Adipose triglyceride lipase suppresses noncanonical inflammasome by hydrolyzing LPS.

Intracellular recognition of lipopolysaccharide (LPS) by mouse caspase-11 or human caspase-4 is a vital event for the activation of the noncanonical inflammasome. Whether negative regulators are involved in intracellular LPS sensing is still elusive. Here we show that adipose triglyceride lipase (ATGL) is a negative regulator of the noncanonical inflammasome. Through screening for genes participating in the noncanonical inflammasome, ATGL is identified as a negative player for intracellular LPS signaling. ATGL binds LPS and catalyzes the removal of the acylated side chains that contain ester bonds. LPS with under-acylated side chains no longer activates the inflammatory caspases. Cells with ATGL deficiency exhibit enhanced immune responses when encountering intracellular LPS, including an elevated secretion of interleukin-1ß, decreased cell viability and increased cell cytotoxicity. Moreover, ATGL-deficient mice show exacerbated responses to endotoxin challenges. Our results uncover that ATGL degrades cytosolic LPS to suppress noncanonical inflammasome activation.

Meeting Report on the 2nd Chinese American Society for Mass Spectrometry Conference: Advancing Biological and Pharmaceutical Mass Spectrometry.

The 2nd CASMS conference was held virtually through Gather. Town platform from October 17 to 21, 2022, with a total of 363 registrants including an outstanding and diverse group of scientists at the forefront of their research fields from both academia and industry worldwide, especially in the United States and China. The conference offered a 5-day agenda with an exciting scientific program consisting of two plenary lectures, 14 parallel symposia, and 4 special sessions in which a total of 97 invited speakers presented technological innovations and their applications in proteomics & biological mass spectrometry and metabo-lipidomics & pharmaceutical mass spectrometry. In addition, 18 invited speakers/panelists presented at 3 research-focused and 2 career development workshops. Moreover, 144 posters, 54 lightning talks, 5 sponsored workshops, and 14 exhibitions were presented, from which 20 posters and 8 lightning talks received presentation awards. Furthermore, the conference featured 1 MCP lectureship and 5 young investigator awardees for the first time to highlight outstanding mid-career and early-career rising stars in mass spectrometry from our society. The conference provided a unique scientific platform for young scientists (i.e., graduate students, postdocs and junior faculty/investigators) to present their research, meet with prominent scientists, and learn about career development and job opportunities (http://casms.org).

Lipid bilayer induces contraction of the denatured state ensemble of a helical-bundle membrane protein.

Defining the denatured state ensemble (DSE) and disordered proteins is essential to understanding folding, chaperone action, degradation, and translocation. As compared with water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we measure the DSE of the helical membrane protein GlpG of Escherichia coli (E. coli) in native-like lipid bilayers. The DSE was obtained using our steric trapping method, which couples denaturation of doubly biotinylated GlpG to binding of two streptavidin molecules. The helices and loops are probed using limited proteolysis and mass spectrometry, while the dimensions are determined using our paramagnetic biotin derivative and double electron-electron resonance spectroscopy. These data, along with our Upside simulations, identify the DSE as being highly dynamic, involving the topology changes and unfolding of some of the transmembrane (TM) helices. The DSE is expanded relative to the native state but only to 15 to 75% of the fully expanded condition. The degree of expansion depends on the local protein packing and the lipid composition. E. coli's lipid bilayer promotes the association of TM helices in the DSE and, probably in general, facilitates interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes.

Capillary zone electrophoresis-high field asymmetric ion mobility spectrometry-tandem mass spectrometry for top-down characterization of histone proteoforms.

Characterization of histone proteoforms with various post-translational modifications (PTMs) is critical for a better understanding of functions of histone proteoforms in epigenetic control of gene expression. Mass spectrometry (MS)-based top-down proteomics (TDP) is a valuable approach for delineating histone proteoforms because it can provide us with a bird's-eye view of histone proteoforms carrying diverse combinations of PTMs. Here, we present the first example of coupling capillary zone electrophoresis (CZE), ion mobility spectrometry (IMS), and MS for online multi-dimensional separations of histone proteoforms. Our CZE-high-field asymmetric waveform IMS (FAIMS)-MS/MS platform identified 366 (ProSight PD) and 602 (TopPIC) histone proteoforms from a commercial calf histone sample using a low microgram amount of histone sample as the starting material. CZE-FAIMS-MS/MS improved the number of histone proteoform identifications by about 3 folds compared to CZE-MS/MS alone (without FAIMS). The results indicate that CZE-FAIMS-MS/MS could be a useful tool for comprehensive characterization of histone proteoforms with high sensitivity.

