Comprehensive proteomic analyses are indispensable for elucidating protein regulatory networks in microbial chassis cells and accelerating the advancement of cellular factory designs and systems biology models. However, conventional methodologies for generating industrial microbial proteomic data sets remain heavily reliant on labor-intensive, manual workflows that are not only time-inefficient and costly but also inherently prone to human error. In this study, we developed an automated high-throughput proteomics sample preparation workflow (AutoDEP) tailored for industrial microorganisms. By integration of a highly efficient sodium dodecyl sulfate (SDS)-based lysis buffer with a commercially available 96-well DNA extraction plate on the Biomek i7 liquid handling system, AutoDEP enables fully automated processing of 96 samples from cell lysis to clean peptide generation. We rigorously benchmarked the performance of AutoDEP, achieving an excellent sample preparation efficiency with a zero missed cleavage rate exceeding 81%. The intraplate coefficient of variation (CV) showed a median below 7%, while interplate variability demonstrated that over 91% of proteins had CVs less than 20%. Additionally, the interday Pearson correlation coefficient (R2) exceeded 0.98, indicating high reproducibility. Notably, AutoDEP effectively handles protein loading amounts ranging from 5 to 200 μg, showcasing its robust applicability. Collectively, this work demonstrates the potential of AutoDEP to accelerate the construction of cell models based on industrial chassis cells. Furthermore, its robust performance suggests promising applications in large-scale studies, including clinical sample analysis.

Formaldehyde serves as a crucial intermediate metabolite during C1 compounds biotransformation. To optimize C1 utilization efficiency, it is essential to identify genes related to formaldehyde tolerance and enhance microbial resistance. Hence, we developed an evolved Corynebacterium glutamicum strain, FM-3, capable of withstanding 2.6 mM formaldehyde-a significant improvement over the parental strain. Integrated transcriptomic and proteomic analyses revealed that the enhanced formaldehyde tolerance correlated with the upregulation of cell wall biosynthesis proteins and DNA repair machinery. Genetic mutations identified in the evolved strains indicated that mutations in Cgl1199 (transcription termination factor Rho), and Cgl1590 (putative gluconeogenesis factor) played a pivotal role in formaldehyde tolerance. Further studies showed that Cgl1590 was involved in cell morphology regulation. This study enriches the understanding of formaldehyde tolerance mechanisms in C. glutamicum and provides guidance for enhancing strain tolerance to formaldehyde.

Pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenases (MDHs), the periplasmic metalloenzymes in Gram-negative methylotrophic bacteria, play a pivotal role in methane and methanol bio-utilization. Although the structures of many PQQ-dependent MDHs have been resolved, including the canonical heterotetrameric enzymes composed of two MxaF and two MxaI subunits with a molecule of PQQ and a calcium ion in the active site in MxaF, the biogenesis of these enzymes remains elusive. Here, we characterize a chaperone, MxaJ, responsible for PQQ incorporation by reconstructing a PQQ-dependent MDH assembly system in Escherichia coli. Using cryo-electron microscopy, we capture the structures of the intermediate complexes formed by the chaperone MxaJ and catalytic subunit MxaF during PQQ-dependent MDH maturation, revealing a chaperone-mediated molecular mechanism of cofactor incorporation. These findings not only advance our understanding on the biogenesis of PQQ-dependent MDH, but also provide an alternative engineering way for methane and methanol bioconversion.

How the brain reconciles dynamism with stability to balance learning and reliable memory storage has not yet been fully understood. To address the critical question, we longitudinally recorded place cells in the hippocampal dorsal CA1 region over 7 to 56 days, utilizing multiple goal-oriented navigation paradigms across various environments. We found that over 80% of place cells displayed multiple fields, undergoing complex evolution events including field disappearance, formation, and retention. Place fields from the same neuron showed limited coordination (~5%), with a preference for synchronized changes. We further uncovered the single field evolution rule: the longer a field remains active, the more likely it is to continue being active; conversely, the longer a field remains inactive, the less likely it is to recover the future fate of a place field depends on its past activity. Mathematical modeling revealed that this rule sufficiently demonstrates the growing stability of the dCA1 spatial representation at the population level. ### Competing Interest Statement The authors have declared no competing interest.

