Integrated Platform for Large-Scale Quantitative Profiling of Phosphotyrosine Signaling Complexes Based on Cofractionation/Mass Spectrometry and Complex-Centric Algorithm.

The scarcity and dynamic nature of phosphotyrosine (pTyr)-modified proteins pose a challenge for researching protein complexes with pTyr modification, which are assembled through multiple protein-protein interactions. We developed an integrated complex-centric platform for large-scale quantitative profiling of pTyr signaling complexes based on cofractionation/mass spectrometry (CoFrac-MS) and a complex-centric algorithm. We initially constructed a trifunctional probe based on pTyr superbinder (SH2-S) for specifically binding and isolation of intact pTyr protein complexes. Then, the CoFrac-MS strategy was employed for the identification of pTyr protein complexes by integrating ion exchange chromatography in conjunction with data independent acquisition mass spectrometry. Furthermore, we developed a novel complex-centric algorithm for quantifying protein complexes based on the protein complex elution curve. Utilizing this algorithm, we effectively quantified 216 putative protein complexes. We further screened 21 regulated pTyr protein complexes related to the epidermal growth factor signal. Our study engenders a comprehensive framework for the intricate examination of pTyr protein complexes and presents, for the foremost occasion, a quantitative landscape delineating the composition of pTyr protein complexes in HeLa cells.

[Impurity profile analysis of amphotericin B using on-line two-dimensional high performance liquid chromatography-quadrupole time-of-flight mass spectrometry].

Amphotericin B (AmB) is a polyene-macrolide antimicrobial drug with a broad antibacterial spectrum and remarkable efficacy against deep fungal infections. It binds to ergosterol on the fungal cell membrane and alters its permeability, thereby destroying the membrane. AmB is a multicomponent antimicrobial medication that contains a wide range of impurities, rendering quality analysis extremely difficult. In the current Chinese Pharmacopoeia (Edition 2020) and European Pharmacopoeia (EP10.3), high performance liquid chromatography (HPLC) is applied to examine related substances in AmB. However, this technique presents a number of issues. For instance, the mobile phases used in the HPLC method described in both references contain nonvolatile inorganic salts, which cannot be coupled with a mass spectrometry (MS) detector. In addition, because the mobile phases used have a low pH, the component/impurities of AmB drug can easily be degraded or interconverted during the analytical process, leading to reduced analytical accuracy. Therefore, the accuracy and sensitivity of this method must be improved. In this study, a method based on on-line two-dimensional high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (2D HPLC-Q TOF/MS) was developed to analyze the impurity profile of AmB in accordance with the Chinese Pharmacopoeia (Edition 2020) and European Pharmacopoeia (EP10.3). The method combines on-line dilution and a multiple-capture HPLC system to achieve the efficient separation of AmB component/impurities. It also resolves the issue of poor solvent compatibility in 2D HPLC, increases the analytical flux, enhances the automation capability, reduces the mutual conversion of AmB and its impurities during the analytical process, and increases the detection sensitivity of the method. MS was also used to determine the structural inference of unstable components and impurities. An XBridge Shield C18 column (250 mm×4.6 mm, 3 µm) was used for first-dimensional-liquid chromatography with gradient elution using methanol-acetonitrile-4.2 g/L citric acid monohydrate solution (10∶30∶60, v/v/v, pH 4.7) as mobile phase A and methanol-acetonitrile-4.2 g/L citric acid monohydrate solution (12∶68∶20, v/v/v, pH 3.9) as mobile phase B. An Xtimate C8 column (10 mm×2.1 mm, 5 µm) was used as the trap column, and trapping and desalting were performed using 10 mmol/L ammonium formate aqueous solution containing 0.1% formic acid-acetonitrile (95∶5, v/v). An Xtimate C8 column (250 mm×2.1 mm, 5 µm) was used for second-dimensional-liquid chromatography with gradient elution using 10 mmol/L ammonium formate aqueous solution containing 0.1% formic acid-acetonitrile (95∶5, v/v) and 10 mmol/L ammonium formate aqueous solution containing 0.1% formic acid-acetonitrile (5∶95, v/v) as mobile phases. The data were collected in positive-ion mode. In this study, the structures of six impurities in amphotericin B were inferred, according to the fragmentation, the MS and MS2 spectra of each impurity. The developed method can be used to quickly and sensitively analyze the impurity profile of AmB. Furthermore, the research results on impurity profiles can be applied to guide improvements in AmB production.

