The novel function of an orphan pheromone receptor reveals the sensory specializations of two potential distinct types of sex pheromones in noctuid moth.

Sex pheromones play crucial role in mating behavior of moths, involving intricate recognition mechanisms. While insect chemical biology has extensively studied type I pheromones, type II pheromones remain largely unexplored. This study focused on Helicoverpa armigera, a representative species of noctuid moth, aiming to reassess its sex pheromone composition. Our research unveiled two previously unidentified candidate type II sex pheromones-3Z,6Z,9Z-21:H and 3Z,6Z,9Z-23:H-in H. armigera. Furthermore, we identified HarmOR11 as an orphan pheromone receptor of 3Z,6Z,9Z-21:H. Through AlphaFold2 structural prediction, molecular docking, and molecular dynamics simulations, we elucidated the structural basis and key residues governing the sensory nuances of both type I and type II pheromone receptors, particularly HarmOR11 and HarmOR13. This study not only reveals the presence and recognition of candidate type II pheromones in a noctuid moth, but also establishes a comprehensive structural framework for PRs, contributing to the understanding of connections between evolutionary adaptations and the emergence of new pheromone types.

Time-Resolved Ultraviolet Photodissociation Mass Spectrometry Probes the Mutation-Induced Alterations in Protein Stability and Unfolding Dynamics.

How mutations impact protein stability and structure dynamics is crucial for understanding the pathological process and rational drug design. Herein, we establish a time-resolved native mass spectrometry (TR-nMS) platform via a rapid-mixing capillary apparatus for monitoring the acid-initiated protein unfolding process. The molecular details in protein structure unfolding are further profiled by a 193 nm ultraviolet photodissociation (UVPD) analysis of the structure-informative photofragments. Compared with the wild-type dihydrofolate reductase (WT-DHFR), the M42T/H114R mutant (MT-DHFR) exhibits a significant stability decrease in TR-nMS characterization. UVPD comparisons of the unfolding intermediates and original DHFR forms indicate the special stabilization effect of cofactor NADPH on DHFR structure, and the M42T/H114R mutations lead to a significant decrease in NADPH-DHFR interactions, thus promoting the structure unfolding. Our study paves the way for probing the mutation-induced subtle changes in the stability and structure dynamics of drug targets.

Panda-UV Unlocks Deeper Protein Characterization with Internal Fragments in Ultraviolet Photodissociation Mass Spectrometry.

Ultraviolet photodissociation (UVPD) mass spectrometry unlocks insights into the protein structure and sequence through fragmentation patterns. While N- and C-terminal fragments are traditionally relied upon, this work highlights the critical role of internal fragments in achieving near-complete sequencing of protein. Previous limitations of internal fragment utilization, owing to their abundance and potential for random matching, are addressed here with the development of Panda-UV, a novel software tool combining spectral calibration, and Pearson correlation coefficient scoring for confident fragment assignment. Panda-UV showcases its power through comprehensive benchmarks on three model proteins. The inclusion of internal fragments boosts identified fragment numbers by 26% and enhances average protein sequence coverage to a remarkable 93% for intact proteins, unlocking the hidden region of the largest protein carbonic anhydrase II in model proteins. Notably, an average of 65% of internal fragments can be identified in multiple replicates, demonstrating the high confidence of the fragments Panda-UV provided. Finally, the sequence coverages of mAb subunits can be increased up to 86% and the complementary determining regions (CDRs) are nearly completely sequenced in a single experiment. The source codes of Panda-UV are available at https://github.com/PHOENIXcenter/Panda-UV.

A critical role of the endothelial S-phase kinase-associated protein 2/phosphatase and tensin homologue axis in angiogenesis and psoriasis.

