First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study

Abstract

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.

Figure 1

Overview of data sets provided by the participants of this study: 32 groups participated in this study, yielding 58 separate cross-linking workflows. Nine data sets had to be excluded due to missing replicates and nonuniform conditions, resulting in a total of 49 data sets that were further considered. Several workflows contain both insolution (47 samples) as well as in-gel digestion (10 samples) as processing methods. The samples were considered only once during a workflow analysis.

Figure 2

(a) Cross-linking reagents used in this study; noncleavable cross-linkers are presented in red, MS-cleavable cross-linkers are shown in blue, (b) reactivity, and (c) spacer length. The cross-linkers used in this study are BS³ (bis(sulfosuccinimidyl)suberate, DSS (disuccinimidylsuberate), DSP (dithiobis(succinimidylpropionate)), DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride) with and without PDH (pimelic acid dihydrazide), sulfo-SDA (sulfosuccinimidyl 4,4′-azipentanoate), CBSS (carboxy-benzophenone sulfosuccinimide), DSSO (disuccinimidylsulfoxide), DSBU (disuccinimidyldibutyric urea), BDP-NHP (N-hydroxyphthalamide ester of biotin aspartate proline), CBDPS (cyanurbiotindimercaptopropionyl succinimide), DC4 (1,4-bis(4-((2,5-dioxopyrrolidin-1-yl)oxy)-4-oxobutyl)-1,4-diazabicyclo[2.2.2]octane-1,4-diium), and MC4 (N,N′-bis(4-((2,5-dioxopyrrolidin-1-yl)oxy)-4-oxobutyl)-morpholine).

Figure 3

(a) Time, temperature, and cross-linker excess (XL-fold) were set as variable parameters, presented as gray spheres. The colored dots are projections of the 3D space onto 2D planes. (b) pH values of the cross-linking reactions ranged between 7.0 and 8.2.

Figure 4

LC/MS/MS conditions applied. (a) MS instrumentation, (b) MS resolving power, and (c) MS/MS resolving power. Resolving power is defined at m/z 200 for orbitrap instruments, while for ICR instruments it is defined at m/z 400. Please note that several research groups generated data sets with different instruments and settings.

Figure 5

(a) Software tools used in this study (a complete summary is found in Table S2, Supporting Information). Red bars indicate that the software is applicable only for noncleavable cross-linkers; blue bars indicate that the software can be used for MS-cleavable cross-linkers. (b) False discovery rates. (c) Mass tolerance MS. (d) Mass tolerance MS/MS. For the Proteome Discoverer, data analysis was performed using the XlinkX software node.

Figure 6

Number of BSA cross-links identified. The numbers of cross-links are plotted for (a) in-solution and (b) in-gel digestion workflows. The different cross-linkers are shown as symbols; abbreviations of the cross-linkers are according to Figure 2. The maximum distances are given for each cross-linker, indicating the number of overlength cross-links. Every point is a sum of three replicate measurements; replicates of the entire experiment are shown in blue, and replicates of the LC/MS analyses are shown in red; the average number and reproducibility of unique cross-links are shown in yellow.

Figure 7

Comparison of unique cross-links. “Cross-link” denotes the unique amino acid residues that are connected by homobifunctional, amine-reactive cross-linkers. (a) Number of cross-links with respect to their reproducibility among the data sets. (b) Linear extrapolation of all (red) or a linear subset (blue) of cross-links resulted in a maximum cross-linking number between 73 and 88. (c) Plot summarizes the intercepts with the y-axis (red) and the correlation coefficient × 100 (blue) of the respective linear extrapolations of part a. The linear extrapolation was calculated as shown in part b by successively removing the data points starting from the lowest reproducible value.