Separation and structural characterization of unknown impurity in vancomycin by two-dimensional preparative liquid chromatography, LC-MS and NMR.

Vancomycin is an effective antibiotic used for the treatment of Gram-positive bacterial infections. During the analysis of vancomycin, an unknown impurity at the level of 0.5% was detected by high-performance liquid chromatography (HPLC). To characterize the structure of the impurity, a new two-dimensional preparative liquid chromatography (2D-Prep-LC) method was developed to separate the impurity from the vancomycin sample. After further analysis including liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy, the structure of the unknown impurity was identified as a vancomycin analog in which the N-Methyl-leucine residue on the side chain is replaced by an N-methylmethionine residue. In this study, we established a reliable and efficient method for separating and identifying vancomycin impurities, which will provide a valuable contribution to the field of pharmaceutical analysis and quality control.

Facile Preparation of Three-Dimensional Wafer with Interconnected Porous Structure for High-Performance Capture and Nondestructive Release of Circulating Tumor Cells.

Efficient isolation and downstream bioinformation analysis of circulating tumor cells (CTCs) in whole blood contribute to the early diagnosis of cancer and investigation of cancer metastasis. However, the separation and release of CTCs remain a great challenge due to the extreme rarity of CTCs and severe interference from other cells in complex clinical samples. Herein, we developed a low-cost and easy-to-fabricate aptamer-functionalized wafer with a three-dimensional (3D) interconnected porous structure by grafting polydopamine (PDA), poly(ethylene glycol) (PEG), and aptamer in sequence (Ni@PDA-PEG-Apt) for the capture and release of CTCs. The Ni@PDA-PEG-Apt wafer integrated the features of Ni foam with a 3D interconnected porous structure offering enough tunnels for cells to flow through and enhancing aptamer-cell contact frequency, the spacer PEG with flexible and high hydrophilic property increasing anti-interference ability and providing the wafer with more binding sites for aptamer, which result in an enhanced capture specificity and efficiency for CTCs. Because of these advantages, the Ni@PDA-PEG-Apt wafer achieved a high capture efficiency of 78.25%. The captured cancer cells were mildly released by endonuclease with up to 61.85% efficiency and good proliferation. Furthermore, tumor cells were injected into mice and experienced circulation in vivo. In blood samples after circulation, 65% of target tumor cells can be efficiently captured by the wafer, followed by released and recultured cells with high viability. Further downstream metabolomics analysis showed that target cancer cells remained with high biological activity and can be well separated from MCF-10A cells based on metabolic profiles by the PCA analysis, indicating the great potential of our strategy for further research on the progression of cancer metastasis. Notably, not only is the wafer cheap with a cost of only 3.58 U.S. dollars and easily prepared by environmental-friendly reagents but also the process of capturing and releasing tumor cells can be completed within an hour, which is beneficial for large-scale clinical use in the future.

Integrated Pipeline of Rapid Isolation and Analysis of Human Plasma Exosomes for Cancer Discrimination Based on Deep Learning of MALDI-TOF MS Fingerprints.

Plasma exosomes have shown great potential for liquid biopsy in clinical cancer diagnosis. Herein, we present an integrated strategy for isolating and analyzing exosomes from human plasma rapidly and then discriminating different cancers excellently based on deep learning fingerprints of plasma exosomes. Sequential size-exclusion chromatography (SSEC) was developed efficiently for separating exosomes from human plasma. SSEC isolated plasma exosomes, taking as less as 2 h for a single sample with high purity such that the discard rates of high-density lipoproteins and low/very low-density lipoproteins were 93 and 85%, respectively. Benefitting from the rapid and high-purity isolation, the contents encapsulated in exosomes, covered by plasma proteins, were well profiled by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS). We further analyzed 220 clinical samples, including 79 breast cancer patients, 57 pancreatic cancer patients, and 84 healthy controls. After MS data pre-processing and feature selection, the extracted MS feature peaks were utilized as inputs for constructing a multi-classifier artificial neural network (denoted as Exo-ANN) model. The optimized model avoided overfitting and performed well in both training cohorts and test cohorts. For the samples in the independent test cohort, it realized a diagnosed accuracy of 80.0% with an area under the curve of 0.91 for the whole group. These results suggest that our integrated pipeline may become a generic tool for liquid biopsy based on the analysis of plasma exosomes in clinics.