Aging-correlated cognitive declines, including deficiencies in spatial orientation and memory, may reflect dysfunction in the hippocampus and medial entorhinal cortex (MEC). However, aging-related changes in MEC at the cellular and molecular levels remain unclear. In this study, we found fewer grid cells with reduced spatial stability in old mice. We compared gene expression profiles between young and old mice using 10x Genomics Visium technology. Among 1664 differentially expressed genes, we discovered Bglap3, a marker gene for subpopulation in MEC Layer III with decreased cell number with age. Silencing of Bglap3+ neurons in young mice impaired the spatial tuning of neurons in MEC and the spatial learning of a new platform location in water maze. These findings help us to understand the cellular and molecular changes in the MEC in healthy aging animals and the changes of Bglap3+ cells in old mice indicating a possible cause of aging-related MEC deficiency. ### Competing Interest Statement The authors have declared no competing interest.

Hippocampal place cells represent the position of a rodent within an environment. In addition, recent experiments show that the CA1 subfield of a passive observer also represents the position of a conspecific performing a spatial task. However, whether this representation is allocentric, egocentric or mixed is less clear. In this study we investigated the representation of others during free behavior and in a task where female mice learned to follow a conspecific for a reward. We found that most cells represent the position of others relative to self-position (social-vector cells) rather than to the environment, with a prevalence of purely egocentric coding modulated by context and mouse identity. Learning of a pursuit task improved the tuning of social-vector cells, but their number remained invariant. Collectively, our results suggest that the hippocampus flexibly codes the position of others in multiple coordinate systems, albeit favoring the self as a reference point. How the position of conspecifics is represented in the brain is not fully understood. Here authors show that the position of conspecifics is represented relative to self-position in the hippocampus of female mice, which is modulated by context and identity and improved through learning.

With fast growth, synthetic biology powers us with the capability to produce high commercial value products in an efficient resource/energy-consuming manner. Comprehensive knowledge of the protein regulatory network of a bacterial host chassis, e.g., the actual amount of the given proteins, is the key to building cell factories for certain target hyperproduction. Many talent methods have been introduced for absolute quantitative proteomics. However, for most cases, a set of reference peptides with isotopic labeling (e.g., SIL, AQUA, QconCAT) or a set of reference proteins (e.g., commercial UPS2 kit) needs to be prepared. The higher cost hinders these methods for large sample research. In this work, we proposed a novel metabolic labeling-based absolute quantification approach (termed nMAQ). The reference Corynebacterium glutamicum strain is metabolically labeled with 15N, and a set of endogenous anchor proteins of the reference proteome is quantified by chemically synthesized light (14N) peptides. The prequantified reference proteome was then utilized as an internal standard (IS) and spiked into the target (14N) samples. SWATH-MS analysis is performed to obtain the absolute expression levels of the proteins from the target cells. The cost for nMAQ is estimated to be less than 10 dollars per sample. We have benchmarked the quantitative performance of the novel method. We believe this method will help with the deep understanding of the intrinsic regulatory mechanism of C. glutamicum during bioengineering and will promote the process of building cell factories for synthetic biology.

A novel xylose regulatory mechanism mediated by the transcription factor IpsA was revealed. A synergistic effect on carbon metabolism and energy supply was found to endow C. glutamicum with the efficient xylose utilization and rapid growth phenotype.

