Michael J. Trnka

Crosslinking mass spectrometry (XL-MS) enables residue-level characterization of protein-protein interactions (PPIs) in both purified assemblies and complex biological systems. Cleavable crosslinkers such as DSSO are widely used to facilitate analysis of complex samples, yet there remains limited consensus on how best to leverage their properties in both acquisition and data analysis workflows. In part, this reflects differences in scoring, classification, and false discovery rate (FDR) estimation across analysis pipelines. Therefore, an integrated XL-MS data analysis pipeline that enables confident identification of MS-cleavable cross-linked peptides through improved scoring and rigorous FDR assessment is essential for systematically comparing and benchmarking workflows across different sample preparations. To this end, we implemented a new capability within the Protein Prospector search engine to improve MS2-based identification of MS-cleavable cross-linked peptides. Next, we developed Touchstone, a framework for re-scoring, classification, FDR-controlled summarization, and visualization of Prospector XL-MS search results. Using this approach, we evaluate the contributions of DSSO-specific fragmentation features and compare multiple acquisition strategies across datasets ranging from purified macromolecular assemblies to proteome-scale samples. We find that the primary advantage of DSSO arises from enhanced backbone fragmentation of both linked peptides, rather than from diagnostic ion formation, and that incorporating diagnostic ion pairs into MS2-based scoring or filtering does not improve identification performance. Benchmarking against established datasets shows that Prospector-Touchstone performs comparably to leading methods for non-cleavable crosslinkers and provides improved performance for DSSO analysis. We further compare acquisition strategies and find that for purified protein assemblies, MS2.stHCD characterizes more residue-pairs than ETD-based, MS3-based, and hybrid methods. In contrast, for proteome-scale XL-MS, MS3-based acquisition yields similar numbers of inter-protein PPIs with lower error rates despite reduced sampling depth. Together, these results highlight complementary strengths of MS2- and MS3-based workflows and provide practical guidance for MS-cleavable XL-MS experiments with varying sample complexities.

In a community effort, we audited error control in twelve crosslinking mass spectrometry pipelines using two ground-truth designs that expose a lower bound on false identifications. Some pipelines passed, whereas for others these verifiably false identifications alone exceeded the requested false discovery rate up to 29-fold at the protein–protein interaction level. The audit also tracked improvement between software versions, supporting its use for regression testing.

Significance: RAS proteins control many aspects of cellular biology in response to extracellular stimuli. These essential nodes of signal transduction interact with effector proteins that contain conserved RAS-binding domains. Mutations in RAS proteins are a common cause of cancer; hence, understanding the function of their downstream effectors is important for the development of novel therapies. In this study, we characterize the interaction between RAS and the effector protein Sin1, a critical component of the mTORC2, through biochemical and structural approaches. We discover that Sin1 contains an atypical RAS-binding domain and assess the role of this interaction in cells and mice. Moreover, we find that this interaction is dispensable for mTORC2 activity and assembly, suggesting an alternative function for Sin1 in cells.

Unique among metazoan repressive histone methyltransferases, G9a and GLP, which chiefly target histone 3 lysine 9 (H3K9), require dimerization for productive H3K9 mono (me1)- and dimethylation (me2) in vivo . Intriguingly, even though each enzyme can independently methylate H3K9, the predominant active form in vivo is a heterodimer of G9a and GLP. How dimerization influences the central H3K9 methyl binding (“reading”) and deposition (“writing”) activity of G9a and GLP, and why heterodimerization is essential in vivo remains opaque. Here, we examine the H3K9me “reading” and “writing” activities of defined, recombinantly produced homo- and heterodimers of G9a and GLP. We find that both reading and writing are significantly enhanced in the heterodimer. Compared to the homodimers, the heterodimer has higher recognition of H3K9me2, and a striking ∼ 10-fold increased turnover rate for nucleosomal substrates under multiple turnover conditions, which is not evident on histone tail peptide substrates. Crosslinking Mass Spectrometry suggests that differences between the homodimers and the unique activity of the heterodimer may be encoded in altered ground state conformations, as each dimer displays different domain contacts. Our results indicate that heterodimerization may be required to relieve autoinhibition of H3K9me reading and chromatin methylation evident in G9a and GLP homodimers. Relieving this inhibition may be particularly important in early differentiation when large tracts of H3K9me2 are deposited by G9a-GLP, which may require a more active form of the enzyme.

Membrane proteins with multiple transmembrane domains play critical roles in cell physiology, but little is known about the machinery coordinating their biogenesis at the endoplasmic reticulum. Here we describe a ~ 360 kDa ribosome-associated complex comprising the core Sec61 channel and five accessory factors: TMCO1, CCDC47 and the Nicalin-TMEM147-NOMO complex. Cryo-electron microscopy reveals a large assembly at the ribosome exit tunnel organized around a central membrane cavity. Similar to protein-conducting channels that facilitate movement of transmembrane segments, cytosolic and luminal funnels in TMCO1 and TMEM147, respectively, suggest routes into the central membrane cavity. High-throughput mRNA sequencing shows selective translocon engagement with hundreds of different multi-pass membrane proteins. Consistent with a role in multi-pass membrane protein biogenesis, cells lacking different accessory components show reduced levels of one such client, the glutamate transporter EAAT1. These results identify a new human translocon and provide a molecular framework for understanding its role in multi-pass membrane protein biogenesis.

During cell division, remodelling of the nuclear envelope enables chromosome segregation by the mitotic spindle¹. The reformation of sealed nuclei requires ESCRTs (endosomal sorting complexes required for transport) and LEM2, a transmembrane ESCRT adaptor^2,3,4. Here we show how the ability of LEM2 to condense on microtubules governs the activation of ESCRTs and coordinated spindle disassembly. The LEM motif of LEM2 binds BAF, conferring on LEM2 an affinity for chromatin^5,6, while an adjacent low-complexity domain (LCD) promotes LEM2 phase separation. A proline–arginine-rich sequence within the LCD binds to microtubules and targets condensation of LEM2 to spindle microtubules that traverse the nascent nuclear envelope. Furthermore, the winged-helix domain of LEM2 activates the ESCRT-II/ESCRT-III hybrid protein CHMP7 to form co-oligomeric rings. Disruption of these events in human cells prevented the recruitment of downstream ESCRTs, compromised spindle disassembly, and led to defects in nuclear integrity and DNA damage. We propose that during nuclear reassembly LEM2 condenses into a liquid-like phase and coassembles with CHMP7 to form a macromolecular O-ring seal at the confluence between membranes, chromatin and the spindle. The properties of LEM2 described here, and the homologous architectures of related inner nuclear membrane proteins^7,8, suggest that phase separation may contribute to other critical envelope functions, including interphase repair^{8,9,10,11,12,13} and chromatin organization^14,15,16,17.