Pilot Evaluation of the Long-Term Reproducibility of Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Top-Down Proteomics of a Complex Proteome Sample.

Mass spectrometry (MS)-based top-down proteomics (TDP) has revolutionized biological research by measuring intact proteoforms in cells, tissues, and biofluids. Capillary zone electrophoresis-tandem MS (CZE-MS/MS) is a valuable technique for TDP, offering a high peak capacity and sensitivity for proteoform separation and detection. However, the long-term reproducibility of CZE-MS/MS in TDP remains unstudied, which is a crucial aspect for large-scale studies. This work investigated the long-term qualitative and quantitative reproducibility of CZE-MS/MS for TDP for the first time, focusing on a yeast cell lysate. Over 1000 proteoforms were identified per run across 62 runs using one linear polyacrylamide (LPA)-coated separation capillary, highlighting the robustness of the CZE-MS/MS technique. However, substantial decreases in proteoform intensity and identification were observed after some initial runs due to proteoform adsorption onto the capillary inner wall. To address this issue, we developed an efficient capillary cleanup procedure using diluted ammonium hydroxide, achieving high qualitative and quantitative reproducibility for the yeast sample across at least 23 runs. The data underscore the capability of CZE-MS/MS for large-scale quantitative TDP of complex samples, signaling its readiness for deployment in broad biological applications. The MS RAW files were deposited in ProteomeXchange Consortium with the data set identifier of PXD046651.

Recent advances (2019-2021) of capillary electrophoresis-mass spectrometry for multilevel proteomics.

Multilevel proteomics aims to delineate proteins at the peptide (bottom-up proteomics), proteoform (top-down proteomics), and protein complex (native proteomics) levels. Capillary electrophoresis-mass spectrometry (CE-MS) can achieve highly efficient separation and highly sensitive detection of complex mixtures of peptides, proteoforms, and even protein complexes because of its substantial technical progress. CE-MS has become a valuable alternative to the routinely used liquid chromatography-mass spectrometry for multilevel proteomics. This review summarizes the most recent (2019-2021) advances of CE-MS for multilevel proteomics regarding technological progress and biological applications. We also provide brief perspectives on CE-MS for multilevel proteomics at the end, highlighting some future directions and potential challenges.

Bounding the Amount of Entanglement from Witness Operators.

We present an approach to estimate the operational distinguishability between an entangled state and any separable state directly from measuring an entanglement witness. We show that this estimation also implies bounds on a variety of other well-known entanglement quantifiers. This approach for entanglement estimation is then extended to both the measurement-device-independent scenario and the fully device-independent scenario, where we obtain nontrivial but suboptimal bounds. The procedure requires no numerical optimization and is easy to compute. It offers ways for experimenters to not only detect, but also quantify, entanglement from the standard entanglement witness procedure.

Meeting Report on the 3rd Chinese American Society for Mass Spectrometry Conference-Advancing Biological and Pharmaceutical Mass Spectrometry.

Following the highly successful Chinese American Society for Mass Spectrometry (CASMS) conferences in the previous 2 years, the 3rd CASMS Conference was held virtually on August 28-31, 2023, using the Gather.Town platform to bring together scientists in the MS field. The conference offered a 4-day agenda with a scientific program consisting of two plenary lectures, and 14 parallel symposia in which a total of 70 speakers presented technological innovations and their applications in proteomics and biological MS and metabo-lipidomics and pharmaceutical MS. In addition, 16 invited speakers/panelists presented at two research-focused and three career development workshops. Moreover, 86 posters, 12 lightning talks, 3 sponsored workshops, and 11 exhibitions were presented, from which 9 poster awards and 2 lightning talk awards were selected. Furthermore, the conference featured four young investigator awardees to highlight early-career achievements in MS from our society. The conference provided a unique scientific platform for young scientists (i.e. graduate students, postdocs, and junior faculty/investigators) to present their research, meet with prominent scientists, learn about career development, and job opportunities (http://casms.org).