To gain a deep understanding of yeast-cell response to heat stress, multiple laboratory strains have been intensively studied via genome-wide expression analysis for the mechanistic dissection of classical heat-shock response (HSR). However, robust industrial strains of Saccharomyces cerevisiae have hardly been explored in global analysis for elucidation of the mechanism of thermotolerant response (TR) during fermentation. Herein, we employed data-independent acquisition and sequential window acquisition of all theoretical mass spectra based proteomic workflows to characterize proteome remodeling of an industrial strain, ScY01, responding to prolonged thermal stress or transient heat shock. By comparing the proteomic signatures of ScY01 in TR versus HSR as well as the HSR of the industrial strain versus a laboratory strain, our study revealed disparate response mechanisms of ScY01 during thermotolerant growth or under heat shock. In addition, through proteomics data-mining for decoding transcription factor interaction networks followed by validation experiments, we uncovered the functions of two novel transcription factors, Mig1 and Srb2, in enhancing the thermotolerance of the industrial strain. This study has demonstrated that accurate and high-throughput quantitative proteomics not only provides new insights into the molecular basis for complex microbial phenotypes but also pinpoints upstream regulators that can be targeted for improving the desired traits of industrial microorganisms.

The traditional Chinese drug Bombyx Batryticatus (BB), which is also named the white stiff silkworm, has been widely used in Chinese clinics for thousands of years. It is famous for its antispasmodic and blood circulation-promoting effects. Cardiomyocyte hypertrophy, interstitial cell hyperplasia, and myocardial fibrosis are closely related to the N-glycosylation of key proteins. To examine the alterations of N-glycosylation that occur in diabetic myocardium during the early stage of the disease, and to clarify the therapeutic effect of 1-Deoxynojirimycin (1-DNJ) extracted from BB, we used the db/db (diabetic) mouse model and an approach based on hydrophilic chromatography solid-phase extraction integrated with an liquid Chromatograph Mass Spectrometer (LC-MS) identification strategy to perform a site-specific N-glycosylation analysis of left ventricular cardiomyocyte proteins. Advanced glycation end products (AGEs), hydroxyproline, connective tissue growth factor (CTGF), and other serum biochemical indicators were measured with enzyme-linked immunosorbent assays (ELISA). In addition, the α-1,6-fucosylation of N-glycans was profiled with lens culinaris agglutinin (LCA) lectin blots and fluorescein isothiocyanate (FITC)-labelled lectin affinity histochemistry. The results indicated that 1-DNJ administration obviously downregulated myocardium protein N-glycosylation in db/db mice. The expression levels of serum indicators and fibrosis-related cytokines were reduced significantly by 1-DNJ in a dose-dependent manner. The glycan α-1,6-fucosylation level of the db/db mouse myocardium was elevated, and the intervention effect of 1-DNJ administration on N-glycan α-1,6-fucosylation was significant. To verify this result, the well-known transforming growth factor-β (TGF-β)/Smad2/3 pathway was selected, and core α-1,6-fucosylated TGF-β receptor II (TGFR-βII) was analysed semi-quantitatively with western blotting. The result supported the conclusions obtained from LCA lectin affinity histochemistry and lectin blot analysis. The expression level of α-1,6-fucosyltransferase (FUT8) mRNA was also detected, and the results showed that 1-DNJ administration did not cause obvious inhibitory effects on FUT8 expression. Therefore, the mechanism of 1-DNJ for relieving diabetic cardiomyopathy (DCM)-associated fibrosis can be concluded as the inhibition of N-acetylglucosamine (N-GlcNAc) formation and the reduction of substrate concentration.

Mass spectrometry (MS) technology has a special advantage in species determination for protein-rich samples which requires identification of species-specific peptides. However, for species discrimination of highly homologous proteins, it remains challenging to select the species unique peptides with routine proteomics approaches. In this work, we chose keratins and keratin-associated proteins (KAPs) present in cashmere fibers from goat and wool fibers from sheep as targets, to develop a dual-protease digestion workflow based on in-silico and experimental analysis. Combined usage of Glu-C and trypsin proteases showed the best digestion performance for MS identification of keratins and KAPs from different species. The parallel reaction monitoring (PRM) technique was implemented to validate and quantify the selected species discriminable peptides. The fiber composition of both blended animal hair fibers and industrial textile fabrics were successfully determined with the PRM assay. Furthermore, we identified over 360 peptides from the cashmere fiber beyond the current Uniprot goat proteome database. We expect our new workflow would improve the identification and quantification of keratin and KAPs, and provide inspiration for distinguishing other highly homologous proteins. We also anticipate the set of species-specific peptides from keratin or KAPs validated in this work would benefit the quality assessment for industrial fiber materials and textile products.

