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PubMed2026

Library-based, multiplexed strategy for mapping protein interaction networks via crosslinking.

BACKGROUND: Protein-protein interactions are fundamental to cellular function, yet resolving their interaction interfaces and dynamic behaviors in native biological contexts remains challenging, particularly for weak or transient interactions. Crosslinking strategies based on noncanonical amino acids offer an effective means to capture such interactions; however, traditional single-site incorporation provides limited coverage and may overlook critical interaction hotspots. RESULTS: By employing a mutagenesis library, multiple interaction partners and cross-linking sites of a target protein can be simultaneously screened in a single experiment, without prior knowledge of its precise structural or functional features, enabling effective and unbiased analysis of its interaction network. In this study, we constructed an amber codon-scanning mutagenesis library of PSMD10, facilitating independent incorporation of the photocrosslinking ncAA p-azido-phenylalanine at multiple distinct residues. This approach allowed us to systematically interrogate and precisely map potential interaction regions across the protein surface. Coupled with crosslinking mass spectrometry, we identified multiple residues involved in intermolecular interactions, as well as previously unreported interaction partners, including T2FA, TBA1C, and ATRIP. CONCLUSIONS: These findings expand our understanding of PSMD10-associated proteasome interactome, demonstrate a multiplexed strategy for in situ mapping of protein interaction interfaces with broad coverage, and offer a valuable platform for developing therapeutics that target protein-protein interactions.

Protein Interaction Mapping↗

PubMed2026

Blood Plasma Analysis in Ovarian Cancer Patients Using an AFM/MS Approach: Effect of Sample Dilution on Proteome Depth.

Early detection of ovarian cancer remains challenging because of the lack of sensitive and reproducible blood-based biomarkers. A major challenge in plasma proteomics is the extremely wide dynamic range of protein concentrations, which prevents simultaneous detection of both high- and low abundance proteins and limits the identification of disease-associated signals. In this study, we applied a combined atomic force microscopy and mass spectrometry (AFM/MS) approach to investigate how sample dilution affects plasma proteome coverage and the detection of differences between healthy donors and patients with stage I and stage III ovarian cancer. Plasma samples were analyzed at two dilution levels (1:100 and 1:10,000). At 1:100 dilution, a total of 235 proteins were identified across all samples, representing the union of all replicates and groups. The reproducible CORE proteome comprised 169 proteins in the Healthy group, 183 in the Stage I group, and 193 in the Stage III group. Differential analysis revealed distinct, non-overlapping protein sets at each dilution level. At 1:100 dilution, most altered proteins were decreased in patients and corresponded to major plasma components, including complement proteins and protease inhibitors. At 1:10,000 dilution, most altered proteins were increased and were predominantly immunoglobulin-related proteins, along with complement regulatory components. These findings show that sample dilution determines which fraction of the plasma proteome is observable. Here, proteome depth refers to the total number of non-redundant proteins accessible within the analytical workflow. When CORE sets from all groups were combined, 216 proteins were identified at 1:100 and 149 at 1:10,000, with 133 shared between the two dilution conditions. The higher dilution contributed 16 additional CORE proteins not detected in the 1:100 CORE union, increasing the combined CORE set to 232 proteins. Thus, higher dilution alone did not increase proteome depth, but provided complementary protein identifications that increased cumulative proteome depth when both dilution conditions were considered together. This effect reflects dilution-dependent selectivity in the composition of the detectable protein subset.

Humans↗

PubMed2026

ChromID: A Protocol for Mapping Protein Chromatin Interactions in Living Cells.

Chromatin modifications regulate genome function by recruiting proteins that control transcription, genome organization, and DNA repair. Identifying the proteins associated with specific chromatin modifications is therefore essential for understanding how these regulatory processes operate. Traditional approaches, including chromatin immunoprecipitation and affinity purification coupled to mass spectrometry, have uncovered many chromatin-associated proteins. However, they often rely on crosslinking and chromatin fragmentation, which can disrupt native chromatin architecture and limit the detection of transient interactions. Here, we describe a proximity-labeling protocol for identifying the chromatin-dependent protein interactome associated with specific chromatin marks, termed ChromID. ChromID uses engineered chromatin readers (eCRs) fused to a promiscuous biotin ligase, which labels proteins in the immediate vicinity of the targeted chromatin mark. The protocol includes in vivo biotin labeling, nuclear extract preparation, streptavidin-based enrichment, and tryptic digestion for downstream LC-MS/MS analysis. The protocol has been validated across multiple cell types and chromatin contexts and can be extended to other chromatin-associated proteins, providing a versatile approach to profile chromatin-associated proteomes within their native cellular environment. Key features • Maps proteins associated with different chromatin modifications in living cells using engineered chromatin readers fused to TurboID, BASU, or other promiscuous biotin ligases. • Preserves native chromatin organization and captures transient chromatin-associated interactions that are often lost during conventional affinity purification workflows. • Validated across multiple chromatin contexts, including histone modifications, DNA methylation, transcription factors, RNA polymerase II, and DNA damage-associated chromatin states. • Applicable to diverse cell types and organisms and adaptable to other chromatin-associated proteins, including transcription factors and chromatin regulators.

Biotin proximity labeling↗

PubMed2026

Crosstalk between cysteine and lysine modifications: Integrating redox and metabolic regulation.

Protein post-translational modifications (PTMs) on amino acid residues enable dynamic cellular responses to changes in metabolic and redox state. Cysteine and lysine are among the most extensively modified amino acid residues, with both undergoing a diversity of acylation and oxidative modifications. Indeed, proximal (<10 Å) cysteine and lysine residues may form integration nodes for crosstalk between metabolism and redox homeostasis pathways. This review highlights the interaction of proximal Cys-Lys residues, including influence on residue pKa by local electrostatics, cysteine-to-lysine transfer of PTM moieties, and covalent crosslinking. We discuss candidate Cys-Lys regulatory pairs in proteins involved in redox regulation, proteostasis, metabolic adaptation and inflammation. We further utilize computational modeling to identify proximity between cysteine and lysine residues in proteins known to be regulated by acylation and oxidative PTMs, and to demonstrate changes in these distances and local electrostatic potential due to lysine acetylation. Finally, we review how mass spectrometry-based proteomics and machine-learning PTM predictive tools can enable the identification, validation, and interpretation of proximal Cys-Lys interactions that regulate cellular responses to oxidative challenge and metabolic flux.

Cysteine↗

PubMed1979

Photodesmosine, an isomer of desmosine obtained by photolysis of this amino acid in ultraviolet light.

Desmosine and isodesmosine are two isomers representing the main crosslinks of elastin. We describe a new isomer, photodesmosine, which is produced by the photolysis of desmosine at 254 nm. The mechanism of this photolysis is described and is shown to consist of two competing paths. After opening of the pyridinium ring to give a tetrasubstituted aminoketone, this compound can either be hydrolysed to give lysine and a trisubstituted analogue of glutaconic aldehyde or undergo a recyclisation and rearomatisation to give a pyridinium compound substituted in positions 1, 2, 3 and 4. An understanding of this mechanism is important in order to use photolysis as a specific method to break elastin cross-links. Although only desmosine and isodesmosine have been reported in purified elastin, the chromatographic properties of photodesmosine suggests that if other natural isomers exist in this protein they could be eluted from an ion-exchange resin at much earlier times than those observed in the case of the two already described cross-links.

Amino Acids↗