Differential Tandem Mass Spectrometry-Based Cross-Linker: A New Approach for High Confidence in Identifying Protein Cross-Linking

Abstract

Chemical cross-linking and mass spectrometry are now widely used to analyze large-scale protein-protein interactions. The major challenge in cross-linking approaches is the complexity of the mass spectrometric data. New approaches are required that can identify cross-linked peptides with high-confidence and establish a user-friendly analysis protocol for the biomedical scientific community. Here, we introduce a novel cross-linker that can be selectively cleaved in the gas phase using two differential tandem mass-spectrometric fragmentation methods, such as collision-induced or electron transfer dissociation (CID and ETD). This technique produces two signature mass spectra of the same cross-linked peptide, thereby producing high confidence in identifying the sites of interaction. Further tandem mass spectrometry can also give additional confidence on the peptide sequences. We demonstrate a proof-of-concept for this method using standard peptides and proteins. Peptides and proteins were cross-linked and their fragmentation characteristics were analyzed using CID and ETD tandem mass spectrometry. Two sequential cleavages unambiguously identified cross-linked peptides. In addition, the labeling efficiency of the new cross-linker was evaluated in macrophage immune cells after stimulation with the microbial ligand lipopolysaccharide and subsequent pulldown experiments with biotin-avidin affinity chromatography. We believe this strategy will help advance insights into the structural biology and systems biology of cell signaling.

Cross-linking mass spectrometry: methods and applications in structural, molecular and systems biology

“…Consequently, considerable effort has been expended on creating crosslinkers that are cleavable in the mass spectrometer so that spectra simply correspond to two modified peptides [94][95][96][97][98][99][100][101][102][103][104][105] .…”

Inflammatory Proteomic Network Analysis of Statin-treated and Lipopolysaccharide-activated Macrophages

“…According to Chakrabarty et al . 55 , proteins were reduced and alkylated, then digested with Trypsin (MS Grade) at a 1:50 enzyme/protein concentration for 16 h at 37 °C. Formic acid (pH < 3) was added to the resulting peptides for acidifying the sample.…”

Cross-linking Proteomics Indicates Effects of Simvastatin on the TLR2 Interactome and Reveals ACTR1A as a Novel Regulator of the TLR2 Signal Cascade

“…Cells were treated with 10 M simvastatin (Sigma) for 24 h, then stimulated with 1 g/ml Pam3CSK4 (P3C; InvivoGen) for 24 h in fresh medium. The cells were then treated with Dual Cleavable Cross-linking Technology (DUCCT) (29) or commercial bissulfosuccinimidyl suberate (BS3) cross-linker (XL), added at a final concentration of 1mol/ml for 30 min, followed by quenching the reaction with 50 mM Tris-HCl, pH 8.0. For IP-pull down for proteomics, the cells were lysed with immunoprecipitation (IP)-lysis buffer supplemented with protease inhibitors at 4°C for 15 mins, then sonicated for another 15 mins.…”