ABSTRACT
CID-MS/MS cleavable cross-linkers hold an enormous potential for an automated analysis of cross-linked products, which is essential for conducting structural proteomics studies. The created characteristic fragment ion patterns can easily be used for an automated assignment and discrimination of cross-linked products. To date, there are only a few software solutions available that make use of these properties, but none allows for an automated analysis of cleavable cross-linked products. The MeroX software fills this gap and presents a powerful tool for protein 3D-structure analysis in combination with MS/MS cleavable cross-linkers. We show that MeroX allows an automatic screening of characteristic fragment ions, considering static and variable peptide modifications, and effectively scores different types of cross-links. No manual input is required for a correct assignment of cross-links and false discovery rates are calculated. The self-explanatory graphical user interface of MeroX provides easy access for an automated cross-link search platform that is compatible with commonly used data file formats, enabling analysis of data originating from different instruments. The combination of an MS/MS cleavable cross-linker with a dedicated software tool for data analysis provides an automated workflow for 3D-structure analysis of proteins. MeroX is available at www.StavroX.com .
Subject(s)
Proteins/chemistry , Sequence Analysis, Protein/methods , Tandem Mass Spectrometry/methods , Cross-Linking Reagents , Models, Molecular , Proteomics , Software , User-Computer InterfaceABSTRACT
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.
Subject(s)
Chromatography, Ion Exchange/methods , Cross-Linking Reagents/isolation & purification , Glutathione Transferase/isolation & purification , Peptides/isolation & purification , Serum Albumin, Bovine/isolation & purification , Spectrometry, Mass, Electrospray Ionization/methods , Animals , Cattle , Cross-Linking Reagents/chemistry , Escherichia coli/enzymology , Glutathione Transferase/chemistry , Models, Molecular , Peptides/chemistry , Serum Albumin, Bovine/chemistry , Succinates/chemistry , Succinimides/chemistry , Tandem Mass Spectrometry/methodsABSTRACT
Chemical crosslinking in combination with mass spectrometry has matured into an alternative approach to derive low-resolution structural information of proteins and protein complexes. Yet, one of the major drawbacks of this strategy remains the lack of software that is able to handle the large MS datasets that are created after chemical crosslinking and enzymatic digestion of the crosslinking reaction mixtures. Here, we describe a software, termed StavroX, which has been specifically designed for analyzing highly complex crosslinking datasets. The StavroX software was evaluated for three diverse biological systems: (1) the complex between calmodulin and a peptide derived from Munc13, (2) an N-terminal ß-laminin fragment, and (3) the complex between guanylyl cyclase activating protein-2 and a peptide derived from retinal guanylyl cyclase. We show that the StavroX software is advantageous for analyzing crosslinked products due to its easy-to-use graphical user interface and the highly automated analysis of mass spectrometry (MS) and tandem mass spectrometry (MS/MS) data resulting in short times for analysis. StavroX is expected to give a further push to the chemical crosslinking approach as a routine technique for protein interaction studies.
