Mass spectrometry-based draft of the mouse proteome.

The laboratory mouse ranks among the most important experimental systems for biomedical research and molecular reference maps of such models are essential informational tools. Here, we present a quantitative draft of the mouse proteome and phosphoproteome constructed from 41 healthy tissues and several lines of analyses exemplify which insights can be gleaned from the data. For instance, tissue- and cell-type resolved profiles provide protein evidence for the expression of 17,000 genes, thousands of isoforms and 50,000 phosphorylation sites in vivo. Proteogenomic comparison of mouse, human and Arabidopsis reveal common and distinct mechanisms of gene expression regulation and, despite many similarities, numerous differentially abundant orthologs that likely serve species-specific functions. We leverage the mouse proteome by integrating phenotypic drug (n > 400) and radiation response data with the proteomes of 66 pancreatic ductal adenocarcinoma (PDAC) cell lines to reveal molecular markers for sensitivity and resistance. This unique atlas complements other molecular resources for the mouse and can be explored online via ProteomicsDB and PACiFIC.

C/EBPε and its acetylation in PMN enhance the tolerance to trauma.

Severe trauma can lead to numerous serious complications, threating the well-being and vitality of the afflicted. The quantity and functionality of PMNs undergo rapid transformations in response to severe trauma, playing a pivotal role in the trauma response. The absence of CCAAT/enhancer-binding protein ε (C/EBPε) profoundly impairs the functionality of polymorphonuclear neutrophils (PMNs), a function of paramount importance in trauma. In this study, by generating mice with C/EBPε knocked out or overexpressed, we substantiate that C/EBPε ensures the restoration of PMN function, enhancing the expression of antimicrobial proteins and thereby promoting trauma recovery. Furthermore, diminished expression of C/EBPε is observed in trauma patients, with levels displaying a negative correlation with ISS and APACHE II scores, suggesting its potential as a prognostic indicator for clinical treatment. Mechanistically, we uncover the upregulation of SIRT1 and the inhibition of P300 participating in the suppression of C/EBPε acetylation, consequently reducing the resilience of mice to trauma. As therapeutic interventions, whether through the sole administration of PMN, NAM treatment, or their combination, all result in an increased survival rate in traumatic mice. In conclusion, our study elucidates the role of C/EBPε in enhancing the resilience to trauma and identifies C/EBPε acetylation as a critical regulatory mechanism, offering potential therapeutic approaches involving PMN transfusion and NAM treatment.

Carnosic acid: an effective phenolic diterpenoid for prevention and management of cancers via targeting multiple signaling pathways.

Cancer is a serious global public health issue, and a great deal of research has been made to treat cancer. Of these, discovery of promising compounds that effectively fight cancer always has been the main point of interest in pharmaceutical research. Carnosic acid (CA) is a phenolic diterpenoid compound widely present in Lamiaceae plants such as Rosemary (Rosmarinus officinalis L.). In recent years, there has been increasing evidence that CA has significant anti-cancer activity, such as leukaemia, colorectal cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, stomach cancer, lymphoma, prostate cancer, oral cancer, etc. The potential mechanisms involved by CA, including inhibiting cell proliferation, inhibiting metastasis, inducing cell apoptosis, stimulating autophagy, regulating the immune system, reducing inflammation, regulating the gut microbiota, and enhancing the effects of other anti-cancer drugs. This article reviews the biosynthesis, pharmacokinetics and metabolism, safety and toxicity, as well as the molecular mechanisms and signaling pathways of the anticancer activity of CA. This will contribute to the development of CA or CA-containing functional foods for the prevention and treatment of cancer, providing important advances in the advancement of cancer treatment strategies.

Meltome atlas-thermal proteome stability across the tree of life.

