Differential alterations of CXCR3, CXCR5 and CX3CR1 in patients with immune thrombocytopenia.

As a versatile element for maintaining homeostasis, the chemokine system has been reported to be implicated in the pathogenesis of immune thrombocytopenia (ITP). However, research pertaining to chemokine receptors and related ligands in adult ITP is still limited. The states of several typical chemokine receptors and cognate ligands in the circulation were comparatively assessed through various methodologies. Multiple variable analyses of correlation matrixes were conducted to characterize the correlation signatures of various chemokine receptors or candidate ligands with platelet counts. Our data illustrated a significant decrease in relative CXCR3 expression and elevated plasma levels of CXCL4, 9-11, 13, and CCL3 chemokines in ITP patients with varied platelet counts. Flow cytometry assays revealed eminently diminished CXCR3 levels on T and B lymphocytes and increased CXCR5 on cytotoxic T cell (Tc) subsets in ITP patients with certain platelet counts. Meanwhile, circulating CX3CR1 levels were markedly higher on T cells with a concomitant increase in plasma CX3CL1 level in ITP patients, highlighting the importance of aberrant alterations of the CX3CR1-CX3CL1 axis in ITP pathogenesis. Spearman's correlation analyses revealed a strong positive association of peripheral CXCL4 mRNA level, and negative correlations of plasma CXCL4 concentration and certain chemokine receptors with platelet counts, which might serve as a potential biomarker of platelet destruction in ITP development. Overall, these results indicate that the differential expression patterns and distinct activation states of peripheral chemokine network, and the subsequent expansion of circulating CXCR5+ Tc cells and CX3CR1+ T cells, may be a hallmark during ITP progression, which ultimately contributes to thrombocytopenia in ITP patients.

High-Sensitivity Detection toward SARS-CoV-2 S1 Glycoprotein by Parallel Reaction Monitoring Mass Spectrometry.

The outbreak of coronavirus disease 2019 (COVID-19) has overwhelmed the global economy and human well-being. On account of the sharp increase in test demand, there is a need for an accurate and alternative diagnosis method for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, with the aim to specifically identify the trace SARS-CoV-2 S1 glycoprotein, we developed a high-sensitivity and high-selectivity diagnostic method based on the targeted parallel reaction monitoring (PRM) assay of eight selected peptides. This study emphasizes the outstanding detection sensitivity of 0.01 pg of the SARS-CoV-2 S1 glycoprotein even in the interference of other structural proteins, which to our knowledge is the current minimum limit of detection for the SARS-CoV-2 S1 glycoprotein. This technology could further identify 0.01 pg of the SARS-CoV-2 S1 glycoprotein in a spike pseudovirus, revealing its practical effectiveness. All our preliminary results throw light on the capability of the mass spectrometry-based targeted PRM assay to identify SARS-CoV-2 as a practicable orthogonal diagnostic tool. Furthermore, this technology could be extended to other pathogens (e.g., MERS-CoV S1 protein or SARS-CoV S1 protein) by quickly adjusting the targeted peptides of MS data acquisition. In summary, this strategy is universal and flexible and could be quickly adjusted to detect and discriminate different mutants and pathogens.

Coexistence of tet(A) and blaKPC-2 in the ST11 hypervirulent tigecycline- and carbapenem-resistant Klebsiella pneumoniae isolated from a blood sample.

