Hierarchical pore structure with a confined resonant mode for improving the solar energy utilizing efficiency of ultra-thin perovskite solar cells.

The perovskite solar cell (PSC) has the benefits of flexibility, inexpensiveness, and high efficiency, and has important prospective applications. However, serious optical losing and low solar energy-utilizing efficiency remain a challenge for the ultra-thin PSCs because of the interface reflection of traditional planar structure. In this study, a hierarchical pore structure with a confined resonant mode is introduced and optimized by electromagnetic theory to improve the solar energy absorbing and utilizing efficiency of ultra-thin PSCs. The large pores in the top layer that support a whispering gallery mode can focus and guide the incident light into the solar cell. The small pores in the bottom layer enable backward scattering of the unabsorbed light and can improve the effective absorption of active layer. The finite-difference time-domain method is employed to optimize the geometric parameters of hierarchical pore structure to improve the light absorption of PSCs. The proposed resonant hierarchical pore structure can greatly improve sunlight absorption of ultra-thin PSCs, and the effective light absorption and photocurrent of PSCs with a hierarchical pore structure is 20.7% higher than that of PSCs with traditional planar structure. This work can offer a beneficial guideline for improving solar energy utilizing efficiency of various thin-film solar cells.

"Warm in Winter and Cool in Summer": Scalable Biochameleon Inspired Temperature-Adaptive Coating with Easy Preparation and Construction.

The highly reflective solar radiation of passive daytime radiative cooling (PDRC) increases heating energy consumption in the cold winter. Inspired by the temperature-adaptive skin color of chameleon, we efficiently combine temperature-adaptive solar absorption and PDRC technology to achieve "warm in winter and cool in summer". The temperature-adaptive radiative cooling coating (TARCC) with color variability is designed and fabricated, achieving 41% visible light regulation capability. Comprehensive seasonal outdoor tests confirm the reliability of the TARCC: in summer, the TARCC exhibits high solar reflectance (∼93%) and atmospheric transmission window emittance (∼94%), resulting in a 6.5 K subambient temperature. In the winter, the TARCC's dark color strongly absorbs solar radiation, resulting in a 4.3 K temperature rise. Compared with PDRC coatings, the TARCC can save up to 20% of annual energy in midlatitude regions and increase suitable human hours by 55%. With its low cost, easy preparation, and simple construction, the TARCC shows promise for achieving sustainable and comfortable indoor environments.

Quantum digital signature with unidimensional continuous-variable against the measurement angular error.

The continuous-variable quantum digital signature (CV-QDS) scheme relies on the components of quantum key generation protocol (KGP) to negotiate classical signature, which is more compatible with optical fibers. Nevertheless, the measurement angular error of heterodyne detection or homodyne detection will cause security issues when performing KGP in the distribution stage. For that, we propose to utilize unidimensional modulation in KGP components, which only requires to modulate single quadrature and without the process of basis choice. Numerical simulation results show that the security under collective attack, repudiation attack and forgery attack can be guaranteed. We expect that the unidimensional modulation of KGP components could further simplify the implementation of CV-QDS and circumvent the security issues caused by the measurement angular error.

Metformin synergistically enhances the antitumour activity of Lenvatinib in hepatocellular carcinoma by altering AKT-FOXO3 signalling pathway.