We have used a mass spectrometry-based proteomic approach to compile an atlas of the thermal stability of 48,000 proteins across 13 species ranging from archaea to humans and covering melting temperatures of 30-90 °C. Protein sequence, composition and size affect thermal stability in prokaryotes and eukaryotic proteins show a nonlinear relationship between the degree of disordered protein structure and thermal stability. The data indicate that evolutionary conservation of protein complexes is reflected by similar thermal stability of their proteins, and we show examples in which genomic alterations can affect thermal stability. Proteins of the respiratory chain were found to be very stable in many organisms, and human mitochondria showed close to normal respiration at 46 °C. We also noted cell-type-specific effects that can affect protein stability or the efficacy of drugs. This meltome atlas broadly defines the proteome amenable to thermal profiling in biology and drug discovery and can be explored online at http://meltomeatlas.proteomics.wzw.tum.de:5003/ and http://www.proteomicsdb.org.

ZnF2-Assisted Synthesis of Highly Luminescent InP/ZnSe/ZnS Quantum Dots for Efficient and Stable Electroluminescence.

High-quality InP-based quantum dots (QDs) have become very promising, environmentally benign light emitters for display applications, but their synthesis generally entails hazardous hydrofluoric acid. Here, we present a highly facile route to InP/ZnSe/ZnS core/shell/shell QDs with a near-unity photoluminescence quantum yield. As the key additive, the inorganic salt ZnF2 mildly reacts with carboxylic acid at a high temperature and in situ generates HF, which eliminates surface oxide impurities, thus facilitating epitaxial shell growth. The resulting InP/ZnSe/ZnS QDs exhibit a narrower emission line width and better thermal stability in comparison with QDs synthesized with hydrofluoric acid. Light-emitting diodes using large-sized InP/ZnSe/ZnS QDs without replacing original ligands achieve the highest peak external quantum efficiency of 22.2%, to the best of our knowledge, along with a maximum brightness of >110 000 cd/m2 and a T95 lifetime of >32 000 h at 100 cd/m2. This safe approach is anticipated to be applied for a wide range of III-V QDs.

Robust Capillary- and Micro-Flow Liquid Chromatography-Tandem Mass Spectrometry Methods for High-Throughput Proteome Profiling.

Capillary- and micro-flow liquid chromatography-tandem mass spectrometry (capLC-MS/MS and µLC-MS/MS) is becoming a valuable alternative to nano-flow LC-MS/MS due to its high robustness and throughput. The systematic comparison of capLC-MS/MS and µLC-MS/MS systems for global proteome profiling has not been reported yet. Here, the capLC-MS/MS (150 µm i.d. column, 1 µL/min) and µLC-MS/MS (1 mm i.d. column, 50 µL/min) systems were both established based on UltiMate 3000 RSLCnano coupled to an Orbitrap Exploris 240 by integrating with different flowmeters. We evaluated both systems in terms of sensitivity, analysis throughput, separation efficiency, and robustness. capLC-MS/MS was about 10 times more sensitive than µLC-MS/MS at different gradient lengths. Compared with capLC-MS/MS, µLC-MS/MS was able to achieve higher analysis throughput and separation efficiency. During the 7 days' long-term performance test, both systems showed good reproducibility of chromatographic full width (RSD < 3%), retention time (RSD < 0.4%), and protein identification (RSD < 3%). These results demonstrate that capLC-MS/MS is more suitable for high-throughput analysis of clinical samples with a limited starting material. When enough samples are available, µLC-MS/MS is preferred. Together, capLC and µLC coupled to Orbitrap Exploris 240 with moderate sensitivity should well meet the needs of large-cohort clinical proteomic analysis.

On the potential of micro-flow LC-MS/MS in proteomics.

INTRODUCTION: Due to its excellent sensitivity, nano-flow liquid chromatography tandem mass spectrometry (LC-MS/MS) is the mainstay in proteome research; however, this comes at the expense of limited throughput and robustness. In contrast, micro-flow LC-MS/MS enables high-throughput, robustness, quantitative reproducibility, and precision while retaining a moderate degree of sensitivity. Such features make it an attractive technology for a wide range of proteomic applications. In particular, large-scale projects involving the analysis of hundreds to thousands of samples. AREAS COVERED: This review summarizes the history of chromatographic separation in discovery proteomics with a focus on micro-flow LC-MS/MS, discusses the current state-of-the-art, highlights advances in column development and instrumentation, and provides guidance on which LC flow best supports different types of proteomic applications. EXPERT OPINION: Micro-flow LC-MS/MS will replace nano-flow LC-MS/MS in many proteomic applications, particularly when sample quantities are not limited and sample cohorts are large. Examples include clinical analyses of body fluids, tissues, drug discovery and chemical biology investigations, plus systems biology projects across all kingdoms of life. When combined with rapid and sensitive MS, intelligent data acquisition, and informatics approaches, it will soon become possible to analyze large cohorts of more than 10,000 samples in a comprehensive and fully quantitative fashion.