Carbapenem-resistant Klebsiella pneumoniae are distributed worldwide. This study aimed to characterize a hypervirulent tigecycline-resistant and carbapenem-resistant Klebsiella pneumoniae strain, XJ-K2, collected from a patient's blood. We tested antimicrobial susceptibility, virulence, and whole-genome sequencing (WGS) on strain XJ-K2. WGS data were used to identify virulence and resistance genes and to perform multilocus sequence typing (MLST) and phylogenetic analysis. Three novel plasmids, including a pLVPK-like virulence plasmid (pXJ-K2-p1) and two multiple resistance plasmids (pXJ-K2-KPC-2 and pXJ-K2-p3), were discovered in strain XJ-K2. The IncFII(pCRY) plasmid pXJ-K2-p3 carried the dfrA14, sul2, qnrS1, blaLAP-2, and tet(A) resistance genes. The IncFII(pHN7A8)/IncR plasmid pXJ-K2-KPC-2 also carried a range of resistance elements, containing rmtB, blaKPC-2, blaTEM-1, blaCTX-M-65, and fosA3. MLST analysis revealed that strain XJ-K2 belonged to sequence type 11 (ST11). Seven complete phage sequences and many virulence genes were found in strain XJ-K2. Meanwhile, antimicrobial susceptibility tests and G. mellonella larval infection models confirmed the extensively drug resistance (XDR) and hypervirulence of KJ-K2. To our knowledge, this is the first observation and description of the ST11 hypervirulent tigecycline- and carbapenem-resistant K. pneumoniae strain co-carrying blaKPC-2 and the tet(A) in a patient's blood in China. Further investigation is needed to understand the resistance and virulence mechanisms of this significant hypervirulent tigecycline- and carbapenem-resistant strain.

Surface Nanosieving Polyether Sulfone Particles with Graphene Oxide Encapsulation for the Negative Isolation toward Extracellular Vesicles.

Extracellular vesicles (EVs) contain specific biomarkers for disease diagnosis. Current EV isolation methods are hampered in important biological applications due to their low recovery and purity. Herein, we first present a novel EV negative isolation strategy based on surface nanosieving polyether sulfone particles with graphene oxide encapsulation (SNAPs) by which the coexisting proteins are irreversibly adsorbed by graphene oxide (GO) inside the particles, while EVs with large sizes are excluded from the outside due to the well-defined surface pore sizes (10-40 nm). By this method, the purity of the isolated EVs from urine could be achieved 4.91 ± 1.01e10 particles/µg, 40.9-234 times higher than those obtained by the ultracentrifugation (UC), size-exclusion chromatography (SEC), and PEG-based precipitation. In addition, recovery ranging from 90.4 to 93.8% could be obtained with excellent reproducibility (RSD < 6%). This was 1.8-4.3 times higher than those obtained via SEC and UC, comparable to that obtained by PEG-based precipitation. Taking advantage of this strategy, we further isolated urinary EVs from IgA nephropathy (IgAN) patients and healthy donors for comparative proteome analysis, by which significantly regulated EV proteins were found to distinguish IgAN patients from healthy donors. All of the results indicated that our strategy would provide a new avenue for highly efficient EV isolation to enable many important clinical applications.

Comprehensive Analysis of Protein N-Terminome by Guanidination of Terminal Amines.

Protein N-termini and their modifications not only represent different protein isoforms but also relate to the functional annotation and proteolytic activities. Currently, negative selection methods, such as terminal amine isotopic labeling of substrates (TAILS), are the most popular strategy to analyze the protein N-terminome, in which dimethylation or acetylation modification is commonly used to block the free amines of proteome samples. However, after tryptic digestion, the generated long peptides, caused by the missing cleavage of blocked lysine, could hardly be identified by MS, which hindered the deep-coverage analysis of N-terminome. Herein, to solve this problem, we developed an approach, named terminal amine guanidination of substrates (TAGS). 1H-Pyrazole-1-carboxamidine was used to effectively guanidinate lysine ε-amines and N-terminal α-amines, followed by tryptic digestion to generate N-terminal peptides without free amines and internal peptides with free amines. Then, the internal peptides with free amines were removed by hyperbranched polyglycerol-aldehyde polymers (HPG-ALDs) to achieve the negative enrichment of N-terminome. By TAGS, not only the cleavage rate of blocked lysine could be improved, but also the ionization efficiency of tryptic peptides was increased. In comparison, 1814 and 1620 protein N-termini were, respectively, identified by TAGS and TAILS in Saccharomyces cerevisiae (S. cerevisiae). Among them, 1012 N-termini were uniquely identified in TAGS. Furthermore, by the combination of TAGS and the stable isotope labeling with amino acids in cell culture (SILAC)/label-free quantitative method, we not only identified the known N-terminal cleavage fragment of gasdermin D but also identified some new cleavage sites during Val-boroPro-induced pyroptosis. All these results demonstrated that our developed approach, TAGS, might be of great promise for the comprehensive analysis of N-terminome and beneficial for promoting the identification of protein isoforms and studying in-depth the proteolytic activity of proteins.