Microliter-level multi-channel fraction collector for high-throughput separation system.

High-performance liquid chromatography (HPLC) plays an important role in analytical applications. To perform high-throughput analysis, especially multi-channel separation, numerous fractions need to be collected. However, multi-channel fraction collector has not been commercialized. Therefore, here we present a multi-channel fraction collector fabricated by 3D-printing technology that can adapt to various kinds of HPLC applications. The collector can perform high accuracy microliter-level fraction cutting for narrow-bore or capillary columns as well as conventional columns. Hundreds of fractions can be collected in a single LC run within 1 hour to meet the demands of high-throughput separation. The collector mainly consists of several environmental-friendly 3D-printed parts and other parts are also easy to purchase, making it possible for researchers to construct it in any kind of lab at a very low price. The automated integrated controller and programs are also introduced to fit different collecting and further analysis requirements. In this work, the structure, functions and automation process of the collector are described in detail, which offers a powerful tool for further development on high-throughput separation.

Strategy for high-throughput identification of protein complexes by array-based multi-dimensional liquid chromatography-mass spectrometry.

Comprehensive elucidation of the composition of multiprotein complexes in model organisms is essential to understand conserved biological systems, but large-scale mapping physical association networks is still challenging due to limited throughput of present methods. In this work, a strategy coupling array-based online two-dimensional liquid chromatography (array-based 2D-LC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was demonstrated for high throughput and in-depth identification of protein complexes from cultured human HeLa cell extracts. Mixed-bed ion-exchange column was employed as the first dimensional (1stD) separating mode and an array consisting of eight reversed phase columns was developed as the second dimensional (2ndD) mode. Taking advantage of array parallel strategy, this online system showed an 8-fold increase in throughput. After array-based online 2D-LC separation, altogether 256 × 2ndD fractions were collected for further LC-MS/MS analysis. Public databases of protein-protein interaction (PPI) and co-elution curves identified by LC-MS were applied to reconstruct the protein complexes. A rigorous inspection was operated by cataloging the protein complexes into chromatographic fractions to minimize the number of false positives. As result, a total number of 4,436 proteins were identified and 26,092 elution curves were graphed. A network consisting of 47,745 PPIs was established among 2,201 proteins and presented 1,530 putative protein complexes with high confidence. Most of the identified PPIs were linked to diverse biological processes and may reveal further disease mechanism and therapeutic strategy.

A new strategy of studying protein-protein interactions: Integrated strong anion exchange/reversed-phase chromatography/immunoprecipitation coupled with mass spectrometry for large-scale identification of proteins interact with immunoglobulin G in HeLa cells.

Recently, significant research efforts have been devoted to the development of technology for large-scale analysis of protein-protein interactions. Herein, a comprehensive method by coupling the first-dimension strong anion exchange chromatography with the second-dimension reversed-phase liquid chromatography via immunoprecipitation technique and high-resolution mass spectrometry analysis was developed for analyzing protein-protein interactions. After two-dimensional liquid chromatography separation, 108 fractions were obtained in one experiment. Immunoglobulin G from human serum was used as a model of an interacting protein. As a result, 919 proteins in these fractions were identified to interact with immunoglobulin G. By searching STRING database and data analysis, 27 of 919 proteins were inferred to directly interact with immunoglobulin G. Moreover, important target proteins that interacted with immunoglobulin G were mapped in the two-dimensional liquid chromatography system, which facilitated selection of these proteins from specific fractions. These results demonstrated that the proposed method can be useful for large-scale investigation of protein-protein interactions and as an advanced tool for the isolation of target proteins.

Deconstruction of Heterogeneity of Size-Dependent Exosome Subpopulations from Human Urine by Profiling N-Glycoproteomics and Phosphoproteomics Simultaneously.

