Research Interests

Protein-protein interactions (PPIs) are fundamental to the formation and assembly of protein complexes. In recent years, cross-linking mass spectrometry (XL-MS) has become an emerging technology for mapping protein interactomes at the systems level and elucidating structural topologies of large protein complexes. In comparison to other MS-based structural tools, XL-MS is unique owing to its capability to simultaneously capture PPIs from native environments and uncover interaction contacts though identification of cross-linked peptides, thus permitting the determination of both identity and connectivity of PPIs in cells. Given that each linker carries a defined length, the resulting cross-links can be utilized as distance constraints for various applications, ranging from structure validation and integrative modeling. In comparison to conventional methods such as X-ray crystallography and NMR, XL-MS offers distinct advantages including speed, accuracy, sensitivity and versatility, especially for the study of heterogeneous and dynamic protein complexes. To facilitate the detection and identification of cross-linked peptides, we have developed a suite of sulfoxide-containing MS-cleavable cross-linkers (Table 1) and demonstrated their effectiveness in profiling PPIs in vitro and in vivo. Currently, we continue our efforts on developing XL-MS technologies to further advance PPI profiling in living systems and to probe structural dynamics of protein complexes. In addition, we plan to develop analytical strategies to enable spatial and temporal characterization of PPIs under different physiological, pathological and pharmacological perturbations. Moreover, we want to establish a robust pipeline by coupling XL-MS data with AI-based structural prediction and high-resolution structural tools such as Cryo-EM to elucidate cellular network topologies and understand structural systems biology.

Table 1. Published sulfoxide-containing MS-cleavable cross-linkers.

Cross-linker	Structure	Features	Reference(s)
DSSO (Disuccinimidyl sulfoxide)		• Lysine-targeting • Low-energy CID cleavage • Symmetrical	Kao, 2011; Thermo Scientific
d0/d10-DMDSSO (d0/d10-Dimethyl disuccinimidyl sulfoxide)		• Lysine-targeting • Isotope-coded (d0/d10) • Low-energy CID cleavage • Symmetrical	Yu, 2014
Azide/Alkyne-A-DSBSO (Azide/Alkyne-tagged, acid-cleavable, disuccinimidyl bisulfoxide)		• Lysine-targeting • Enrichable via click chemistry • Low-energy CID cleavage • Symmetrical	Kaake, 2014; Burke, 2015
DHSO (Dihydrazide sulfoxide)		• Acid residue (D/E)-targeting • DMTMM/EDC-compatible • Low-energy CID cleavage • Symmetrical	Gutierrez, 2016
BMSO (Bismaleimide sulfoxide)		• Cysteine-targeting • Low-energy CID cleavage • Symmetrical	Gutierrez, 2018

Understanding Proteasomal Biology

The 26S proteasome is a macromolecular machine responsible for ubiquitin/ATP dependent protein degradation in both cytosol and nucleus. Of the two distinct sub-complexes, the 20S proteasome complex is responsible for various proteolytic activities while the 19S regulatory complex assists ATP-dependent proteolysis of ubiquitinated substrates through multiple biochemical functions. Disregulation of proteasomal degradation has been implicated in neurodegeneration and tumorigenesis; however, underlying mechanisms remain elusive. To better understand the involvement of proteasomal degradation during disease development and its potential for improved therapeutics, we aim to employ advanced proteomic approaches to comprehensively characterize the dynamic proteome of the 26S proteasome complex in various biological and pathological processes. Specially, we are interested in dissecting stress- and aggregation-mediated regulation of proteasomes to obtain new means for early detection and prevention of neurological disorders such as Huntington’s and Alzheimer’s’ diseases.

Regulation of Protein Ubiquitination

Protein ubiquitination is the essential step prior to protein degradation by the 26S proteasome. Cullin–RING ubiquitin E3 ligases (CRLs) represent the largest superfamily of multi-subunit E3s, which orchestrate ~20% of protein degradation in the UPS. The assembly and function of CRLs are regulated by a multisubunit deneddylase complex, i.e. the COP9 signalosome (CSN) that is critical for controlling diverse cellular and developmental processes in animals and plants. Aberrations in CRL and CSN complexes have been shown in various human diseases, and both complexes have displayed great potential as drug targets for better cancer therapy. We aim to develop and employ biochemical and proteomic approaches to uncover molecular details underlying regulation and function of CSN and CRL, and define their roles in human health and medicine.

General Method Developments for Proteomic Research

• Proteome profiling
• Quantitative mass spectrometry
• Mapping posttranslational modifications (phosphorylation, ubiquitination, etc)
• Peptide and protein separation

For proteomics service provided by UCI high-end Mass Spectrometry Facility, please visit the UCI High-end Mass Spectrometry Facility.