BACKGROUND: Psoriasis is a common chronic skin disorder. Pathologically, it features abnormal epidermal proliferation, infiltrating inflammatory cells and increased angiogenesis in the dermis. Aberrant expression of E3 ubiquitin ligase and a dysregulated protein ubiquitination system are implicated in the pathogenesis of psoriasis. OBJECTIVES: To examine the potential role of S-phase kinase-associated protein 2 (Skp2), an E3 ligase and oncogene, in psoriasis. METHODS: Gene expression and protein levels were evaluated with quantitative reverse transcriptase polymerase chain reaction, Western blotting, immunohistochemistry and immunofluorescence staining of skin samples from patients with psoriasis vulgaris and an imiquimod (IMQ)-induced mouse model, as well as from cultured endothelial cells (ECs). Protein interaction, substrate ubiquitination and degradation were examined using co-immunoprecipitation, Western blotting and a cycloheximide chase assay in human umbilical vein ECs. Angiogenesis was measured in vitro using human dermal microvascular ECs (HDMECs) for BrdU incorporation, migration and tube formation. In vivo angiogenesis assays included chick embryonic chorioallantoic membrane, the Matrigel plug assay and quantification of vasculature in the mouse lesions. Skp2 gene global knockout (KO) mice and endothelial-specific conditional KO mice were used. RESULTS: Skp2 was increased in skin samples from patients with psoriasis and IMQ-induced mouse lesions. Immunofluorescent double staining indicated a close association of Skp2 expression with excessive vascularity in the lesional dermal papillae. In HDMECs, Skp2 overexpression was enhanced, whereas Skp2 knockdown inhibited EC proliferation, migration and tube-like structure formation. Mechanistically, phosphatase and tensin homologue (PTEN), which suppresses the phosphoinositide 3-kinase/Akt pathway, was identified to be a novel substrate for Skp2-mediated ubiquitination. A selective inhibitor of Skp2 (C1) or Skp2 small interfering RNA significantly reduced vascular endothelial growth factor-triggered PTEN ubiquitination and degradation. In addition, Skp2-mediated ubiquitination depended on the phosphorylation of PTEN by glycogen synthase kinase 3ß. In the mouse model, Skp2 gene deficiency alleviated IMQ-induced psoriasis. Importantly, tamoxifen-induced endothelial-specific Skp2 KO mice developed significantly ameliorated psoriasis with diminished angiogenesis of papillae. Furthermore, topical use of the Skp2 inhibitor C1 effectively prevented the experimental psoriasis. CONCLUSIONS: The Skp2/PTEN axis may play an important role in psoriasis-associated angiogenesis. Thus, targeting Skp2-driven angiogenesis may be a potential approach to treating psoriasis.

The utility of proteases in proteomics, from sequence profiling to structure and function analysis.

In mass spectrometry (MS)-based bottom-up proteomics, protease digestion plays an essential role in profiling both proteome sequences and post-translational modifications (PTMs). Trypsin is the gold standard in digesting intact proteins into small-size peptides, which are more suitable for high-performance liquid chromatography (HPLC) separation and tandem MS (MS/MS) characterization. However, protein sequences lacking Lys and Arg cannot be cleaved by trypsin and may be missed in conventional proteomic analysis. Proteases with cleavage sites complementary to trypsin are widely applied in proteomic analysis to greatly improve the coverage of proteome sequences and PTM sites. In this review, we survey the common and newly emerging proteases used in proteomics analysis mainly in the last 5 years, focusing on their unique cleavage features and specific proteomics applications such as missing protein characterization, new PTM discovery, and de novo sequencing. In addition, we summarize the applications of proteases in structural proteomics and protein function analysis in recent years. Finally, we discuss the future development directions of new proteases and applications in proteomics.

Ultraviolet Photodissociation Reveals the Molecular Mechanism of Crown Ether Microsolvation Effect on the Gas-Phase Native-like Protein Structure.

Maintaining the protein high-order structures and interactions during the transition from aqueous solution to gas phase is essential to the structural analysis of native mass spectrometry (nMS). Herein, we systematically interrogate the effects of charge state and crown ether (CE) complexation on the gas-phase native-like protein structure by integrating nMS with 193 nm ultraviolet photodissociation (UVPD). The alterations of photofragmentation yields of protein residues and the charge site distribution of fragment ions reveal the specific sites and sequence regions where charge and CE take effect. Our results exhibit the CE complexation on protonated residues can largely alleviate the structure disruption induced by the intramolecular solvation of charged side chains. The influences of CE complexation and positive charge on gas-phase protein structure exhibit generally opposite trends because the CE microsolvation avoids the hydrogen-bonding formation between the charged side chains with backbone carbonyls. Thus, CE complexation leads to a more stable and native-like protein structure in the gas phase.