Large-scale top-down proteomics of the Arabidopsis thaliana leaf and chloroplast proteomes.

We present a large-scale top-down proteomics (TDP) study of plant leaf and chloroplast proteins, achieving the identification of over 4700 unique proteoforms. Using capillary zone electrophoresis coupled with tandem mass spectrometry analysis of offline size-exclusion chromatography fractions, we identify 3198 proteoforms for total leaf and 1836 proteoforms for chloroplast, with 1024 and 363 proteoforms having post-translational modifications, respectively. The electrophoretic mobility prediction of capillary zone electrophoresis allowed us to validate post-translational modifications that impact the charge state such as acetylation and phosphorylation. Identified modifications included Trp (di)oxidation events on six chloroplast proteins that may represent novel targets of singlet oxygen sensing. Furthermore, our TDP data provides direct experimental evidence of the N- and C-terminal residues of numerous mature proteoforms from chloroplast, mitochondria, endoplasmic reticulum, and other sub-cellular localizations. With this information, we suggest true transit peptide cleavage sites and correct sub-cellular localization signal predictions. This large-scale analysis illustrates the power of top-down proteoform identification of post-translational modifications and intact sequences that can benefit our understanding of both the structure and function of hundreds of plant proteins.

Coupling High-Field Asymmetric Waveform Ion Mobility Spectrometry with Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Top-Down Proteomics.

Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has emerged as an essential technique for top-down proteomics (TDP), providing superior separation efficiency and high detection sensitivity for proteoform analysis. Here, we aimed to further enhance the performance of CZE-MS/MS for TDP via coupling online gas-phase proteoform fractionation using high-field asymmetric waveform ion mobility spectrometry (FAIMS). When the compensation voltage (CV) of FAIMS was changed from -50 to 30 V, the median mass of identified proteoforms increased from less than 10 kDa to about 30 kDa, suggesting that FAIMS can efficiently fractionate proteoforms by their size. CZE-FAIMS-MS/MS boosted the number of proteoform identifications from a yeast sample by nearly 3-fold relative to CZE-MS/MS alone. It particularly benefited the identification of relatively large proteoforms, improving the number of proteoforms in a mass range of 20-45 kDa by 6-fold compared to CZE-MS/MS alone. FAIMS fractionation gained nearly 20-fold better signal-to-noise ratios of randomly selected proteoforms than no FAIMS. We expect that CZE-FAIMS-MS/MS will be a useful tool for further advancing the sensitivity and coverage of TDP. This work shows the first example of coupling CE with ion mobility spectrometry (IMS) for TDP.

Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Top-Down Proteomics of Mouse Brain Integral Membrane Proteins.

Mass spectrometry (MS)-based top-down characterization of integral membrane proteins (IMPs) is crucial for understanding their functions in biological processes. However, it is technically challenging due to their low solubility in typical MS-compatible buffers. In this work, for the first time, we developed an efficient capillary zone electrophoresis (CZE)-tandem MS (MS/MS) method for the top-down proteomics (TDP) of IMPs enriched from mouse brains. Our technique employs a sample buffer containing 30% (v/v) formic acid and 60% (v/v) methanol for solubilizing IMPs and utilizes a separation buffer of 30% (v/v) acetic acid and 30% (v/v) methanol for maintaining the solubility of IMPs during CZE separation. Single-shot CZE-MS/MS identified 51 IMP proteoforms from the mouse brain sample. Coupling size exclusion chromatography (SEC) to CZE-MS/MS enabled the identification of 276 IMP proteoforms from the mouse brain sample containing 1-4 transmembrane domains. This proof-of-concept work demonstrates the high potential of CZE-MS/MS for the large-scale TDP of IMPs.

TopFD: A Proteoform Feature Detection Tool for Top-Down Proteomics.