BACKGROUND AND AIMS: Lenvatinib is a first-line drug commonly used in the treatment of advanced hepatocellular carcinoma (HCC). However, its clinical efficacy is very limited due to drug resistance. Therefore, there is a great need to explore its combination with other agents to achieve better therapeutic effects. Metformin has been demonstrated to show an anti-cancer effect. This study aimed to investigate the combined effect of lenvatinib with metformin in HCC cells both in vitro and in vivo and elucidate the possible molecular mechanisms. METHODS: Flow cytometry, colony formation, CCK-8 and transwell assays were used to study the effect of Lenvatinib-Metformin combination on the malignant behaviour of HCC cells in vitro. Constructing an animal model of tumour-bearing to study the effect of combined drugs on HCC in vivo. Western blot experiments were performed to assess the relationship between AKT and FOXO3 and the cellular translocation of FOXO3. RESULTS: Our results suggested that Lenvatinib and Metformin synergistically inhibited HCC growth and motility. Mechanistically, the combination of Lenvatinib and Metformin synergistically suppressed the activation of the AKT signalling pathway, which in turn reduced the phosphorylation level of downstream effector FOXO3 and induced its nuclear aggregation. In vivo studies further confirmed the synergistic suppression of lenvatinib with metformin in HCC growth. CONCLUSION: The Lenvatinib-Metformin combination may provide a potential therapeutic strategy to improve the prognosis of HCC patients.

Safety and feasibility of a modularized procedure for trans-subxiphoid robotic extended thymectomy.

BACKGROUND: The purpose of this study was to introduce an "eight-step modularized procedure (M-RET)" for trans-subxiphoid robotic extended thymectomy for patients with myasthenia gravis (MG). Its safety and feasibility were further verified in this study. MATERIALS AND METHODS: This retrospective study included 87 consecutive MG patients who underwent trans-subxiphoid robotic extended thymectomy at our institution between September 2016 and August 2021. According to different resection models, patients were divided into two groups: traditional trans-subxiphoid robotic extended thymectomy group (T-RET group) and eight-step modularized technique group (M-RET group). Baseline demographic characteristics and operation-related parameters were collected and compared between the two groups. RESULTS: There were 41 (47.1%) patients in the M-RET group and 46 (52.9%) patients in the T-RET group. The M-RET group resected a greater amount of mediastinal adipose tissues and required more dissection time (median and interquartile range: 135.0, 125.0 to 164.0 v. 120.0, 105.0 to 153.8, P = 0.006) compared with the T-RET group. There were no statistically significant differences in terms of the intraoperative blood loss, duration of chest drainage, length of hospital stay, and postoperative complications between the two groups. There was no mortality or conversion in each of the two groups and all patients recovered well upon discharge. CONCLUSION: The eight-step modularized technique of trans-subxiphoid robotic extended thymectomy was verified to be a safe, effective, radical procedure, which offers unique superiority over ectopic thymic tissue resection.

Quercetin inhibits the amphiregulin/EGFR signaling-mediated renal tubular epithelial-mesenchymal transition and renal fibrosis in obstructive nephropathy.

Quercetin is a widely distributed, bioactive flavonoid compound, which displays potential to inhibit fibrosis in several diseases. The purpose of our study was to determine the effect of quercetin treatment on renal fibrosis and investigate the mechanism. Human proximal tubular epithelial cells (HK-2) stimulated by transforming growth factor-ß1 (TGF-ß1) and a rat model of unilateral ureter obstruction (UUO) that contributes to fibrosis were used to investigate the role and molecular mechanism of quercetin. PD153035 (N-[3-Bromophenyl]-6,7-dimethoxyquinazolin-4-amine) was used to inactivate EGFR (epidermal growth factor receptor). The level of fibrosis, proliferation, apoptosis, and oxidative stress in HK-2 were measured. All data are presented as means ± standard deviation (SD). p-value < .05 was considered statistically significant. In UUO rats, quercetin reduced the area of fibrosis as well as inflammation, oxidative stress, and cell apoptosis. In cultured HK-2 cells, quercetin significantly ameliorated the EMT induced by TGF-ß1, which was accompanied by increased amphiregulin (AREG) expression. Moreover, quercetin inhibited AREG binding to the EGFR receptor, thereby further affecting other downstream pathways. Quercetin may alleviate fibrosis in vitro and in vivo by inhibiting the activation of AREG/EGFR signaling indicating a potential therapeutic effect of quercetin in renal fibrosis.

Deubiquitinase Ubp3 enhances the proteasomal degradation of key enzymes in sterol homeostasis.