The traditional Chinese drug Bombyx Batryticatus mori.L (BBm) which is also named the 18 white stiff silkworm has been widely used in Chinese clinics for thousands of years. It is famous for 19 its antispasmodic and blood circulation promoting effects. Cardiomyocyte hypertrophy, interstitial 20 cell hyperplasia and myocardial fibrosis are closely related to the N-glycosylation of key proteins. 21 To examine the alterations of N-glycosylation that occur in diabetic myocardium during the early 22 stage of the disease and clarify the therapeutic effect of 1-DNJ extracted from BBm, we used the 23 db/db mouse model and an approach based on hydrophilic chromatography solid-phase extraction 24 integrated with an LC-MS/MS identification strategy to perform a site-specific N-glycosylation 25 analysis of left ventricular cardiomyocyte proteins. AGEs, hydroxyproline, CTGF and other serum 26 biochemical indicators were measured with ELISA. In addition, the α1,6-fucosylation of N-glycans 27 was profiled with LCA lectin blots and FITC-labelled lectin affinity histochemistry. The results 28 indicated that 1-DNJ administration obviously downregulated myocardium protein 29 N-glycosylation in db/db mice. The expression levels of serum indicators and fibrosis-related 30 cytokines were reduced significantly by 1-DNJ in a dose-dependent manner. The glycan 31 α1,6-fucosylation level of the db/db mouse myocardium was elevated, and the intervention effect 32 of 1-DNJ administration on N-glycan α1,6-fucosylation was significant. To verify this result, the 33 well-known TGF-β/smad2/3 pathway was selected, and core α1,6-fucosylated TGFR-βII was 34 analysed semi-quantitatively with western blotting. The result supported the conclusions obtained 35 from LCA lectin affinity histochemistry and lectin blot analysis. The expression level of FUT8 36 mRNA was also detected, and the results showed that 1-DNJ administration did not cause 37 obvious inhibitory effects on FUT8 expression. Therefore, the mechanism of 1-DNJ to relieve the 38 DCM-associated fibrosis can be concluded as the inhibition of N-GlcNAc formation and the 39 reduction of substrate concentration. 40

The Chinese drug Bombyx Batryticatus mori.L which also named as the white stiff silkworm is widely used in clinics, due to the significant antispasmodic and promotional blood circulation effects. In addition, its hypoglycemic effect is also recognized in recent years. From a pathological point of view, the enzymatic glycosylation and non-enzymatic glycation both have important roles in regulating properties of proteins and are associated with Diabetes. With the db/db mouse model, we examined the alterations of N-glycosylation of diabetic myocardium at primary stage and clarify the differences in glycosylation of myocardium before and after with 1-DNJ treatment. Hydrophilic chromatography solid phase extraction enrichment and LC-MS/MS identification was applied to profile the alternations in protein glycosylation. Meanwhile, N-glycan α1, 6-fucosylation alterations were profiled with LCA lectin blot and FITC-labelled lectin affinity histochemistry. Our results showed that AGES, hydroxyproline, CTGF and other serum indicators and fibrosis related cytokines expressional levels were reduced significantly by 1-DNJ in a dose-dependent manner. In order to verify this result, the well-known pathway of TGF-β/smad2/3 was picked out and α1, 6-core fucosylated TGFR-βⅡwas semi-quantified with western blot method. The result sustained the conclusion from LCA lectin affinity histochemistry and lectin blot analysis. The expressional level of α1, 6-fucosyltransferase mRNA was increased in the myocardium of db/db mice, however, the 1-DNJ administration did not show obvious inhibitory effect on FU8 expression. This unexpected result can be interpreted as 1-DNJ plays the roles by reducing the concentration of substrate rather than inhibiting α1，6-fucose glycosyltransferase expression. Meanwhile, 1-DNJ crude extract from BBm with some flavonoids accompany can also play the roles of anti-oxidant, and all the chemicals protect the diabetic myocardium from hyperglycemia damage commonly.