Top-down liquid chromatography-mass spectrometry (LC-MS) analyzes intact proteoforms and generates mass spectra containing peaks of proteoforms with various isotopic compositions, charge states, and retention times. An essential step in top-down MS data analysis is proteoform feature detection, which aims to group these peaks into peak sets (features), each containing all peaks of a proteoform. Accurate protein feature detection enhances the accuracy in MS-based proteoform identification and quantification. Here, we present TopFD, a software tool for top-down MS feature detection that integrates algorithms for proteoform feature detection, feature boundary refinement, and machine learning models for proteoform feature evaluation. We performed extensive benchmarking of TopFD, ProMex, FlashDeconv, and Xtract using seven top-down MS data sets and demonstrated that TopFD outperforms other tools in feature accuracy, reproducibility, and feature abundance reproducibility.

Pilot investigation of magnetic nanoparticle-based immobilized metal affinity chromatography for efficient enrichment of phosphoproteoforms for mass spectrometry-based top-down proteomics.

Protein phosphorylation is a vital and common post-translational modification (PTM) in cells, modulating various biological processes and diseases. Comprehensive top-down proteomics of phosphorylated proteoforms (phosphoproteoforms) in cells and tissues is essential for a better understanding of the roles of protein phosphorylation in fundamental biological processes and diseases. Mass spectrometry (MS)-based top-down proteomics of phosphoproteoforms remains challenging due to their relatively low abundance. Herein, we investigated magnetic nanoparticle-based immobilized metal affinity chromatography (IMAC, Ti4+, and Fe3+) for selective enrichment of phosphoproteoforms for MS-based top-down proteomics. The IMAC method achieved reproducible and highly efficient enrichment of phosphoproteoforms from simple and complex protein mixtures. It outperformed one commercial phosphoprotein enrichment kit regarding the capture efficiency and recovery of phosphoproteins. Reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) analyses of yeast cell lysates after IMAC (Ti4+ or Fe3+) enrichment produced roughly 100% more phosphoproteoform identifications compared to without IMAC enrichment. Importantly, the phosphoproteoforms identified after Ti4+-IMAC or Fe3+-IMAC enrichment correspond to proteins with much lower overall abundance compared to that identified without the IMAC treatment. We also revealed that Ti4+-IMAC and Fe3+-IMAC could enrich different pools of phosphoproteoforms from complex proteomes and the combination of those two methods will be useful for further improving the phosphoproteoform coverage from complex samples. The results clearly demonstrate the value of our magnetic nanoparticle-based Ti4+-IMAC and Fe3+-IMAC for advancing top-down MS characterization of phosphoproteoforms in complex biological systems.

Evaluation of Machine Learning Models for Proteoform Retention and Migration Time Prediction in Top-Down Mass Spectrometry.

Reversed-phase liquid chromatography (RPLC) and capillary zone electrophoresis (CZE) are two primary proteoform separation methods in mass spectrometry (MS)-based top-down proteomics. Proteoform retention time (RT) prediction in RPLC and migration time (MT) prediction in CZE provide additional information for accurate proteoform identification and quantification. While existing methods are mainly focused on peptide RT and MT prediction in bottom-up MS, there is still a lack of methods for proteoform RT and MT prediction in top-down MS. We systematically evaluated eight machine learning models and a transfer learning method for proteoform RT prediction and five models and the transfer learning method for proteoform MT prediction. Experimental results showed that a gated recurrent unit (GRU)-based model with transfer learning achieved a high accuracy (R = 0.978) for proteoform RT prediction and that the GRU-based model and a fully connected neural network model obtained a high accuracy of R = 0.982 and 0.981 for proteoform MT prediction, respectively.

Automated Capillary Isoelectric Focusing-Mass Spectrometry with Ultrahigh Resolution for Characterizing Microheterogeneity and Isoelectric Points of Intact Protein Complexes.