Sterol homeostasis is tightly controlled by molecules that are highly conserved from yeast to humans, the dysregulation of which plays critical roles in the development of antifungal resistance and various cardiovascular diseases. Previous studies have shown that sterol homeostasis is regulated by the ubiquitin-proteasome system. Two E3 ubiquitin ligases, Hrd1 and Doa10, are known to mediate the proteasomal degradation of 3-hydroxy-3-methylglutaryl-CoA reductase Hmg2 and squalene epoxidase Erg1 with accumulation of the toxic sterols in cells, but the deubiquitinases (DUBs) involved are unclear. Here, we screened for DUBs responsible for sterol homeostasis using yeast strains from a DUB-deletion library. The defective growth observed in ubp3-deleted (ubp3Δ) yeast upon fluconazole treatment suggests that lack of Ubp3 disrupts sterol homeostasis. Deep-coverage quantitative proteomics reveals that ergosterol biosynthesis is rerouted into a sterol pathway that generates toxic products in the absence of Ubp3. Further genetic and biochemical analysis indicated that Ubp3 enhances the proteasome's ability to degrade the ergosterol biosynthetic enzymes Erg1 and Erg3. The retardation of ergosterol enzyme degradation in the ubp3Δ strain resulted in the severe accumulation of the intermediate lanosterol and a branched toxic sterol, and ultimately disrupted sterol homeostasis and led to the fluconazole susceptibility. Our findings uncover a role for Ubp3 in sterol homeostasis and highlight its potential as a new antifungal target.

Analysis of the genetic diversity and population structure of Monochasma savatieri Franch. ex Maxim using novel EST-SSR markers.

BACKGROUND: Monochasma savatieri Franch. ex Maxim is a medicinally valuable herb. However, the collection and protection of the wild germplasm resources of M. savatieri are still insufficient, and their genetic diversity and population structure have been poorly studied. RESULTS: We collected and examined 46 M. savatieri individuals from Fujian, Hunan, Jiangxi, and Zhejiang provinces for genetic diversity and population structure, using 33 newly developed expressed sequence tag-simple sequence repeat (EST-SSR) markers. Applying these markers, we detected a total of 208 alleles, with an average of 6.303 alleles per locus. The polymorphic information content varied from 0.138 to 0.884 (average: 0.668), indicating a high level of polymorphism. At the population level, there was a low degree of genetic diversity among populations (I = 0.535, He = 0.342), with Zhejiang individuals showing the highest genetic diversity among the four populations (Fst = 0.497), which indicated little gene flow within the M. savatieri populations (Nm = 0.253). Mantel test analysis revealed a significant positive correlation between geographical and genetic distance among populations (R2 = 0.3304, p < 0.05), and structure and principal coordinate analyses supported classification of populations into three clusters, which was consistent with the findings of cluster analysis. CONCLUSIONS: As a rare medicinal plants, the protection of M. savatieri does not look optimistic, and accordingly, protective efforts should be beefed up on the natural wild populations. This study provided novel tools and insights for designing effective collection and conservation strategies for M. savatieri.

Rab31, a receptor of advanced glycation end products (RAGE) interacting protein, inhibits AGE induced pancreatic ß-cell apoptosis through the pAKT/BCL2 pathway.

Receptor of advanced glycation end products (RAGE) mediates diverse signal transduction following ligand stimulation and plays an important role in diabetes complications and aging associated disease. We have previously verified that advanced glycation end products (AGE) bind to RAGE to cause pancreatic ß-cell apoptosis through the mitochondrial pathway. However, the direct interacting protein(s) of RAGE in ß cells has never been appreciated. In the present study, we utilized GST pull-down assay combined with mass spectrometry to identify the interacting proteins of the RAGE intracellular domain (C-terminal 43 amino acid of RAGE). Overall four RAGE interacting proteins, including Rab31, were identified with scores over 160. Rab31 was detected in three ß-cell lines and confirmed to have interacted with RAGE via co-immunoprecipitation and immunostaining assays. This interaction was further enhanced by glycation-serum (GS) stimulation due to membrane distribution of Rab31 following treatment with GS. We further confirmed that Rab31 promoted RAGE endocytosis and inhibited GS-induced ß-cell apoptosis through the pAKT/BCL2 pathway. These findings reveal a new RAGE interaction protein Rab31 that prevents AGE/RAGE-induced pancreatic ß-cell apoptosis. Rab31 is therefore a promising therapeutic target for preserving functional ß cells under diabetes conditions.