Site-Specific Quantification of Persulfidome by Combining an Isotope-Coded Affinity Tag with Strong Cation-Exchange-Based Fractionation.

Protein persulfidation is one of the most important oxidative translational modifications and plays vital roles in various important biological processes. However, the proteome-wide identification of persulfidation sites is a great challenge because of the difficulties in accurately differentiating persulfide groups with disulfide and thiol groups in proteins as well as the extremely low abundance of persulfidated peptides. By current approaches, the persulfidated peptides were often identified by the cleavage of their persulfide groups by reductants prior to MS analysis; therefore, it would bring about a false positive identification and was unable to identify persulfidation sites accurately for a single peptide with multiple cysteine residues. In this study, a novel strategy for the site-specific quantification of persulfidome (SSQPer) was developed. By this strategy, the persulfidated proteins were first labeled with cleavable isotope-coded affinity tag (c-ICAT) reagents. After digestion, the labeled persulfidated peptides were selectively enriched with streptavidin beads and fractionated by strong cation exchange chromatography, followed by LC-MS/MS identification. To evaluate the performance of SSQPer, the persulfidated BSA digests with 20 persulfidation sites identified were used to spike HeLa cell digests with mass ratios of 1:100 and 1:1000, and 16 and 13 persulfidated sites were respectively identified. We applied SSQPer to the site-specific quantification of persulfidome in the epithelial-mesenchymal transition (EMT) process, and 226 endogenous persulfidation sites were identified, of which 74.3% were newly discovered. All of these results demonstrated that the SSQPer strategy would provide a promising tool to profile the site-specific persulfidome and pave the way for future investigation to expand our knowledge of persulfidation.

Advances in exosome isolation methods and their applications in proteomic analysis of biological samples.

Exosomes are membrane-bound vesicles secreted by cells, and contain various important biological molecules, such as lipids, proteins, messenger RNAs, microRNAs, and noncoding RNAs. Emerging evidence demonstrates that proteomic analysis of exosomes is of great significance in studying metabolic diseases, tumor metastasis, immune regulation, and so forth. However, exosome proteomic analysis has high requirements with regard to the purity of collected exosomes. Here recent advances in the methods for isolating exosomes and their applications in proteomic analysis are summarized. Graphical abstract.

Enzymatic Reactor with Trypsin Immobilized on Graphene Oxide Modified Polymer Microspheres To Achieve Automated Proteome Quantification.

Protein digestion and isotope labeling are two critical steps in proteome quantification. However, the conventional in-solution protocol unavoidably suffers from disadvantages such as time-consuming, low labeling efficiency, and tedious off-line manual operation, which might affect the quantification accuracy, reproducibility, and throughput. To address these problems, we developed a fully automated proteome quantification platform, in which an ultraperformance immobilized microreactor (upIMER) with graphene-oxide-modified polymer microspheres as the matrix was developed, to achieve not only the simultaneous protein digestion and 18O labeling, but also the online integration with nano-high-pressure liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoHPLC-ESI-MS/MS). Compared to the conventional off-line protocols, such a platform exhibits obviously improved digestion and 18O labeling efficiency (only 8% peptides with missed cleavage sites, 99% labeling efficiency, and 2.5 min reaction time), leading to the increased quantification coverage, accuracy, precision and throughput. All the results demonstrated that our developed fully automated platform should provide new opportunities to improve the accuracy, reproducibility, and throughput for proteome quantification.

Depletion of internal peptides by site-selective blocking, phosphate labeling, and TiO2 adsorption for in-depth analysis of C-terminome.