The medial entorhinal cortex (MEC) contains several discrete classes of GABAergic interneurons, but their specific contributions to spatial pattern formation in this area remain elusive. We employed a pharmacogenetic approach to silence either parvalbumin (PV)- or somatostatin (SOM)-expressing interneurons while MEC cells were recorded in freely moving mice. PV-cell silencing antagonized the hexagonally patterned spatial selectivity of grid cells, especially in layer II of MEC. The impairment was accompanied by reduced speed modulation in colocalized speed cells. Silencing SOM cells, in contrast, had no impact on grid cells or speed cells but instead decreased the spatial selectivity of cells with discrete aperiodic firing fields. Border cells and head direction cells were not affected by either intervention. The findings point to distinct roles for PV and SOM interneurons in the local dynamics underlying periodic and aperiodic firing in spatially modulated cells of the MEC. VIDEO ABSTRACT.

New types of modifications of histones keep emerging. Recently, histone H4K8 2-hydroxyisobutyrylation (H4K8(hib)) was identified as an evolutionarily conserved modification. However, how this modification is regulated within a cell is still elusive, and the enzymes adding and removing 2-hydroxyisobutyrylation have not been found. Here, we report that the amount of H4K8(hib) fluctuates in response to the availability of carbon source in Saccharomyces cerevisiae and that low-glucose conditions lead to diminished modification. The removal of the 2-hydroxyisobutyryl group from H4K8 is mediated by the histone lysine deacetylase Rpd3p and Hos3p in vivo. In addition, eliminating modifications at this site by alanine substitution alters transcription in carbon transport/metabolism genes and results in a reduced chronological life span (CLS). Furthermore, consistent with the glucose-responsive H4K8(hib) regulation, proteomic analysis revealed that a large set of proteins involved in glycolysis/gluconeogenesis are modified by lysine 2-hydroxyisobutyrylation. Cumulatively, these results established a functional and regulatory network among K-hib, glucose metabolism, and CLS.

The core-fucosylated (CF) glycoproteins are widely distributed in mammalian tissues and regulated under pathological conditions, especially in cancer progression. The Food and Drug Administration (FDA) has approved the core-fucosylated α-fetoprotein as a biomarker for the early diagnosis of hepatocellular carcinoma (HCC). An approach for identifying CF glycoproteins has significantly practical value. Here we introduce a novel method for identification of CF glycoproteome in human plasma. The method integrates tandem glycopeptide enrichment, stepped fragmentation, and "glycan diagnostic ion"-based spectrum refinement. With this method, the productivity of identifying CF glycopeptides will be significantly improved. We anticipate that this method could be widely utilized to explore the CF glycoproteins and their regulation under physiological or pathological condition.

Cashmere is regarded as a specialty and luxury fiber due to its scarcity and high economic value. For fiber quality assessment, it is technically very challenging to distinguish and quantify the cashmere fiber from yak or wool fibers because of their highly similar physical appearance and substantial protein sequence homology. To address this issue, we propose a workflow combining untargeted and targeted proteomics strategies for selecting, verifying and quantifying biomarkers for cashmere textile authentication. Untargeted proteomic surveys were first applied to identify 174, 157, and 156 proteins from cashmere, wool and yak fibers, respectively. After marker selection at different levels, peptides turned out to afford much higher selectivity than proteins for fiber species discrimination. Subsequently, parallel reaction monitoring (PRM) methods were developed for ten selected peptide markers. The PRM-based targeted analysis of peptide markers enabled accurate determination of fiber species and cashmere percentages in different fiber mixtures. Furthermore, collective use of these peptide makers allowed us to discriminate and quantify cashmere fibers in commercial finished fabrics that have undergone heavy chemical treatments. Cashmere proportion measurement in fabric samples using our proteomic approach was in good agreement with results from traditional light microscopy, yet our method can be more readily standardized to become an objective and robust assay for assessing authenticity of fibers and textiles. We anticipate that the proteomic strategies presented in our study could be further implicated in discovery of quality trait markers for other products containing highly homologous proteomes.