Perioperative Clinical Features of Mediastinal Parathyroid Adenoma: A Case Series.

Rare ectopic mediastinal parathyroid adenoma can result in persistent or recurrent hyperparathyroidism. In this article, we summarized the perioperative outcomes of six patients with mediastinal parathyroid adenoma. All patients underwent minimally invasive surgery (MIS). Abnormal accumulation of sestamibi was observed in four of five patients for preoperative localization of adenoma. Postoperatively, the blood calcium dropped quickly. In addition, we found adenoma function was negatively related to adenoma volume in these patients. In conclusion, although MIS is feasible for parathyroid adenoma, blood calcium should be monitored in a timely manner to avoid hypocalcemia postoperatively. In addition, sestamibi might be a potential pitfall when locating parathyroid adenoma.

Biomimetic hierarchical structure for enhancing concentrated solar energy converting and utilizing efficiency.

Concentrated solar technology has the problems of local overheating, inadequate light absorption, and insufficient diffusion of solar irradiation energy flow. In this study, the idea of using biomimetic hierarchical structure is proposed to solve these problems through radiation regulating and photon managing, thereby enhancing the conversion and utilization efficiency of concentrated solar energy. The performance of uniform porous structure and biomimetic hierarchical structure are comparatively investigated. The geometrical parameters (diameters, ratio, and filling factor) of the biomimetic hierarchical structure are investigated and optimized by finite-difference time-domain (FDTD) method combined with particle swarm optimization algorithm. The results indicate that the biomimetic hierarchical structure can enhance the effective light absorption and energy flow diffusion efficiency of concentrated solar energy, an increasing pore size is better for the biomimetic hierarchical structure than a decreasing pore size, and the feature sizes which serve as the boundary between large and small pores are found. For the effective light absorption, the biomimetic hierarchical structure has an enhancement of 3.4%, and for the energy flow diffusion efficiency, this structure has an enhancement of over 25%, compared with the uniform porous structure. This research can provide general and valuable guidance for enhancing solar energy utilizing efficiency of high-temperature solar thermochemical reactors, solar cells, and photocatalytic carriers, based on the biomimetic hierarchical structure.

Wrinkled surface microstructure for enhancing the infrared spectral performance of radiative cooling.

Radiative cooling is a passive cooling method that does not consume additional energy and has broad application prospects. In recent studies, the surface microstructure was found to have a significant influence on improving the emissivity in infrared spectra for radiative cooling. Accordingly, in this paper, an innovative wrinkled surface microstructure without any periodicity is proposed for enhancing the infrared spectral performance of radiative cooling. The effects of the height and number of wrinkles as well as the radius and volume fraction of particles on the infrared spectral performance of radiative cooling are investigated. The radiative cooling performances of the plane, pyramid, moth-eye, and wrinkled microstructures are comparatively investigated using the finite-difference time-domain (FDTD) method. The results show that the mean emissivity of innovative radiative cooling films with the wrinkled surface microstructure reaches 99.58% in the "atmospheric window" wavelength range. The mean emissivity of the wrinkled microstructure is improved by 19%, 22.16%, and 8.41% over those of the plane, pyramid, and moth-eye microstructures, respectively. This indicates that the wrinkled microstructure exhibits a better performance for radiative cooling than single periodic surface microstructures. Furthermore, the wrinkled microstructure has no periodicity so it has low production cost, which makes it possible to replace other periodic surface microstructures.