The heterogeneous populations of exosomes with distinct nanosize have impeded our understanding of their corresponding function as intercellular communication agents. Profiling signaling proteins packaged in each size-dependent subtype can disclose this heterogeneity of exosomes. Herein, new strategy was developed for deconstructing heterogeneity of distinct-size urine exosome subpopulations by profiling N-glycoproteomics and phosphoproteomics simultaneously. Two-dimension size exclusion liquid chromatography (SEC) was utilized to isolate large exosomes (L-Exo), medium exosomes (M-Exo), and small exosomes (S-Exo) from human urine samples. Then, hydrophilic carbonyl-functionalized magnetic zirconium-organic framework (CFMZOF) was developed as probe for capturing the two kinds of post-translational modification (PTM) peptides simultaneously. Finally, liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with database search was used to characterize PTM protein contents. We identified 144 glycoproteins and 44 phosphoproteins from L-Exo, 156 glycoproteins, and 46 phosphoproteins from M-Exo and 134 glycoproteins and 10 phosphoproteins from S-Exo. The ratio of the proteins with simultaneous glycosylation and phosphorylation is 11%, 9%, and 3% in L-Exo, M-Exo, and S-Exo, respectively. Based on label-free quantification intensity results, both principal component analysis and Pearson's correlation coefficients indicate that distinct-size exosome subpopulations exist significant differences in PTM protein contents. Analysis of high abundance PTM proteins in each exosome subset reveals that the preferentially packaged PTM proteins in L-Exo, M-Exo, and S-Exo are associated with immune response, biological metabolism, and molecule transport processes, respectively. Our PTM proteomics study based on size-dependent exosome subtypes opens a new avenue for deconstructing the heterogeneity of exosomes.

Investigating the proteomic expression profile of tobacco (Nicotiana tabacum) leaves during four growth stages using the iTRAQ method.

Despite the importance of tobacco (Nicotiana tabacum) in agriculture and model organism investigations, the proteomic changes that occur in the tobacco leaf as it matures remain to be explored. In this study, an isobaric tags for relative and absolute quantification (iTRAQ) strategy was applied to investigate the proteomic profiles of K326 and Honghua Dajinyuan (HD) tobacco leaves at four growth stages. The proteomic profile varied with growth stage in both K326 and HD. Gene ontology (GO) classification was used to identify the biological processes that showed the greatest changes in protein expression between growth stages of HD and K326. Moreover, the number of differentially expressed proteins was greater in HD than in K326, especially during the rosette growth stage and the fast-growing stage. The galactose metabolism and glycosphingolipid biosynthesis-globo series pathways appeared only during the rosette growth stage of HD. It therefore appears that these pathways may be correlated with tobacco mosaic disease. The identification of these pathways should prove useful in investigations of the pathogenesis of tobacco mosaic virus. Graphical abstract ᅟ.

Integrated Proteome Analysis Device for Fast Single-Cell Protein Profiling.

In our previous work, we have demonstrated an integrated proteome analysis device (iPAD-100) to analyze proteomes from 100 cells. (1) In this work, for the first time, a novel integrated device for single-cell analysis (iPAD-1) was developed to profile proteins in a single cell within 1 h. In the iPAD-1, a selected single cell was directly sucked into a 22 µm i.d. capillary. Then the cell lysis and protein digestion were simultaneously accomplished in the capillary in a 2 nL volume, which could prevent protein loss and excessive dilution. Digestion was accelerated by using elevated temperature with ultrasonication. The whole time of cell treatment was 30 min. After that, single-cell digest peptides were transferred into an LC column directly through a true zero dead volume union, to minimize protein transfer loss. A homemade 22 µm i.d. nano-LC packing column with 3 µm i.d. ESI tip was used in the device to achieve ultrasensitive detection. A 30 min elution program was applied to analysis of the single-cell proteome. Therefore, the total time needed for a single-cell analysis was only 1 h. In an analysis of 10 single HeLa cells, a maximum of 328 proteins were identified in one cell by using an Orbitrap Fusion Tribrid MS instrument, and the detection limit was estimated at around 1.7-170 zmol. Such a sensitivity of the iPAD-1 was ∼120-fold higher than that of our previously developed iPAD-100 system. (1) Prominent cellular heterogeneity in protein expressive profiling was observed. Furthermore, we roughly estimated the phases of the cell cycle of tested HeLa cells by the amount of core histone proteins.