Crosslinking mass spectrometry unveils novel interactions and structural distinctions in the model green alga Chlamydomonas reinhardtii

Interactomics is an emerging field that seeks to identify both transient and complex-bound protein interactions that are essential for metabolic functions. Crosslinking mass spectrometry (XL-MS) has enabled untargeted global analysis of these protein networks, permitting largescale simultaneous analysis of protein structure and interactions. Increased commercial availability of highly specific, cell permeable crosslinkers has propelled the study of these critical interactions forward, with the development of MS-cleavable crosslinkers further increasing confidence in identifications. Herein, the global interactome of the unicellular alga Chlamydomonas reinhardtii was analyzed via XL-MS by implementing the MS-cleavable disuccinimidyl sulfoxide (DSSO) crosslinker and enriching for crosslinks using strong cation exchange chromatography. Gentle lysis via repeated freeze-thaw cycles facilitated in vitro analysis of 157 protein-protein crosslinks (interlinks) and 612 peptides linked to peptides of the same protein (intralinks) at 1% FDR throughout the C. reinhardtii proteome. The interlinks confirmed known protein relationships across the cytosol and chloroplast, including coverage on 42% and 38% of the small and large ribosomal subunits, respectively. Of the 157 identified interlinks, 92% represent the first empirical evidence of interaction observed in C. reinhardtii. Several of these crosslinks point to novel associations between proteins, including the identification of a previously uncharacterized Mg-chelatase associated protein (Cre11.g477733.t1.2) bound to seven distinct lysines on Mg-chelatase (Cre06.g306300.t1.2). Additionally, the observed intralinks facilitated characterization of novel protein structures across the C. reinhardtii proteome. Together, these data establish a framework of protein-protein interactions that can be further explored to facilitate understanding of the dynamic protein landscape in C. reinhardtii.

Automated summary

Interactomics is an emerging field focused on identifying essential protein interactions for metabolic functions. Crosslinking mass spectrometry (XL-MS) has enabled global analysis of protein networks, with the commercial availability of specific crosslinkers driving research forward. Analysis of the unicellular alga Chlamydomonas reinhardtii revealed novel protein associations and structural insights that enhance understanding of protein interactions in this organism.