Vital roles of soil microbes in driving terrestrial nitrogen immobilization.

Nitrogen immobilization usually leads to nitrogen retention in soil and, thus, influences soil nitrogen supply for plant growth. Understanding soil nitrogen immobilization is important for predicting soil nitrogen cycling under anthropogenic activities and climate changes. However, the global patterns and drivers of soil nitrogen immobilization remain unclear. We synthesized 1350 observations of gross soil nitrogen immobilization rate (NIR) from 97 articles to identify patterns and drivers of NIR. The global mean NIR was 8.77 ± 1.01 mg N kg-1  soil day-1 . It was 5.55 ± 0.41 mg N kg-1  soil day-1 in croplands, 15.74 ± 3.02 mg N kg-1  soil day-1 in wetlands, and 15.26 ± 2.98 mg N kg-1  soil day-1 in forests. The NIR increased with mean annual temperature, precipitation, soil moisture, soil organic carbon, total nitrogen, dissolved organic nitrogen, ammonium, nitrate, phosphorus, and microbial biomass carbon. But it decreased with soil pH. The results of structural equation models showed that soil microbial biomass carbon was a pivotal driver of NIR, because temperature, total soil nitrogen, and soil pH mostly indirectly influenced NIR via changing soil microbial biomass. Moreover, microbial biomass carbon accounted for most of the variations in NIR among all direct relationships. Furthermore, the efficiency of transforming the immobilized nitrogen to microbial biomass nitrogen was lower in croplands than in natural ecosystems (i.e., forests, grasslands, and wetlands). These findings suggested that soil nitrogen retention may decrease under the land use change from forests or wetlands to croplands, but NIR was expected to increase due to increased microbial biomass under global warming. The identified patterns and drivers of soil nitrogen immobilization in this study are crucial to project the changes in soil nitrogen retention.

Precision De Novo Peptide Sequencing Using Mirror Proteases of Ac-LysargiNase and Trypsin for Large-scale Proteomics.

De novo peptide sequencing for large-scale proteomics remains challenging because of the lack of full coverage of ion series in tandem mass spectra. We developed a mirror protease of trypsin, acetylated LysargiNase (Ac-LysargiNase), with superior activity and stability. The mirror spectrum pairs derived from the Ac-LysargiNase and trypsin treated samples can generate full b and y ion series, which provide mutual complementarity of each other, and allow us to develop a novel algorithm, pNovoM, for de novo sequencing. Using pNovoM to sequence peptides of purified proteins, the accuracy of the sequence was close to 100%. More importantly, from a large-scale yeast proteome sample digested with trypsin and Ac-LysargiNase individually, 48% of all tandem mass spectra formed mirror spectrum pairs, 97% of which contained full coverage of ion series, resulting in precision de novo sequencing of full-length peptides by pNovoM. This enabled pNovoM to successfully sequence 21,249 peptides from 3,753 proteins and interpreted 44-152% more spectra than pNovo+ and PEAKS at a 5% FDR at the spectrum level. Moreover, the mirror protease strategy had an obvious advantage in sequencing long peptides. We believe that the combination of mirror protease strategy and pNovoM will be an effective approach for precision de novo sequencing on both single proteins and proteome samples.

Unambiguous Phosphosite Localization through the Combination of Trypsin and LysargiNase Mirror Spectra in a Large-Scale Phosphoproteome Study.