Protein complexes are the functional machines in the cell and are heterogeneous due to protein sequence variations and post-translational modifications (PTMs). Here, we present an automated nondenaturing capillary isoelectric focusing-mass spectrometry (ncIEF-MS) methodology for uncovering the microheterogeneity of intact protein complexes. The method exhibited superior separation resolution for protein complexes than conventional native capillary zone electrophoresis (nCZE-MS). In our study, ncIEF-MS achieved liquid-phase separations and MS characterization of seven different forms of a streptavidin homotetramer with variations of N-terminal methionine removal, acetylation, and formylation and four forms of the carbonic anhydrase-zinc complex arising from variations of PTMs (succinimide, deamidation, etc.). In addition, ncIEF-MS resolved different states of an interchain cysteine-linked antibody-drug conjugate (ADC1) as a new class of anticancer therapeutic agents that bears a distribution of varied drug-to-antibody ratio (DAR) species. More importantly, ncIEF-MS enabled precise measurements of isoelectric points (pIs) of protein complexes, which reflect the surface electrostatic properties of protein complexes. We studied how protein sequence variations/PTMs modulate the pIs of protein complexes and how drug loading affects the pIs of antibodies. We discovered that keeping the N-terminal methionine residue of one subunit of the streptavidin homotetramer decreased its pI by 0.1, adding one acetyl group onto the streptavidin homotetramer reduced its pI by nearly 0.4, incorporating one formyl group onto the streptavidin homotetramer reduced its pI by around 0.3, and loading two more drug molecules on one ADC1 molecule increased its pI by 0.1. The data render the ncIEF-MS method a valuable tool for delineating protein complexes.

Capillary Zone Electrophoresis-Tandem Mass Spectrometry As an Alternative to Liquid Chromatography-Tandem Mass Spectrometry for Top-down Proteomics of Histones.

Top-down proteomics (TDP) is an ideal approach for deciphering the histone code and it routinely employs reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS). Because of the extreme complexity of histones regarding the number of proteoforms, new analytical tools with high-capacity separation and highly sensitive detection of proteoforms are required for TDP of histones. Here we present capillary zone electrophoresis (CZE)-MS/MS via the electro-kinetically pumped sheath-flow CE-MS interface for large-scale top-down delineation of histone proteoforms. CZE-MS/MS identified a comparable number of proteoforms to RPLC-MS/MS from a calf histone sample with more than 30-fold less sample consumption (75-ng vs. Three µg), indicating its substantially higher sensitivity. We identified about 400 histone proteoforms from the calf histone sample using two-dimensional size-exclusion chromatography (SEC)-CZE-MS/MS with less than 300-ng proteins consumed. We identified histone proteoforms carrying various tentative post-translational modifications (PTMs), for example, acetylation, methylation (mono-, di-, and tri-), phosphorylation, and succinylation. The electrophoretic mobility (µef) of unmodified histone proteoforms can be predicted accurately (R2 = 0.98) with an optimized semiempirical model based on our recent work. The results render CZE-MS/MS as a useful tool for deciphering the histone code in a proteoform-specific manner and on a global scale.

Nanoparticle-Aided Nanoreactor for Nanoproteomics.

Large-scale bottom-up proteomics of few even single cells is crucial for a better understanding of the roles played by cell-to-cell heterogeneity in disease and development. Novel proteomic methodologies with extremely high sensitivity are required for few even single-cell proteomics. Sample processing with high recovery and no contaminants is one key step. Here we developed a nanoparticle-aided nanoreactor for nanoproteomics (Nano3) technique for processing low-nanograms of mammalian cell proteins for proteome profiling. The Nano3 technique employed nanoparticles packed in a capillary channel to form a nanoreactor (≤30 nL) for concentrating, cleaning, and digesting proteins originally in a lysis buffer containing sodium dodecyl sulfate (SDS), followed by nanoRPLC-MS/MS analysis. The Nano3 method identified a 40-times higher number of proteins based on MS/MS from 2-ng mouse brain protein samples compared to the SP3 (single-pot solid-phase-enhanced sample preparation) method, which performed the sample processing using the nanoparticles in a 10 µL solution in an Eppendorf tube. The data indicates a drastically higher sample recovery of the Nano3 compared to the SP3 method for processing mass-limited proteome samples. In this pilot study, the Nano3 method was further applied in processing 10-1000 HeLa cells for bottom-up proteomics, producing 441 ± 263 (n = 4) (MS/MS) and 983 ± 292 (n = 4) [match between runs (MBR)+MS/MS] protein identifications from only 10 HeLa cells using a Q-Exactive HF mass spectrometer. The preliminary results render the Nano3 method a useful approach for processing few mammalian cells for proteome profiling.

