alexa Optimizing the enrichment of cross-linked products for mass spectrometric protein analysis.
Bioinformatics & Systems Biology

Bioinformatics & Systems Biology

Journal of Proteomics & Bioinformatics

Author(s): Fritzsche R, Ihling CH, Gtze M, Sinz A

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Abstract RATIONALE: Chemical cross-linking in combination with a mass spectrometric analysis of the created cross-linked products is an area of growing interest for deriving low-resolution structural information of proteins and protein complexes. One of the greatest challenges is the complexity of the created cross-linking mixtures, which can be met by a charge-based enrichment of cross-linked peptides after proteolytic digestion using strong cation-exchange (SCX) chromatography. METHODS: SCX chromatography was used for the enrichment of cross-linked peptides with the N-hydroxysuccinimide ester bis(sulfosuccinimidyl)succinate (BS(3)) prior to a mass spectrometric analysis by nano-HPLC/nano-ESI-LTQ-Orbitrap-MS/MS. Bovine serum albumin (BSA) and glutathione S-transferase (GST) were employed as model proteins. RESULTS: Conditions for SCX enrichment were optimized for obtaining as many interpeptide cross-linked peptides as possible in order to maximize the amount of structural information from a single experiment. With an SCX-based enrichment step of cross-linked products within BSA using the cross-linker BS(3), 154 interpeptidal cross-linking products were identified during nano-HPLC/nano-ESI-MS/MS analyses, whereas analyses without a prior SCX enrichment allowed the identification of merely 20 cross-linked products. The application of the SCX enrichment strategy for the analysis of cross-linked products of GST with BS(3) allowed the identification of 26 interpeptidal cross-linked products compared with 16 without SCX enrichment. CONCLUSIONS: For both proteins investigated herein, BSA and GST, the introduction of an SCX-based enrichment step prior to nano-HPLC/nano-ESI-MS/MS of cross-linked products led to a considerable gain in structural information. Copyright © 2012 John Wiley & Sons, Ltd. This article was published in Rapid Commun Mass Spectrom and referenced in Journal of Proteomics & Bioinformatics

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