

Kim Jong-Seo JS Dai Ziyu Z Aryal Uma K UK Moore Ronald J RJ Camp David G DG Baker Scott E SE Smith Richard D RD Qian Wei-Jun WJ

Analytical chemistry 20130627 14

A resin-assisted enrichment method has been developed for specific isolation of protein N-terminal peptides to facilitate LC-MS/MS characterization of proteolytic processing, a major form of posttranslational modifications. In this method, protein thiols are blocked by reduction and alkylation, and protein lysine residues are converted to homoarginines. Protein N-termini are selectively converted to reactive thiol groups, and the thiol-containing N-terminal peptides are then captured by a thiol- ...[more]

PMID: 23772796

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Project description:S-Glutathionylation (SSG) is an important regulatory posttranslational modification on protein cysteine (Cys) thiols, yet the role of specific cysteine residues as targets of modification is poorly understood. We report a novel quantitative mass spectrometry (MS)-based proteomic method for site-specific identification and quantification of S-glutathionylation across different conditions. Briefly, this approach consists of initial blocking of free thiols by alkylation, selective reduction of glutathionylated thiols, and covalent capture of reduced thiols using thiol affinity resins, followed by on-resin tryptic digestion and isobaric labeling with iTRAQ (isobaric tags for relative and absolute quantitation) for MS-based identification and quantification. The overall approach was initially validated by application to RAW 264.7 mouse macrophages treated with different doses of diamide to induce glutathionylation. A total of 1071 Cys sites from 690 proteins were identified in response to diamide treatment, with ~90% of the sites displaying >2-fold increases in SSG modification compared to controls. This approach was extended to identify potential SSG-modified Cys sites in response to H2O2, an endogenous oxidant produced by activated macrophages and many pathophysiological stimuli. The results revealed 364 Cys sites from 265 proteins that were sensitive to S-glutathionylation in response to H2O2 treatment, thus providing a database of proteins and Cys sites susceptible to this modification under oxidative stress. Functional analysis revealed that the most significantly enriched molecular function categories for proteins sensitive to SSG modifications were free radical scavenging and cell death/survival. Overall the results demonstrate that our approach is effective for site-specific identification and quantification of SSG-modified proteins. The analytical strategy also provides a unique approach to determining the major pathways and cellular processes most susceptible to S-glutathionylation under stress conditions.

Project description:Nymphaea lotus L. is the medicinal plant that has long been used for food, cosmetics and traditional medicines in Africa and Asia since ancient times. Its flavonoids and other interesting phytochemical compounds from rhizome, leaf and the whole flowers have been reported in the previous published research. However, stamens, which are essential for reproductive functions, may also represent new alternative sources of potential antioxidant flavonoids, as investigated in this study. The innovative green chemistry methods, i.e., ultrasound-assisted extraction (UAE) as well as a macroporous resin (MPR) purification procedure, were employed in this current research. Using a full factorial design coupled to three-dimensional (3D) surface plot methodology, the influence of three variables, namely aqEtOH concentration (ranging from 50 to 100% (v/v), US frequency (ranging from 0 (no US applied) to 45 kHz), and the extraction duration (ranging from 20 to 60 min), were evaluated. Five MPRs with different surface areas, average pore diameters, matrix types and polarities were also investigated for the purification of total flavonoids. The optimal UAE condition is 90% (v/v) aqEtOH with 34.65 khz ultrasonic frequency and 46 min of extraction duration. Compared with the conventional heat reflux extraction (HRE) method, a significant 1.35-fold increase in total flavonoids content was obtained using optimized UAE conditions (169.64 for HRE vs. 235.45 mg/g dry weight for UAE), causing a 2.80-fold increase when this UAE associated with MPR purification (475.42 mg/g dry weight). In vitro cell free antioxidant activity of N. lotus stamen extracts and in cellulo antioxidant investigation using yeast model showed the same trend, indicating that the best antioxidant flavonoid can be found in UAE coupled with MPR purification. Moreover, in the yeast model, the expression of key antioxidant genes such as SIR2 and SOD2 were expressed at the highest level in yeast cells treated with the extract from UAE together with MPR purification. Consequently, it can be seen that the UAE combined with MPR purification can help enhance the flavonoid antioxidant potential of the stamens extract from this medicinal species.

Dataset Information

Resin-assisted enrichment of N-terminal peptides for characterizing proteolytic processing.

Publications

Resin-assisted enrichment of N-terminal peptides for characterizing proteolytic processing.

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