The analysis of protein C-termini is of great importance, because it not only provides valuable information about protein function, but also facilitates the elucidation of proteolytic processing. However, even with the recent methods for the global profiling of protein C-termini, the identification of C-termini is still far behind that of N-termini due to the lack of basic residue and low reactive carboxyl group. Therefore, an unbiased and complementary method for C-termini profiling is imperative. In this work, we developed a negative enrichment strategy to achieve the in-depth analysis of C-terminome. Proteins were firstly amidated to block carboxyl groups, followed by lysyl endoproteinase (LysC) digestion to generate C-terminal peptides with α-amines and internal peptides bearing both α- and ε-amines. After the α-amines were blocked by site-selective dimethylation or succinylation, the remaining ε-amines on internal peptides were labeled with phosphate groups. Finally, internal peptides were depleted by TiO2, leaving exclusively the fraction of C-terminal peptides for LC-MS/MS analysis. With Escherichia coli (E. coli) digests as the sample, the efficiency of amidation, dimethylation/succinylation, phosphate labeling and TiO2 depletion was proved high. With the combination of dimethyl and succinic blocking strategy, our method enabled the identification of 477 unique C-terminal peptides in E. coli. In comparison with the C-terminal amine-based isotope labeling of substrates (C-TAILS) method, 83 C-termini were identified by both methods, whereas 369 C-termini were unique to C-TAILS and 394 to our dataset. The method proposed is therefore efficient and possibly promotes the comprehensive profiling of C-termini. Graphical Abstract Negative isolation of C-terminal peptides with combination of site-selective blocking, phosphate labeling, and TiO2 adsorption.

Integrated platform with a combination of online digestion and (18)O labeling for proteome quantification via an immobilized trypsin microreactor.

A novel automated integrated platform for quantitative proteome analysis was established with a combination of online digestion of proteins and in situ(18)O labeling by an immobilized enzyme reactor (IMER); digests were captured and desalted by a C18 trap column, and peptides were analyzed by nanoRPLC-ESI-MS/MS. Bovine serum albumin (BSA) was used to evaluate the performance of the developed platform. Compared with traditional offline methods, not only the digestion and labeling time was shortened from 36 h to just 1 h, but also the labeling efficiency was improved from 95% to 99%. Furthermore, the back-exchange from (18)O to (16)O could also be efficiently avoided by the use of IMER. The platform was further evaluated by the quantitative analysis of 100 ng (18)O and (16)O online labeled yeast sample with a mixing ratio of 1 : 1, and the results showed significantly improved sensitivity and reproducibility, as well as improved quantitative accuracy than offline method. With these advantages, the integrated platform was finally applied to the quantitative profiling of 100 ng proteins extracted from two mouse hepatocarcinoma ascites syngeneic cell lines with high and low lymph node metastases rates, and ten differentially expressed proteins were successfully found, most of which were related to tumorigenesis and tumor metastasis. All these results demonstrate that the developed integrated platform can provide a new way for high efficiency (18)O labeling and the quantitative analysis of trace amounts of sample with high accuracy and high reproducibility.

1-Dodecyl-3-methylimidazolium chloride-assisted sample preparation method for efficient integral membrane proteome analysis.

Due to their extremely hydrophobic nature, the analysis of integral membrane proteins (IMPs) is of great challenge. Although various additives have been applied to improve the solubility of IMPs, they still suffer from low solubilization efficiency, incompatibility with trypsin digestion, or interference with MS detection. Herein, the systematic study on the effect of ionic liquid structure on membrane protein solubilization and trypsin biocompatibility was performed, based on which 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) was selected for the sample preparation of IMPs. Compared with other commonly used additives, such as sodium dodecyl sulfate (SDS), Rapigest, and methanol, C12Im-Cl showed the best performance. In addition, with a strong cation exchange trap column, it could be easily removed after trypsin digestion, which not only was beneficial to avoid protein precipitation during digestion but also had no adverse effect on LC-MS-based separation and detection. Such a C12Im-Cl-assisted sample preparation method was further applied to the membrane proteome analysis of rat brain. Compared with the SDS-assisted method, 1.4 and 3.5 times improvement on the identified IMP and hydrophobic peptide number were achieved (251 vs 178, and 982 vs 279). All these results demonstrated that the C12Im-Cl-assisted sample preparation method is of great promise to promote the large-scale membrane proteome profiling.