Understanding of the kinase-guided signaling pathways requires the identification and analysis of phosphosites. Mass spectrometry (MS)-based phosphoproteomics is a rapid and highly sensitive approach for high-throughput identification of phosphosites. However, phosphosite determination from MS data with a single protease is more likely to be ambiguous, regardless of the strategy used for phosphopeptide detection. Here, we explored the application of LysargiNase, which was recently reported to mirror trypsin in specificity to cleave arginine and lysine residues exclusively at the N-terminal side. We found that the combination of trypsin and LysargiNase mirror spectra resulted in higher ion coverage in MS2 spectra. The median ion coverage values of b ions in tryptic spectra, LysargiNase spectra, and combined spectra are 8.3, 20.5, and 25.0%, respectively. As for the median ion coverage of y ions, these values are 27.8, 10.0, and 32.3%. Higher ion coverage was helpful to pinpoint the precise phosphosites. Compared to trypsin alone, the combined use of trypsin and LysargiNase mirror spectra enabled 67.1% of mirror spectra with unreliable scores (confidence score <0.75) to become reliable (confidence score ≥ 0.75). Meanwhile, all of the mirror peptide-spectrum matches (PSMs) with multiple potential phosphosites from trypsin and LysargiNase digests could be assigned one precise phosphosite after applying the combination strategy. Besides, the combination strategy could identify more novel phosphosites than the union strategy did. We synthesized three phosphopeptides corresponding to the three novel phosphosites and validated the reliability of the identification. Taken together, our data demonstrated the distinctive potential of the combination strategy presented here for unambiguous phosphosite localization (Project accession PXD011178).

Quantitative Proteomics Combined with Two Genetic Strategies for Screening Substrates of Ubiquitin Ligase Hrt3.

Ubiquitin ligases (E3s) serve as key regulators for the ubiquitylation-mediated pathway. The identification of the corresponding relationship between E3 and its substrates is challenging but required for understanding the regulatory network of ubiquitylation. The low abundance of ubiquitinated conjugates and high redundancy of E3 substrate regulation made the screening pretty hard. Herein, we combined SILAC-based quantitative proteomics with two contrary genetic methods (overexpression and knockout) in theory for E3 (Hrt3, the F-box subunit of the SCF complex) substrate screening. The knockout method could not overcome the constraint mentioned above, while the overexpression approach turned on the access to the potential substrates of E3. Subsequently, we obtained 77 candidates, which are involved in many critical biological processes and need to be verified in the future. Within these candidates, we confirmed the relationship between one of the candidates Nce103 and Hrt3 and linked Hrt3 with oxygen sensitivity and oxidative stress response in which Nce103 took part as well. This research is also beneficial for understanding the impact of oxygen supply on regulation of yeast growth through the ubiquitination of Nce103.

Probing the Neutron Skin with Ultrarelativistic Isobaric Collisions.

Particle production in ultrarelativistic heavy ion collisions depends on the details of the nucleon density distributions in the colliding nuclei. We demonstrate that the charged hadron multiplicity distributions in isobaric collisions at ultrarelativistic energies provide a novel approach to determine the poorly known neutron density distributions and thus the neutron skin thickness in finite nuclei, which can in turn put stringent constraints on the nuclear symmetry energy.

Influence of uterine corpus invasion on prognosis in stage IA2-IIB cervical cancer: A multicenter retrospective cohort study.

OBJECTIVE: To determine the associations between the presence and depth of uterine corpus invasion and survival in patients with cervical cancer. METHODS: Clinical data of patients with stage IA2-IIB cervical cancer who underwent radical hysterectomy between 2004 and 2016 were retrospectively reviewed. Uterine corpus invasion was identified from a review of uterine pathology. Independent prognostic factors for 5-year disease-free survival (DFS) and overall survival (OS) were identified using multivariate forward stepwise Cox proportional hazards regression models. RESULTS: A total of 1414 patients with stage IA2-IIB cervical cancer from 11 medical institutions in China were included. Retrospective review of the original pathology reports revealed a missed diagnosis of uterine corpus invasion in 38 (13.4%) patients and a misdiagnosis in 20 (1.8%) patients. Therefore, 284 patients with cervical cancer and uterine corpus invasion (90 [31.7%] patients had endometrial invasion, 105 [37.0%] patients had myometrial invasion <50%, and 89 [31.3%] patients had myometrial invasion ≥50%), and 1130 patients with cervical cancer without uterine corpus invasion were included in the analysis. The 5-year DFS and OS were significantly shorter for patients with uterine corpus invasion compared to patients with no uterine corpus invasion. Myometrial invasion ≥50% was an independent prognostic factor associated with decreased 5-year DFS (aHR, 2.307, 95% CI, 1.588-3.351) and 5-year OS (aHR, 2.736, 95% CI, 1.813-4.130), while myometrial invasion <50% or endometrial invasion had no effect on patient outcomes. CONCLUSIONS: Diagnosis of uterine corpus invasion is frequently missed. Myometrial invasion ≥50% within the uterine corpus was an independent factor associated with worse prognosis in patients with cervical cancer, while myometrial invasion <50% or endometrial invasion had no effect on outcomes.

