Optimized Cross-Linking Mass Spectrometry for in Situ Interaction Proteomics

Abstract

Recent development of mass spectrometer cleavable protein cross-linkers and algorithms for their spectral identification now permits large-scale cross-linking mass spectrometry (XL-MS). Here, we optimized the use of cleavable disuccinimidyl sulfoxide (DSSO) cross-linker for labeling native protein complexes in live human cells. We applied a generalized linear mixture model to calibrate cross-link peptide-spectra matching (CSM) scores to control the sensitivity and specificity of large-scale XL-MS. Using specific CSM score thresholds to control the false discovery rate, we found that higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) can both be effective for large-scale XL-MS protein interaction mapping. We found that the coverage of protein-protein interaction maps is significantly improved through the use of multiple proteases. In addition, the use of focused sample-specific search databases can be used to improve the specificity of cross-linked peptide spectral matching. Application of this approach to human chromatin labeled in live cells recapitulated known and revealed new protein interactions of nucleosomes and other chromatin-associated complexes in situ. This optimized approach for mapping native protein interactions should be useful for a wide range of biological problems.

Keywords: BSA; chromatin; cross-linking; database search; false positive discovery; mass spectrometry; protein−protein interactions; proteomics; target-decoy strategy.

Figure 1.

Calibration of cross-link peptide-spectra matching (CSM) scores to control the sensitivity and specificity of large-scale XL-MS. (A) Workflow for preparation of cross-linked BSA peptides and cross-linked BSA peptides spiked into non-cross-linked proteome background peptides. (B) High number of BSA cross-linked peptides (red) and non-BSA cross-linked peptides (black) identified across fragmentation methods and in both cross-linked BSA and cross-linked BSA spiked into background. (C) Fitted Gaussian distributions of cross-link spectra matching score of non-BSA cross-links and BSA cross-links for cross-linked BSA only sample acquired with CID-MS2/HCD-MS2 fragmentation. (D) Percentile plot of Gaussian distributions of CSM scores with 1% and 10% score filter to eliminate 99% and 90% of non-BSA cross-links. (E) Comparison of number of cross-links identified when no score filter is applied and when 1% score filter is applied. (F) Cross-links violating physical distance constraint of cross-linker (red) are eliminated when 1% score filter is applied when mapping cross-links to crystal structure (PDB ID 4f5s). Dotted line indicates maximum Cα–Cα distance of 26 Å for DSSO.

Figure 2.

Improved cross-linked peptide identification using CID-MS2/HCD-MS2 fragmentation. Comparison of 4 different fragmentation methods: CID-MS2/HCD-MS2, CID-MS2/ETD-MS2, CID-MS2/EThcD-MS2, CID-MS2/HCD-MS3, and CID-MS2-MS3/ETD-MS2 and databases: BSA protein specific database or human proteome database with BSA for increasing cross-link peptide identification. (A) Comparison between 5 fragmentation methods for average number of cross-link identifications of cross-linked BSA sample. BSA cross-links colored red and non-BSA cross-links colored black. Error bars represent standard deviation from three biological replicates. (B) Comparison between 5 fragmentation methods for overlap of all unique cross-link identifications. (C) Comparison between 5 fragmentation methods for average number of cross-link identifications of cross-linked BSA peptides spiked into human proteome background. (D) Comparison between 5 fragmentation methods for overlap of all unique cross-link identifications of cross-linked BSA sample spiked into human proteome background. (E) Comparison between 2 databases for average number of cross-link identifications of cross-linked BSA peptides and cross-linked BSA peptides spiked into human proteome background. (F) Comparison between 2 databases for overlap of all unique cross-link identifications of cross-linked BSA peptides and cross-linked BSA peptides spiked into human proteome background.

Figure 3.

Multiple proteases to expand the coverage and density of DSSO XL-MS protein interaction maps. (A) Distribution of CSM score for identified cross-linked peptides from cross-linked BSA digested with trypsin only or sequential LysC and trypsin or GluC only or chymotrypsin only proteases. Cross-linked BSA peptides were spiked into non-cross-linked proteome background digested with the corresponding protease. (B) Overlap of cross-linked lysine sites identified from trypsin only or sequential LysC and trypsin or GluC only or chymotrypsin only protease digestion. (C) Visualization of BSA cross-link sites identified from trypsin only, LysC and trypsin, GluC only or chymotrypsin only protease digestion, as analyzed using CID-MS2/HCD-MS2 fragmentation. Cross-links with a CSM score above the 1% score filter are highlighted in black.

Figure 4.

Native chromatin protein–protein interactions identified using cross-linking mass spectrometry in situ. (A) Purified chromatin bound proteins are cross-linked by cross-linking in situ. (B) Enrichment of histone protein (H3K4me3) with absence of nuclear protein (BRG1) and cytoplasmic protein (GAPDH) from chromatin bound proteins by Western blot. (C) Cross-links identified map onto known high resolution nucleosome structure (PDB ID 3av1). (D) Map of protein–protein interactions between core histone proteins (H2A, H2B, H3, H4) colored according to CSM score. (E) Map of protein–protein interactions involving linker histone H1. Proteins known to bind DNA are marked in blue.

Figure 5.

Chromatin interactions of proteins involved in DNA damage repair and ribonucleoproteins. (A) DNA repair protein PARP1 interacts with Histone H2B. (B) PARP1 cross-links map onto known high resolution structure of PARP1 bound to DNA double stranded break (PDB ID 4dqy). (C) Protein–protein interactions of RNA-binding heterogeneous nuclear ribonucleoproteins (hnRNPs).