Improved accuracy for label-free absolute quantification of proteome by combining the Absolute Protein EXpression profiling algorithm and summed tandem mass spectrometric total ion current.

Proteome scale absolute quantification is fundamental for the quantitative understanding of an organism. The unsatisfactory accuracy for protein abundance estimation of current algorithms has been partially improved by the Absolute Protein EXpression profiling (APEX) algorithm, which implements the prior expectations of peptides' appearances in the calculation of protein abundances. However, the abundance feature (AF) in APEX is the spectral count (SC); an AF suffers from a narrow dynamic range, thus, unsatisfactory accuracy. Therefore, we adopted another tandem mass spectrometric (MS/MS) level AF called Summed MS/MS Total ion current (SMT), which cumulates the MS/MS fragment intensities rather than simply counting the MS/MS spectra, to surmount this particular deficiency. The combination of APEX and SMT (abbreviated as APEX-SMT) is capable of improving the accuracy of absolute quantification by reducing the average relative deviation by ~55-85% compared to that of APEX, through a series of tests on the Universal Proteomics Standard sample with a dynamic range of 5 orders of magnitude (UPS2). The algorithm could also be used for relative quantification. When applied to the relative quantification of a publicly available benchmark dataset, APEX-SMT could provide comparable accuracy to APEX. All these results suggest that APEX-SMT is a promising alternative to APEX for proteome quantification.

Biphasic microreactor for efficient membrane protein pretreatment with a combination of formic acid assisted solubilization, on-column pH adjustment, reduction, alkylation, and tryptic digestion.

Combining good dissolving ability of formic acid (FA) for membrane proteins and excellent complementary retention behavior of proteins on strong cation exchange (SCX) and strong anion exchange (SAX) materials, a biphasic microreactor was established to pretreat membrane proteins at microgram and even nanogram levels. With membrane proteins solubilized by FA, all of the proteomics sample processing procedures, including protein preconcentration, pH adjustment, reduction, and alkylation, as well as tryptic digestion, were integrated into an "SCX-SAX" biphasic capillary column. To evaluate the performance of the developed microreactor, a mixture of bovine serum albumin, myoglobin, and cytochrome c was pretreated. Compared with the results obtained by the traditional in-solution process, the peptide recovery (93% vs 83%) and analysis throughput (3.5 vs 14 h) were obviously improved. The microreactor was further applied for the pretreatment of 14 µg of membrane proteins extracted from rat cerebellums, and 416 integral membrane proteins (IMPs) (43% of total protein groups) and 103 transmembrane peptides were identified by two-dimensional nanoliquid chromatography-electrospray ionization tandem mass spectrometry (2D nano-LC-ESI-MS/MS) in triplicate analysis. With the starting sample preparation amount decreased to as low as 50 ng, 64 IMPs and 17 transmembrane peptides were identified confidently, while those obtained by the traditional in-solution method were 10 and 1, respectively. All these results demonstrated that such an "SCX-SAX" based biphasic microreactor could offer a promising tool for the pretreatment of trace membrane proteins with high efficiency and throughput.

The efficacy and safety of epirubicin and cyclophosphamide combined with pyrotinib in neoadjuvant treatment for HER2-positive breast cancer: A real-world study.