HIF-1α is necessary for activation and tumour-promotion effect of cancer-associated fibroblasts in lung cancer.

Cancer-associated fibroblasts (CAFs) activation is crucial for the establishment of a tumour promoting microenvironment, but our understanding of CAFs activation is still limited. In this study, we found that hypoxia-inducible factor-1α (HIF-1α) was highly expressed in CAFs of human lung cancer tissues and mouse spontaneous lung tumour. Accordingly, enhancing the expression of HIF-1α in fibroblasts via hypoxia induced the conversion of normal fibroblasts into CAFs. HIF-1α-specific inhibitor or HIF-1α knockout (KO) significantly attenuated CAFs activation, which was manifested by the decreased expression of COL1A2 and α-SMA. In vivo, during tumour formation, the expression of Ki-67 and proliferating cell nuclear antigen (PCNA) in the tumour tissue with HIF-1α KO fibroblasts was significantly lower than that of normal fibroblasts. Moreover, HIF-1α in fibroblasts could activate the NF-κB signalling pathway and enhance a subsequent secretion of CCL5, thus promoting the tumour growth. In conclusion, our results suggest that HIF-1α is essential for the activation and tumour-promotion function of CAFs in lung cancer (LC). And targeting HIF-1α expression on CAFs may be a promising strategy for LC therapy.

Identification of 7 000-9 000 Proteins from Cell Lines and Tissues by Single-Shot Microflow LC-MS/MS.

A current trend in proteomics is to acquire data in a "single-shot" by LC-MS/MS because it simplifies workflows and promises better throughput and quantitative accuracy than schemes that involve extensive sample fractionation. However, single-shot approaches can suffer from limited proteome coverage when performed by data dependent acquisition (ssDDA) on nanoflow LC systems. For applications where sample quantities are not scarce, this study shows that high proteome coverage can be obtained using a microflow LC-MS/MS system operating a 1 mm i.d. × 150 mm column, at a flow-rate of 50 µL/min and coupled to an Orbitrap HF-X mass spectrometer. The results demonstrate the identification of ∼9 000 proteins from 50 µg of protein digest from Arabidopsis roots, 7 500 from mouse thymus, and 7 300 from human breast cancer cells in 3 h of analysis time in a single run. The dynamic range of protein quantification measured by the iBAQ approach spanned 5 orders of magnitude and replicate analysis showed that the median coefficient of variation was below 20%. Together, this study shows that ssDDA by µLC-MS/MS is a robust method for comprehensive and large-scale proteome analysis and which may be further extended to more rapid chromatography and data independent acquisition approaches in the future.̀.

Robust Microflow LC-MS/MS for Proteome Analysis: 38 000 Runs and Counting.

Microflow liquid chromatography tandem mass spectrometry (µLC-MS/MS) is becoming a viable alternative to nanoflow LC-MS/MS for the analysis of proteomes. We have recently demonstrated the potential of such a system operating with a 1 mm i.d. × 150 mm column and at a flow rate of 50 µL/min for high-throughput applications. On the basis of the analysis of ∼38 000 samples measured on two instruments over the past two years, we now show that the approach is extremely robust. Up to 1500 analyses were performed within one month, and >14 000 samples could be analyzed on a single column without loss of chromatographic performance. Samples included proteomes of cell lines, tissues, and human body fluids, which were analyzed with or without prior peptide fractionation or stable isotope labeling. We show that the µLC-MS/MS system is capable of measuring 2600 proteins from undepleted human plasma and ∼5000 proteins from crude human urine in 1 day, demonstrating its potential for in-depth as well as high-throughput clinical application.