Detection of proteins containing single amino acid polymorphisms (SAPs) encoded by nonsynonymous SNPs (nsSNPs) can aid researchers in studying the functional significance of protein variants. Most proteogenomic approaches for large-scale SAPs mapping require construction of a sample-specific database containing protein variants predicted from the next-generation sequencing (NGS) data. Searching shotgun proteomic data sets against these NGS-derived databases allowed for identification of SAP peptides, thus validating the proteome-level sequence variation. Contrary to the conventional approaches, our study presents a novel strategy for proteome-wide SAP detection without relying on sample-specific NGS data. By searching a deep-coverage proteomic data set from an industrial thermotolerant yeast strain using our strategy, we identified 337 putative SAPs compared to the reference genome. Among the SAP peptides identified with stringent criteria, 85.2% of SAP sites were validated using whole-genome sequencing data obtained for this organism, which indicates high accuracy of SAP identification with our strategy. More interestingly, for certain SAP peptides that cannot be predicted by genomic sequencing, we used synthetic peptide standards to verify expression of peptide variants in the proteome. Our study has provided a unique tool for proteogenomics to enable proteome-wide direct SAP identification and capture nongenetic protein variants not linked to nsSNPs.

Proteomic analysis with data-independent acquisition (DIA) approaches represented by the sequential window acquisition of all theoretical fragment ion spectra (SWATH) technique has gained intense interest in recent years because DIA is able to overcome the intrinsic weakness of conventional data-dependent acquisition (DDA) methods and afford higher throughout and reproducibility for proteome-wide quantification. Although the raw mass spectrometry (MS) data quality and the data-mining workflow conceivably influence the throughput, accuracy and consistency of SWATH-based proteomic quantification, there lacks a systematic evaluation and optimization of the acquisition and data-processing parameters for SWATH MS analysis. Herein, we evaluated the impact of major acquisition parameters such as the precursor mass range, isolation window width and accumulation time as well as the data-processing variables including peak extraction criteria and spectra library selection on SWATH performance. Fine tuning these interdependent parameters can further improve the throughput and accuracy of SWATH quantification compared to the original setting adopted in most SWATH proteomic studies. Furthermore, we compared the effectiveness of two widely used peak extraction software PeakView and Spectronaut in discovery of differentially expressed proteins in a biological context. Our work is believed to contribute to a deeper understanding of the critical factors in SWATH MS experiments and help researchers optimize their SWATH parameters and workflows depending on the sample type, available instrument and software.

It is very challenging to distinguish and quantify the cashmere fiber from wool fibers due to their highly similar physical appearance and substantial protein sequence homology .To address this issue ,the proteomics research strategies were proposed for selecting ,verifying and quantifying biomarkers for cashmere textiles .Firstly ,the stable isotope labeled quantitative method and LC‐MS/MS were combined to analyze proteins from cashmere and wool fibers ,consequently the candidate markers were se‐lected .Secondly ,several specific‐species biomarkers were verified using targeted MS acquisition method with parallel reaction monitoring (PRM ) ,altogether 4 peptides from wool and 2 peptides from cashmere were considered to be the candidate peptide markers .Finally ,combining dimethyl labeling and PRM strategy ,the standard cures of cashmere candidate peptide markers was established ,and the quantitative method was validated with standard blended samples .As a result ,the relative error between ex‐perimental and theoretical is less than 15% .In conclusion ,a method has been established which can discover and verify biomark‐ers efficiently ,and can provide the objective and robust assay for assessing authenticity of fibers and textiles .The proteomic strategies presented in this work can be further implicated in discovery of biomarkers for other samples containing highly homolo‐gous proteomes ,also provide technical support for commercial textile components identifying or discovering quality trait markers of other products containing highly homologous proteomes .