High Sensitivity Crosslink Detection Coupled With Integrative Structure Modeling in the Mass Spec Studio

Abstract

The Mass Spec Studio package was designed to support the extraction of hydrogen-deuterium exchange and covalent labeling data for a range of mass spectrometry (MS)-based workflows, to integrate with restraint-driven protein modeling activities. In this report, we present an extension of the underlying Studio framework and provide a plug-in for crosslink (XL) detection. To accommodate flexibility in XL methods and applications, while maintaining efficient data processing, the plug-in employs a peptide library reduction strategy via a presearch of the tandem-MS data. We demonstrate that prescoring linear unmodified peptide tags using a probabilistic approach substantially reduces search space by requiring both crosslinked peptides to generate sparse data attributable to their linear forms. The method demonstrates highly sensitive crosslink peptide identification with a low false positive rate. Integration with a Haddock plug-in provides a resource that can combine multiple sources of data for protein modeling activities. We generated a structural model of porcine transferrin bound to TbpB, a membranebound receptor essential for iron acquisition in Actinobacillus pleuropneumoniae. Using mutational data and crosslinking restraints, we confirm the mechanism by which TbpB recognizes the iron-loaded form of transferrin, and note the requirement for disparate sources of restraint data for accurate model construction. The software plugin is freely available at www.msstudio. Integrative methods in structural biology use data from disparate sources to generate accurate models of large protein structures and assemblies (1). In this way, the reach of classical structure providers such as x-ray crystallography and NMR can be extended. Biophysical data with an underlying spatial component can be combined with "building block" structures in a molecular modeling framework, to generate high-fidelity models of systems of impressive size and complexity (2-5). Mass spectrometry can provide rich sets of data in support these activities, in the form of topographical footprints (covalent labeling, CL) 1 (6 -8), conformational dynamics (hydrogen/deuterium exchange, HX) (9, 10) and distance restraints (crosslinking, XL) (11-13). We have built informatics routines within the Mass Spec Studio framework to mine restraints from both CL and HX data (14), to support such data-driven molecular modeling activities. In this study, we describe a new plug-in built into the Studio for identifying crosslinks from LC-MS/MS data sets.Advances in instrumentation, methods and cross-linking protocols have generated renewed interest in what is an older technique. However, useful informatics routines are essential for gaining access to quality crosslinking information as site identification is not a trivial problem (15). Some noteworthy tools that have emerged in the last few years include xQuest (16), Merox (17), Stavrox (18), pLink (20), XlinkX (21), and XiQ (22). The proliferation of such tools is a strong indication that new XL reagents and methods re...

Lactoferrin binding protein B – a bi-functional bacterial receptor protein

“…The ability of LbpB to bind Lf at two different sites complicates the analysis of specific interactions at the individual sites. Our recent success at using XL-MS to probe the known Tf-TbpB interaction [ 21 ] prompted us to use XL-MS to capture individual LbpB:Lf complexes and analyze the composition and interactions involved. Similarly, our prior experience at probing the TbpB:Tf interaction with hydrogen/deuterium exchange coupled to mass spectrometry (HX-MS) [ 18 , 22 , 23 ] prompted us to use this complementary approach to characterize the LbpB:hLf interaction.…”

The substrate specificity of the human TRAPPII complex’s Rab-guanine nucleotide exchange factor activity

“…MS/MS data were acquired at a resolution of 15,000 with 100 ms maximum injection time and a target AGC of 1.0 × 10 5 . All data were analyzed using the cross-linking module (CRIMP) in MS Studio v2.3.0 (www.msstudio.ca) 76 . Parameters were set as follows: charge states 4-8, peptide length 4-60, percent E-value threshold = 50, MS mass tolerance = 5 ppm, MS/MS mass tolerance = 10, elution width = 0.25 min.…”

Structural basis for loading and inhibition of a bacterial T6 SS phospholipase effector by the VgrG spike

“…The 10 most intense ions with at least three charges were selected for HCD fragmentation (NCE 24). NanoLC-MS/MS data were processed automatically using Mass Spec Studio v2.0 (Sarpe et al, 2016) with methionine oxidation as a variable modification and NNP9 modification sites: lysine, serine, threonine and tyrosine. Mass modifications were set to 314.1127 Da for cross-linked peptides and 288.1335 and 331.1393 for dead-end modifications when NNP9 reacted respectively with water or ammonium molecule.…”