Research Progress on Diagnosis and Treatment of Chronic Osteomyelitis.

We review the representatives literatures on chronic osteomyelitis, sum up the new insights in recent years into diagnostic options and treatment regimens, analyze the advantages and disadvantages of various diagnostic approaches and treatment strategies, and propose areas of interest to make current diagnostic and treatment strategies more specific.

SH2 Superbinder Modified Monolithic Capillary Column for the Sensitive Analysis of Protein Tyrosine Phosphorylation.

In this study, we present a method to specifically capture phosphotyrosine (pTyr) peptides from minute amount of sample for the sensitive analysis of protein tyrosine phosphorylation. We immobilized SH2 superbinder on a monolithic capillary column to construct a microreactor to enrich pTyr peptides. It was found that the synthetic pTyr peptide could be specifically enriched by the microreactor from the peptide mixture prepared by spiking of the synthetic pTyr peptide into the tryptic digests of α-casein and ß-casein with molar ratios of 1:1000:1000. The microreactor was further applied to enrich pTyr peptides from pervanadate-treated HeLa cell digests for phosphoproteomics analysis, which resulted in the identification of 796 unique pTyr sites. In contrast, the conventional SH2 superbinder-based method identified 41 pTyr sites for the same sample, only 5.2% of the number achieved by the microreactor. Finally, this microreactor was also applied to analyze the pTyr in Shc1 complex, an immunopurified protein complex, which resulted in the identification of 15 pTyr sites. Together, this technique is best fitted to analyze the pTyr in minute amount of sample and will have broad application in fields where only a limited amount of sample is available.

Diazoxide ameliorates severity of experimental osteoarthritis by activating autophagy via modulation of the osteoarthritis-related biomarkers.

Accumulating evidence suggests that autophagy plays a protective role in chondrocytes and prevents cartilage degeneration in osteoarthritis (OA). The objective of this study was to investigate the effect of diazoxide on chondrocyte death and cartilage degeneration and to determine whether these effects are correlated to autophagy in experimental OA. In this study, a cellular OA model was established by stimulating SW1353 cells with interleukin 1ß. A rat OA model was generated by transecting the anterior cruciate ligament combined with the resection of the medial menisci, followed by treatment with diazoxide or diazoxide combination with 3-methyladenine. The percentage of viable cells was evaluated using calcein-acetoxymethyl/propidium iodide double staining. The messenger RNA expression levels of collagen type II alpha 1 chain (COL2A1), matrix metalloproteinase 13 (MMP-13), TIMP metallopeptidase inhibitor 1 (TIMP-1), and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) were determined using quantitative real-time polymerase chain reaction. The cartilage thickness and joint space were evaluated using ultrasound. SW1353 cell degeneration and autophagosomes were observed using transmission electron microscopy. The expression levels of microtubule-associated protein 1 light chain 3 (LC3), beclin-1, P62, COL2A1, and MMP-13 were evaluated using immunofluorescence staining and Western blot analysis. Diazoxide significantly attenuated articular cartilage degeneration and SW1353 cell death in experimental OA. The restoration of autophagy was observed in the diazoxide-treated group. The beneficial effects of diazoxide were markedly blocked by 3-methyladenine. Diazoxide treatment also modulated the expression levels of OA-related biomarkers. These results demonstrated that diazoxide exerted a chondroprotective effect and attenuated cartilage degeneration by restoring autophagy via modulation of OA-related biomarkers in experimental OA. Diazoxide treatment might be a promising therapeutic approach to prevent the development of OA.

Ultra-deep tyrosine phosphoproteomics enabled by a phosphotyrosine superbinder.