Structural Mass Spectrometry Probes the Inhibitor-Induced Allosteric Activation of CDK12/CDK13-Cyclin K Dissociation.

The rational design and development of effective inhibitors for cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) are largely dependent on the understanding of the dynamic inhibition conformations but are difficult to be achieved by conventional characterization tools. Herein, we integrate the structural mass spectrometry (MS) methods of lysine reactivity profiling (LRP) and native MS (nMS) to systematically interrogate both the dynamic molecular interactions and overall protein assembly of CDK12/CDK13-cyclin K (CycK) complexes under the modulation of small molecule inhibitors. The essential structure insights, including inhibitor binding pocket, binding strength, interfacial molecular details, and dynamic conformation changes, can be derived from the complementary results of LRP and nMS. We find the inhibitor SR-4835 binding can greatly destabilize the CDK12/CDK13-CycK interactions in an unusual allosteric activation way, thereby providing a novel alternative for the kinase activity inhibition. Our results underscore the great potential of LRP combination with nMS for the evaluation and rational design of effective kinase inhibitors at the molecular level.

Integrative analysis of metabolomics and proteomics unravels purine metabolism dysregulation in the SOD1G93A mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with progressive paralysis of limbs and bulb in patients, the cause of which remains unclear. Accumulating studies suggest that motor neuron degeneration is associated with systemic metabolic impairment in ALS. However, the metabolic reprogramming and underlying mechanism in the longitudinal progression of the disease remain poorly understood. In this study, we aimed to investigate the molecular changes at both metabolic and proteomic levels during disease progression to identify the most critical metabolic pathways and underlying mechanisms involved in ALS pathophysiological changes. Utilizing liquid chromatography-mass spectrometry-based metabolomics, we analyzed the metabolites' levels of plasma, lumbar spinal cord, and motor cortex from SOD1G93A mice and wildtype (WT) littermates at different stages. To elucidate the regulatory network underlying metabolic changes, we further analyzed the proteomics profile in the spinal cords of SOD1G93A and WT mice. A group of metabolites implicated in purine metabolism, methionine cycle, and glycolysis were found differentially expressed in ALS mice, and abnormal expressions of enzymes involved in these metabolic pathways were also confirmed. Notably, we first demonstrated that dysregulation of purine metabolism might contribute to the pathogenesis and disease progression of ALS. Furthermore, we discovered that fatty acid metabolism, TCA cycle, arginine and proline metabolism, and folate-mediated one­carbon metabolism were also significantly altered in this disease. The identified differential metabolites and proteins in our study could complement existing data on metabolic reprogramming in ALS, which might provide new insight into the pathological mechanisms and novel therapeutic targets of ALS.

Molecular Structure Characterization of Micro/Nanoplastics by 193 nm Ultraviolet Photodissociation Mass Spectrometry.

The degradation of macroplastics results in micro/nanoplastics (MNPs) in the natural environment, inducing high health risks worldwide. It remains challenging to characterize the accurate molecular structures of MNPs. Herein, we integrate 193 nm ultraviolet photodissociation (UVPD) with mass spectrometry to interrogate the molecular structures of poly(ethylene glycol) terephthalate and polyamide (PA) MNPs. The backbones of the MNP polymer can be efficiently dissociated by UVPD, producing rich types of fragment ions. Compared to high-energy collision dissociation (HCD), the structural informative fragment ions and corresponding sequence coverages obtained by UVPD were all improved 2.3 times on average, resulting in almost complete sequence coverage and precise structural interrogation of MNPs. We successfully determine the backbone connectivity differences of MNP analogues PA6, PA66, and PA610 by improving the average sequence coverage from 26.8% by HCD to 89.4% by UVPD. Our results highlight the potential of UVPD in characterizing and discriminating backbone connectivity and chain end structures of different types of MNPs.