Investigating native capillary zone electrophoresis-mass spectrometry on a high-end quadrupole-time-of-flight mass spectrometer for the characterization of monoclonal antibodies.

Native capillary zone electrophoresis-mass spectrometry (CZE-MS) has attracted attentions for the characterization of monoclonal antibodies (mAbs) due to the potential of CZE for highly efficient separations of mAbs under native conditions as well as its compatibility with native electrospray ionization (ESI)-MS. However, the low sample loading capacity and limited separation resolution of native CZE for large proteins and protein complexes (e.g. mAbs) impede the widespread adoption of native CZE-MS. Here, we present a novel native capillary isoelectric focusing (cIEF)-assisted CZE-MS method for the characterization of mAbs with much larger sample loading capacity and significantly better separation resolution than native CZE-MS alone. The native cIEF-assisted CZE-MS employed separation capillaries with a new carbohydrate-based neutral coating, a commercilized electrokinetically pumped sheathflow CE-MS interface, and a high-end quadrupole-time-of-flight (Q-TOF) mass spectrometer. Using the method, we documented the separations of different proteoforms of the SigmaMAb and the detection of its various glyco-proteoforms and homodimer. The native cIEF-assisted CZE-MS separated the NIST mAb into three peaks with a submicroliter sample loading volume, corresponding to its different proteoforms. We observed that both the NIST mAb and its homodimer had eight glyco-proteoforms, four of which had low abundance. The results demonstrate the potential of our native cIEF-assisted CZE-MS method for advancing the characterization of large proteins and protein complexes under native conditions.

COPS8 in cutaneous melanoma: an oncogene that accelerates the malignant development of tumor cells and predicts poor prognosis.

This study aimed to investigate the roles of COP9 signalosome subunit 8 (COPS8) and its underlying mechanism in cutaneous melanoma. Bioinformatics tools were utilized to analyze the expression of COPS8 in cutaneous melanoma, while Kaplan-Meier analysis was employed to assess the correlation between COPS8 and patients' overall survival. The proliferation, migration, and invasion of cells were estimated by CCK8, colony formation, and Transwell assays. Western blot was used to check the expression of epithelial-mesenchymal transition (EMT)-related proteins. Results showed that COPS8 was up-regulated and predicted a poor clinical outcome for cutaneous melanoma patients. Knockdown of COPS8 inhibited cutaneous melanoma cell proliferation, migration and invasion, whereas overexpression of COPS8 resulted in the opposite outcomes. The up-regulation of E-cadherin and down-regulation of N-cadherin, vimentin, and snail were caused by silencing COPS8 while their expression showed contrary trends in cells with overexpressed COPS8. Collectively, COPS8 is up-regulated and promotes cutaneous melanoma progression via regulating EMT.

Toward a Universal Sample Preparation Method for Denaturing Top-Down Proteomics of Complex Proteomes.

A universal and standardized sample preparation method becomes vital for denaturing top-down proteomics (dTDP) to advance the scale and accuracy of proteoform delineation in complex biological systems. It needs to have high protein recovery, minimum bias, good reproducibility, and compatibility with downstream mass spectrometry (MS) analysis. Here, we employed a lysis buffer containing sodium dodecyl sulfate for extracting proteoforms from cells and, for the first time, compared membrane ultrafiltration (MU), chloroform-methanol precipitation (CMP), and single-spot solid-phase sample preparation using magnetic beads (SP3) for proteoform cleanup for dTDP. The MU method outperformed CMP and SP3 methods, resulting in high and reproducible protein recovery from both Escherichia coli cell (59 ± 3%) and human HepG2 cell (86 ± 5%) samples without a significant bias. Single-shot capillary zone electrophoresis (CZE)-MS/MS analyses of the prepared E. coli and HepG2 cell samples using the MU method identified 821 and 516 proteoforms, respectively. Nearly 30 and 50% of the identified E. coli and HepG2 proteins are membrane proteins. CZE-MS/MS identified 94 histone proteoforms from the HepG2 sample with various post-translational modifications, including acetylation, methylation, and phosphorylation. Our results suggest that combining the SDS-based protein extraction and the MU-based protein cleanup could be a universal sample preparation method for dTDP. The MS raw data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD018248.