We present a new strategy for systematic identification of phosphotyrosine (pTyr) by affinity purification mass spectrometry (AP-MS) using a Src homology 2 (SH2)-domain-derived pTyr superbinder as the affinity reagent. The superbinder allows for markedly deeper coverage of the Tyr phosphoproteome than anti-pTyr antibodies when an optimal amount is used. We identified ∼20,000 distinct phosphotyrosyl peptides and >10,000 pTyr sites, of which 36% were 'novel', from nine human cell lines using the superbinder approach. Tyrosine kinases, SH2 domains and phosphotyrosine phosphatases were preferably phosphorylated, suggesting that the toolkit of kinase signaling is subject to intensive regulation by phosphorylation. Cell-type-specific global kinase activation patterns inferred from label-free quantitation of Tyr phosphorylation guided the design of experiments to inhibit cancer cell proliferation by blocking the highly activated tyrosine kinases. Therefore, the superbinder is a highly efficient and cost-effective alternative to conventional antibodies for systematic and quantitative characterization of the tyrosine phosphoproteome under normal or pathological conditions.

Sensitive, Robust, and Cost-Effective Approach for Tyrosine Phosphoproteome Analysis.

Albeit much less abundant than Ser/Thr phosphorylation (pSer/pThr), Tyr phosphorylation (pTyr) is considered as a hallmark in cellular signal transduction. However, its analysis at the proteome level remains challenging. The conventional immunopurification (IP) approach using antibodies specific to pTyr sites is known to have low sensitivity, poor reproducibility and high cost. Our recent study indicated that SH2 domain-derived pTyr-superbinder is a good replacement of pTyr antibody for the specific enrichment of pTyr peptides for phosphoproteomics analysis. In this study, we presented an efficient SH2 superbinder based workflow for the sensitive analysis of tyrosine phosphoproteome. This new method can identify 41% more pTyr peptides than the previous method. Its excellent performance was demonstrated by the analysis of a variety of different samples. For the highly tyrosine phosphorylated sample, for example, pervanadate-treated Jurkat T cells, it identified over 1800 high confident pTyr sites from only 2 mg of proteins. For the unstimulated Jurkat cells, where the pTyr events rarely occurred, it identified 343 high confident pTyr sites from 5 mg of proteins, which was 31% more than that obtained by the antibody-based method. For the heterogeneous sample of tissue, it identified 197 high confident pTyr sites from 5 mg protein digest of mouse skeletal muscle. In general, it is a sensitive, robust and cost-effective approach and would have wide applications in the study of the regulatory role of tyrosine phosphorylation in diverse physiological and pathological processes.

Kinetics of CO2 Absorption into Aqueous Basic Amino Acid Salt: Potassium Salt of Lysine Solution.

Aqueous amino acid salts are considered as an attractive alternative to alkanolamine solvents (e.g., MEA) for carbon dioxide (CO2) absorption. The kinetics of CO2 into unloaded aqueous solutions of potassium lysinate (LysK) was studied using a wetted wall column at concentrations ranging from 0.25 to 2.0 M and temperatures from 298 to 333 K. Physicochemical properties of aqueous LysK solutions such as density, viscosity, and physical solubility of CO2 were measured to evaluate the reaction rate constants. The reaction pathway is described using zwitterion mechanism taking into account the effect of ionic strength on the reaction rate. Under the fast pseudo-first-order regime, the reaction rate parameters were obtained and correlated in a power-law reaction rate expression. LysK shows higher chemical reactivity toward CO2 than the industrial standard MEA and most of amino acid salts. Its reaction rate constants increase considerably with concentration and temperature. The reaction order is found to be an average value of 1.58 with respect to LysK. The forward second-order kinetic rate constant, k2 0 , are obtained as 31615 and 84822 m3 kmol−1 s−1 at 298 and 313 K, respectively with activation energy of 51.0 kJ mol−1. The contribution of water to the zwitterion deprotonation seems to be more significant than that of LysK for the above-mentioned kinetic conditions

The proteomic analysis improved by cleavage kinetics-based fractionation of tryptic peptides.

Selective enrichment of specific peptides is an effective way to identify low abundance proteins. Fractionation of peptides prior to mass spectrometry is another widely used approach to reduce sample complexity in order to improve proteome coverage.In this study, we designed a multi-stage digestion strategy to generate peptides with different trypsin cleavage kinetics. It was found that each of the collected peptide fractions yielded many new protein identifications compared to the control group due to the reduced complexity. The overlapping peptides identified between adjacent fractions were very low, indicating that each fraction had different sets of peptides. The multi-stage digestion strategy separates tryptic peptides with different cleavage kinetics while RPLC separates peptides with different hydrophobicity. These two separation strategies were highly orthogonal, and showed an effective multidimensional separation to improve proteome coverage.