Chloride-Mediated Peroxide-Free Photochemical Oxidation of Proteins (PPOP) in Mass Spectrometry-Based Structural Analysis.

Ultraviolet (UV) laser photolysis of hydrogen peroxide (H2O2) for the in situ generation of hydroxyl radicals (•OH) is a widely utilized strategy in the oxidation footprinting of native proteins and mass spectrometry (MS)-based structural analysis. However, it remains challenging to realize peroxide-free photochemical oxidation footprinting. Herein, we describe the footprinting of native proteins by chloride-mediated peroxide-free photochemical oxidation of proteins (PPOP). The protein samples are prepared within biocompatible phosphate-buffered saline (PBS) containing 10 mM Gln as radical scavengers and oxidized in a capillary flow reactor directly under a single-pulse (10 ns) irradiation of a 193 nm ArF UV laser. The main oxidized protein residues are CMYWFHLI. We demonstrate that the PPOP-MS strategy is highly sensitive to the protein high-order structures and can be applied to monitor the protein-drug interfaces, which provides a promising footprinting alternative for protein structure-function explorations.

The interfacial interactions of nanomaterials with human serum albumin.

The fates of nanomaterials (NMs) in vivo are greatly dependent on their interactions with human serum proteins. However, the interfacial molecular details of NMs-serum proteins are still difficult to be probed. Herein, the molecular interaction details of human serum albumin (HSA) with Au and SiO2 nanoparticles have been systematically interrogated and compared by using lysine reactivity profiling mass spectrometry (LRP-MS). We demonstrated the biocompatibility of Au is better than SiO2 nanoparticles and the NMs surface charge state played a more important role than particle size in the combination of NMs-HSA at least in the range of 15-40 nm. Our results will contribute to the fundamental mechanism understanding of NMs-serum protein interactions as well as the NMs rational design.

Size-Selective VAILase Proteolysis Provides Dynamic Insights into Protein Structures.

Monitoring the dynamic alterations of protein structures within an aqueous solution remains enormously challenging. In this study, we describe a size-selective VAILase proteolysis (SVP)-mass spectrometry (MS) strategy to probe the protein structure changes without strict control of the proteolysis kinetics. The unique conformation selectivity of SVP depends on the uniform nano-sized entrance pores of the VAILase hexameric cage as well as the six inherent molecular rulers in the VAILase-substrate recognition and cleavage. The dynamic insights into subtle conformation alterations of both myoglobin unfolding transition and Aurora kinase A-inhibitor binding are successfully captured using the SVP strategy, which matches well with the results in the molecular dynamics simulation. Our work provides a new paradigm of size-selective native proteolysis for exploring the aqueous protein structure-function relationships.

Lysine reactivity profiling reveals molecular insights into human serum albumin-small-molecule drug interactions.

Human serum albumin (HSA) is one of the most important serum carrier proteins that deliver small-molecule drugs to their specific targets. Clarifying the molecular mechanism of the interaction between natural HSA and drugs in an aqueous solution has been a hot topic in pharmaceutical chemistry, clinical medicine, and biochemistry in recent years, but it is still challenging. In this paper, the details of molecular interactions of HSA with a variety of therapeutic drugs including ibuprofen, indomethacin, phenylbutazone, and warfarin are systematically investigated using a mass spectrometry (MS)-based lysine reactivity profiling (LRP) strategy. The results reaffirm that the major ligand binding sites (including Sites I and II) of HSA are located in subdomains IIA and IIIA, while several potential drug-binding areas at subdomain IIIB and α helix IIB-IIIA are newly characterized. The MS-LRP strategy may have important application prospects in pharmacodynamics, pharmacokinetics, and safety evaluation of small-molecule drugs.

Probing the Proteomics Dark Regions by VAILase Cleavage at Aliphatic Amino Acids.