Purpose: Long-term survival benefit of anthracyclines for human epidermal growth factor receptor 2 (HER2)-positive breast cancer is clear. In the neoadjuvant treatment, compared with the monoclonal antibody such as trastuzumab and pertuzumab, the clinical benefit of pyrotinib, a new small-molecule tyrosine kinase inhibitor (TKI), as the main anti-HER2 strategy currently requires more research to determine. Our real-world study is the first prospective observational study in China to evaluate the efficacy and safety of epirubicin (E) and cyclophosphamide (C) with pyrotinib as anti-HER2 therapy in the neoadjuvant setting of patients with stage II-III HER2-positive breast cancer. Methods: From May 2019 to December 2021, 44 untreated patients with HER2-positive nonspecific invasive breast cancer who received 4 cycles of neoadjuvant EC with pyrotinib. The primary endpoint was pathological complete response (pCR) rate. Secondary endpoints included the overall clinical response, breast pathological complete response rate (bpCR), the rate of axillary lymph nodes pathological negativity and adverse events (AEs). Other objective indicators were the rate of surgical breast-conserving, the negative conversion ratios of tumor markers. Results: Thirty-seven (84.1%) of 44 patients completed this neoadjuvant therapy, and 35 (79.5%) had surgery and were included in the primary endpoint assessment. The objective response rate (ORR) of 37 patients was 97.3%. Two patients reached clinical complete response, 34 obtained clinical partial response, 1 sustained stable disease, and no one had progressive disease. Eleven (31.4%) of 35 patients who had surgery achieved bpCR and the rate of axillary lymph nodes pathological negativity was 61.3%. The tpCR rate was 28.6% (95% CI: 12.8-44.3%). Safety was evaluated in all 44 patients. Thirty-nine (88.6%) had diarrhea, and 2 developed grade 3 diarrhea. Four (9.1%) patients had grade 4 leukopenia. All grade 3-4 AEs could be improved after symptomatic treatment. Conclusion: The regimen of 4 cycles of EC combined with pyrotinib presented some feasibility in the neoadjuvant setting for HER2-positive breast cancer with manageable safety. New regimens with pyrotinib should be evaluated for higher pCR in future. Trial registration: chictr.org Identifier: ChiCTR1900026061.

On-capillary alkylation micro-reactor: a facile strategy for proteo-metabolome profiling in the same single cells.

Single-cell multi-omics analysis can provide comprehensive insights to study cell-to-cell heterogeneity in normal and disease physiology. However, due to the lack of amplification technique, the measurement of proteome and metabolome in the same cell is challenging. Herein, a novel on-capillary alkylation micro-reactor (OCAM) was developed to achieve proteo-metabolome profiling in the same single cells, by which proteins were first covalently bound to an iodoacetic acid functionalized open-tubular capillary micro-reactor via sulfhydryl alkylation reaction, and metabolites were rapidly eluted, followed by on-column digestion of captured proteins. Compared with existing methods for low-input proteome sample preparation, OCAM exhibited improved efficiency, anti-interference ability and recovery, enabling the identification of an average of 1509 protein groups in single HeLa cells. This strategy was applied to single-cell proteo-metabolome analysis of mouse oocytes at different stages, 3457 protein groups and 171 metabolites were identified in single oocytes, which is the deepest coverage of proteome and metabolome from single mouse oocytes to date, achieving complementary characterization of metabolic patterns during oocyte maturation.

Emodin Ameliorates Severe Acute Pancreatitis-Associated Acute Lung Injury in Rats by Modulating Exosome-Specific miRNA Expression Profiles.

Background: Numerous preclinical investigations have exhibited the beneficial impact of emodin (EMO) on the management of severe acute pancreatitis (SAP)-associated acute lung injury (ALI). However, the potential of EMO to mitigate organ damage through the modulation of exosome (Exo)-specific miRNA expression profiles remains unclear. Methods: The SAP rat model was established by retrograde injection of 5% sodium taurocholate into the pancreatic bile duct. Rats received intragastric administration of EMO at 2 h and 12 h post-modeling. Plasma and bronchoalveolar lavage fluid (BALF)-derived exosomes were isolated and purified from SAP rats treated with EMO. The therapeutic effects of these Exos in SAP rats were assessed using hematoxylin-eosin staining and measurement of inflammatory factor levels. MicroRNA (miRNA) sequencing was conducted on plasma and BALF-derived Exos, and rescue experiments were performed to investigate the function of NOVEL miR-29a-3p in the treatment of SAP using EMO. Results: EMO exhibits ameliorative effects on pancreatic and lung injury and inflammation in rats with SAP. Plasma/BALF-derived Exos from EMO-treated SAP rats also have therapeutic effects on SAP rats. The miRNA expression profile of plasma and BALF-derived Exos in SAP rats underwent significant changes upon exposure to EMO. In particular, 34 differentially expressed miRNAs (DEmiRNAs) were identified when comparing BALF-SAP+EMO-Exo and BALF-SAP-Exo. 39 DEmiRNAs were identified when comparing plasma-SAP+EMO-Exo to plasma-SAP-Exo. We found that SAP rats treated with Exos derived from BALF exhibited a more potent therapeutic response than those treated with Exos derived from plasma. EMO may rely on NOVEL-rno-miR-29a-3p expression to prevent pulmonary injury in SAP rats. Conclusion: The mechanism of action of EMO is observed to have a significant impact on the miRNA expression profile of Exos derived from plasma and BALF in SAP rats. NOVEL-rno-miR-29a-3p, which is specific to Exos, and is derived from BALF, may play a crucial role in the therapeutic efficacy of EMO.