Selective Enrichment of Cysteine-Containing Phosphopeptides for Subphosphoproteome Analysis.

Among the natural amino acids, cysteine is unique since it can form a disulfide bond through oxidation and reduction of sulfhydryl and thus plays a pervasive role in modulation of proteins activities and structures. Crosstalk between phosphorylation and other post-translational modifications has become a recurrent theme in cell signaling regulation. However, the crosstalk between the phosphorylation and the formation and reductive cleavage of disulfide bond has not been investigated so far. To facilitate the study of this crosstalk, it is important to explore the subset of phosphoproteome where phosphorylations are occurred near to cysteine in the protein sequences. In this study, we developed a straightforward sequential enrichment method by combining the thiol affinity chromatography with the immobilized titanium ion affinity chromatography to selectively enrich cysteine-containing phosphopeptides. The high specificity and high sensitivity of this method were demonstrated by analyzing the samples of Jurkat cells. This "divide and conquer" strategy by specific analysis of a subphosphoproteome enables identification of more low abundant phosphosites than the conventional global phosphoproteome approach. Interestingly, amino acid residues surrounding the identified phosphosites were enriched with buried residues (L, V, A, C) while depleted with exposed residues (D, E, R, K). Also, the phosphosites identified by this approach showed a dramatic decrease in locating in disorder regions compared to that identified by conventional global phosphoproteome. Further analysis showed that more proline directed kinases and fewer acidophilic kinases were responsible for the phosphorylation sites of this subphosphoproteome.

Identification of phosphopeptides with unknown cleavage specificity by a de novo sequencing assisted database search strategy.

In theory, proteases with broad cleavage specificity could be applied to digest protein samples to improve the phosphoproteomic analysis coverage. However, in practice this approach is seldom employed. This is because the identification of phosphopeptides without enzyme specificity by conventional database search strategy is extremely difficult due to the huge search space. In this study, we investigated the performance of a de novo sequencing assisted database search strategy for the identification of such phosphopeptides. Firstly, we compared the performance of conventional database search strategy and the de novo sequencing assisted database search strategy for the identification of peptides and phosphopeptides without stetting enzyme specificity. It was found that the identification sensitivity dropped significantly for the conventional one while it was only slightly decreased for the new approach. Then, this new search strategy was applied to identify phosphopeptides generated by Proteinase K digestion, which resulted in the identification of 717 phosphopeptides. Finally, this strategy was utilized for the identification of serum endogenous phosphopeptides, which were generated in vivo by different kinds of proteases and kinases, and the identification of 68 unique serum endogenous phosphopepitdes was successfully achieved.

Large-scale quantification of single amino-acid variations by a variation-associated database search strategy.

Global quantification of the single amino-acid variations (SAAVs) is essential to investigate the roles of SAAVs in disease progression. However, few efforts have been made on this issue due to the lack of high -throughput approach. Here we presented a strategy by integration of the stable isotope dimethyl labeling with variation-associated database search to globally quantify the SAAVs at the first time. A protein database containing 87,745 amino acid variant sequences and 73,910 UniProtKB/Swiss-Prot canonical protein entries was constructed for database search, and higher energy collisional dissociation combined with collision-induced dissociation fragmentation modes were applied to improve the quantification coverage of SAAVs. Compared with target proteomics in which only a few sites could be quantified, as many as 282 unique SAAVs sites were quantified between hepatocellular carcinoma (HCC) and normal human liver tissues by our strategy. The variation rates in different samples were evaluated, and some interesting SAAVs with significant increase normalized quantification ratios, such as T1406N in CPS1 and S197R in HTATIP2, were observed to highly associate with HCC progression. Therefore, the newly developed strategy enables the large-scale comparative analysis of variations at the protein level and holds a promising future in the research related to variations.