Proteomics emerges from the protein identification to protein functional elucidation, which depends to a large extent on the characterization of protein sequences. However, a large part of proteome sequences remains unannotated due to the limitation in proteolytic digestion by golden standard protease trypsin. Herein, we demonstrated that a cyanobacterial protease VAILase could specifically cleave at the short-chain aliphatic amino acids valine, alanine, leucine, isoleucine and threonine with cleavage specificity about 92% in total for proteomic analysis. The unique features of VAILase cleavage facilitate the characterization of most proteins and exhibit high complementarity to trypsin, and 22% of the covered sequences by VAILase are unique. VAILase can greatly improve the coverages of sequences with abundant aliphatic residues that are usually dark regions in conventional proteomic analysis, such as the transmembrane regions within anion exchanger 1 and photosystem II.

Global Quantification of Intact Proteins via Chemical Isotope Labeling and Mass Spectrometry.

Although thousands of intact proteins have been feasibly identified in recent years, global quantification of intact proteins is still challenging. Herein, we develop a high-throughput strategy for global intact protein quantification based on chemical isotope labeling. The isotope incorporation efficiency is as high as 99.2% for complex intact protein samples extracted from HeLa cells. Further, the pTop 2.0 software is developed for automated quantification of intact proteoforms in a high-throughput manner. The high quantification accuracy and reproducibility of this strategy have been demonstrated for both standard and complex cellular protein samples. A total of 2283 intact proteoforms originated from 660 protein accessions are successfully quantified under anaerobic and aerobic conditions and the differentially expressed proteins are observed to be involved in the important biological processes such as stress response.

Analysis of oligonucleotides by ion-pair reversed-phase liquid chromatography coupled with positive mode electrospray ionization mass spectrometry.

Oligonucleotides are usually analyzed by ion-pair reversed-phase liquid chromatography (IP-RPLC) coupled with negative mode electrospray ionization mass spectrometry (ESI-MS) due to their highly negative charged phosphodiester backbones. Herein, the signal suppression effect of triethylamine (TEA) adducts caused the ion-pair reagent TEA/hexafluoroisopropanol (HFIP) is greatly alleviated after improving the in-source energy in positive mode ESI-MS. This strategy is applied for different RNA sequencing through analyzing their formic acid hydrolysates via IP-RPLC MS. Comparing with negative ion mode, we demonstrate that IP-RPLC MS analysis in positive ion mode is more suitable for RNA sequencing with fewer contaminant interferences. Finally, simultaneous online separation and detection of oligonucleotides and protein digests are achieved in positive ion mode IP-RPLC MS analysis with little interference to each other.

Biflavones from Ginkgo biloba as inhibitors of human thrombin.

Ginkgo Biloba leaf extract has been widely used for the prevention and treatment of thrombosis and cardiovascular disease in both eastern and western countries, but the bioactive constituents and the underlying mechanism of anti-thrombosis have not been fully characterized. The purpose of this study was to investigate the inhibitory effects of major constituents in Ginkgo biloba on human thrombin, a key serine protease regulating the blood coagulation cascade and the processes of thrombosis. To this end, a fluorescence-based biochemical assay was used to assay the inhibitory effects of sixteen major constituents from Ginkgo biloba on human thrombin. Among all tested natural compounds, four biflavones (ginkgetin, isoginkgetin, bilobetin and amentoflavone), and five flavonoids (luteolin, apigenin, quercetin, kaempferol and isorhamnetin) were found with thrombin inhibition activity, with the IC50 values ranging from 8.05 µM to 82.08 µM. Inhibition kinetic analyses demonstrated that four biflavones were mixed inhibitors against thrombin-mediated Z-GGRAMC acetate hydrolysis, with the Ki values ranging from 4.12 µM to 11.01 µM. Molecular docking method showed that the four biflavones could occupy the active cavity with strong interactions of salt bridges and hydrogen bonds. In addition, mass spectrometry-based lysine labeling reactivity assay suggested that the biflavones could bind on human thrombin at exosite I rather than exosite II. All these findings suggested that the biflavones in Ginkgo biloba were naturally occurring inhibitors of human thrombin, and these compounds could be used as lead compounds for the development of novel thrombin inhibitors with improved efficacy and high safety profiles.