Competing endogenous RNA network and prognostic nomograms for hepatocellular carcinoma patients who underwent R0 resection.

The prognosis of hepatocellular carcinoma (HCC) after R0 resection is unsatisfactory due to the high rate of recurrence. In this study, we investigated the recurrence-related RNAs and the underlying mechanism. The long noncoding RNA (lncRNA), microRNA (miRNA), and messenger RNA (mRNA) expression data and clinical information of 247 patients who underwent R0 resection patients with HCC were obtained from The Cancer Genome Atlas. Comparing the 1-year recurrence group (n = 56) with the nonrecurrence group (n = 60), we detected 34 differentially expressed lncRNAs (DElncRNAs), five DEmiRNAs, and 216 DEmRNAs. Of these, three DElncRNAs, hsa-mir-150-5p, and 11 DEmRNAs were selected for constructing the competing endogenous RNA (ceRNA) network. Next, two nomogram models were constructed based separately on the lncRNAs and mRNAs that were further selected by Cox and least absolute shrinkage and selection operator regression analysis. The two nomogram models that showed a high prediction accuracy for disease-free survival with the concordance indexes at 0.725 and 0.639. Further functional enrichment analysis of DEmRNAs showed that the mRNAs in the ceRNA network and nomogram models were associated with immune pathways. Hence, we constructed a hsa-mir-150-5p-centric ceRNA network and two effective nomogram prognostic models, and the related RNAs may be useful as potential biomarkers for predicting recurrence in patients with HCC.

Ac-LysargiNase Complements Trypsin for the Identification of Ubiquitinated Sites.

Mass spectrometry (MS)-based identification of ubiquitinated sites requires trypsin digestion prior to MS analysis, and a signature peptide was produced with a diglycine residue attached to the ubiquitinated lysine (K-ε-GG peptide). However, the missed cleavage of modified lysines by trypsin results in modified peptides with increased length and charge, whose detection by MS analysis is suppressed by the vast majority of internally unmodified peptides. LysargiNase, the mirrored trypsin, is reported to cleave before lysine and arginine residues and to be favorable for the identification of methylation and phosphorylation, but its digestive characteristics related to ubiquitination are unclear. Herein, we tested the capacity of the in-house developed acetylated LysargiNase (Ac-LysargiNase) with high activity and stability, for cleaving ubiquitinated sites in both the seven types of ubiquitin chains and their corresponding K-ε-GG peptides. Interestingly, Ac-LysargiNase could efficiently cleave the K63-linked chain but had little effect on the other types of chains. Additionally, Ac-LysargiNase had higher exopeptidase activity than trypsin. Utilizing these features of the paired mirror proteases, a workflow of trypsin and Ac-LysargiNase tandem digestion was developed for the identification of ubiquitinated proteins. Through this method, the charge states and ionization capacity of the unmodified peptides were efficiently reduced, and the identification of modified sites was consequently increased by 30% to 50%. Strikingly, approximately 15% of the modified sites were cleaved by Ac-LysargiNase, resulting in shorter K-ε-GG peptides for better identification. The enzyme Ac-LysargiNase is expected to serve as an option for increasing the efficiency of modified site identification in ubiquitome research.