A multi-omics dataset of human transcriptome and proteome stable reference.

The development of high-throughput omics technology has greatly promoted the development of biomedicine. However, the poor reproducibility of omics techniques limits their application. It is necessary to use standard reference materials of complex RNAs or proteins to test and calibrate the accuracy and reproducibility of omics workflows. The transcriptome and proteome of most cell lines shift during culturing, which limits their applicability as standard samples. In this study, we demonstrated that the human hepatocellular cell line MHCC97H has a very stable transcriptome (r = 0.983~0.997) and proteome (r = 0.966~0.988 for data-dependent acquisition, r = 0.970~0.994 for data-independent acquisition) after 9 subculturing generations, which allows this steady standard sample to be consistently produced on an industrial scale in long term. Moreover, this stability was maintained across labs and platforms. In sum, our study provides omics standard reference material and reference datasets for transcriptomic and proteomics research. This helps to further standardize the workflow and data quality of omics techniques and thus promotes the application of omics technology in precision medicine.

Integrated platform for proteome profiling with combination of microreversed phase based protein and peptide separation via online solvent exchange and protein digestion.

An online integrated platform for proteome profiling was established, with the combination of protein separation by microreversed phase liquid chromatography (µRPLC), online acetonitrile (ACN) removal, and pH adjustment by a hollow fiber membrane interface (HFMI), online digestion by an immobilized enzymatic microreactor (IMER), as well as peptide separation and proteins identification by µRPLC or nano-RPLC-electrospray ionization tandem mass spectrometry (µRPLC-ESI-MS/MS). To evaluate the performance of such a platform, a three-protein mixture with mass ranging from 5 to 500 ng was analyzed automatically. Compared to the offline counterpart, although similar protein sequence coverages were obtained by the integrated platform, the signal intensity of total ion chromatogram was improved by almost 4 times. In addition, such an integrated platform was further applied for the analysis of extracted proteins from rat brain. Compared to the results obtained by offline counterpart and traditional MudPIT approach under similar conditions, by the integrated platform, the identified protein group number was comparable, but the analysis time was shortened to less than half of that taken by the traditional approaches. All these results demonstrated that our developed integrated platform might offer a promising tool for high-throughput and large-scale profiling of proteomes.

Nano-flow multidimensional liquid chromatography platform integrated with combination of protein and peptide separation for proteome analysis.

An integrated multidimensional nano-flow liquid chromatography platform with the combination of protein and peptide separation via online digestion by an immobilized enzymatic reactor was established, and successfully applied for proteome analysis. By this platform, proteins were first separated by a weak anion and weak cation mixed-bed microcolumn under a series of salt steps, online digested by a trypsin immobilized enzymatic reactor, digests trapped and desalted by a C18 precolumn, separated by nano-reversed phase liquid chromatography, and finally identified by electrospray ionization-MS/MS. To evaluate the performance of such a platform, Escherichia coli whole cell lysate proteins were analyzed. Compared with the results obtained by shotgun approach, the identified protein number was increased by 6%, with the consumed time decreased from 38 to 14 h. We also compared with integrate platform based on micro-HPLC, and the required sample amount was decreased to 8 µg. These results demonstrated that such an integrated approach would be an attractive alternative to commonly applied approaches for proteome research.