Identification of pyridoxal phosphate-modified proteins using mass spectrometry.

RATIONALE: Pyridoxal 5'-phosphate (PLP) cooperates with a variety of enzymes in all organisms for many important biological processes. The development of mass spectrometry-based methodology for high-throughput modification analyses could provide an alternative way for PLP identification. The present study aims to identify PLP modification. METHODS: More PLP site-determining information was obtained by introducing multistage activation (MSA)-assisted collision-induced dissociation (CID). We then utilized immobilized metal ion affinity chromatography (IMAC) with Ti4+ to enrich the PLP peptides. In addition, alkaline phosphatase (ALP) was used to remove the phosphoryl group and further confirm the PLP modification site. RESULTS: MSA was able to greatly enhance the identification and localization of PLP modification. We applied this strategy to analyze PLP-modified proteins in Escherichia coli samples and accurately determine PLP site K270 in tryptophanase. CONCLUSIONS: MSA-assisted CID was used to provide better identification of PLP-modified peptides. Furthermore, tryptophanase with PLP modification at K270 in E. coli was identified with Ti4+ -IMAC enrichment followed by ALP treatment. This method provides a promising alternative for investigating biological functions of PLP-modified proteins.

Novel Features of Eukaryotic Photosystem II Revealed by Its Crystal Structure Analysis from a Red Alga.

Photosystem II (PSII) catalyzes light-induced water splitting, leading to the evolution of molecular oxygen indispensible for life on the earth. The crystal structure of PSII from cyanobacteria has been solved at an atomic level, but the structure of eukaryotic PSII has not been analyzed. Because eukaryotic PSII possesses additional subunits not found in cyanobacterial PSII, it is important to solve the structure of eukaryotic PSII to elucidate their detailed functions, as well as evolutionary relationships. Here we report the structure of PSII from a red alga Cyanidium caldarium at 2.76 Å resolution, which revealed the structure and interaction sites of PsbQ', a unique, fourth extrinsic protein required for stabilizing the oxygen-evolving complex in the lumenal surface of PSII. The PsbQ' subunit was found to be located underneath CP43 in the vicinity of PsbV, and its structure is characterized by a bundle of four up-down helices arranged in a similar way to those of cyanobacterial and higher plant PsbQ, although helices I and II of PsbQ' were kinked relative to its higher plant counterpart because of its interactions with CP43. Furthermore, two novel transmembrane helices were found in the red algal PSII that are not present in cyanobacterial PSII; one of these helices may correspond to PsbW found only in eukaryotic PSII. The present results represent the first crystal structure of PSII from eukaryotic oxygenic organisms, which were discussed in comparison with the structure of cyanobacterial PSII.

Enhancing the Mass Spectrometry Sensitivity for Oligonucleotide Detection by Organic Vapor Assisted Electrospray.

There are two challenges in oligonucleotide detection by liquid chromatography coupled with mass spectrometry (LC-MS), the serious ion suppression effects caused by ion-pair reagents and the low detection sensitivity in positive mode MS. In this study, highly concentrated alcohol vapors were introduced into an enclosed electrospray ionization chamber, and oligonucleotides could be well detected in negative mode MS even with 100 mM triethylammonium acetate (TEAA) as an ion-pair reagent. The MS signal intensity was improved 600-fold (for standard oligonucleotide dT15) by the isopropanol vapor assisted electrospray, and effective ion-pair LC separation was feasibly coupled with high-sensitive MS detection. Then, oligonucleotides were successfully detected in positive mode MS with few adducts by propanoic acid vapor assisted electrospray. The signal intensity was enhanced more than 10-fold on average compared with adding acids into the electrospray solution. Finally, oligonucleotides and peptides or histones were simultaneously detected in MS with little interference with each other. Our strategy provides a useful alternative for investigating the biological functions of oligonucleotides.