[Applications of high performance liquid chromatography-mass spectrometry in proteomics].

Proteomics profiling plays an important role in biomedical studies. Proteomics studies are much more complicated than genome research, mainly because of the complexity and diversity of proteomic samples. High performance liquid chromatography-mass spectrometry (HPLC-MS) is a fundamental tool in proteomics research owing to its high speed, resolution, and sensitivity. Proteomics research targets from the peptides and individual proteins to larger protein complexes, the molecular weight of which gradually increases, leading to sustained increases in structural and compositional complexity and alterations in molecular properties. Therefore, the selection of various separation strategies and stationary-phase parameters is crucial when dealing with the different targets in proteomics research for in-depth proteomics analysis. This article provides an overview of commonly used chromatographic-separation strategies in the laboratory, including reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), hydrophobic interaction chromatography (HIC), ion-exchange chromatography (IEC), and size-exclusion chromatography (SEC), as well as their applications and selectivity in the context of various biomacromolecules. At present, no single chromatographic or electrophoretic technology features the peak capacity required to resolve such complex mixtures into individual components. Multidimensional liquid chromatography (MDLC), which combines different orthogonal separation modes with MS, plays an important role in proteomics research. In the MDLC strategy, IEC, together with RPLC, remains the most widely used separation mode in proteomics analysis; other chromatographic methods are also frequently used for peptide/protein fractionation. MDLC technologies and their applications in a variety of proteomics analyses have undergone great development. Two strategies in MDLC separation systems are mainly used in proteomics profiling: the "bottom-up" approach and the "top-down" approach. The "shotgun" method is a typical "bottom-up" strategy that is based on the RPLC or MDLC separation of whole-protein-sample digests coupled with MS; it is an excellent technique for identifying a large number of proteins. "Top-down" analysis is based on the separation of intact proteins and provides their detailed molecular information; thus, this technique may be advantageous for analyzing the post-translational modifications (PTMs) of proteins. In this paper, the "bottom-up" "top-down" and protein-protein interaction (PPI) analyses of proteome samples are briefly reviewed. The diverse combinations of different chromatographic modes used to set up MDLC systems are described, and compatibility issues between mobile phases and analytes, between mobile phases and MS, and between mobile phases in different separation modes in multidimensional chromatography are analyzed. Novel developments in MDLC techniques, such as high-abundance protein depletion and chromatography arrays, are further discussed. In this review, the solutions proposed by researchers when encountering compatibility issues are emphasized. Moreover, the applications of HPLC-MS combined with various sample pretreatment methods in the study of exosomal and single-cell proteomics are examined. During exosome isolation, the combined use of ultracentrifugation and SEC can yield exosomes of higher purity. The use of SEC with ultra-large-pore-size packing materials (200 nm) enables the isolation of exosomal subgroups, and proteomics studies have revealed significant differences in protein composition and function between these subgroups. In the field of single-cell proteomics, researchers have addressed challenges related to reducing sample processing volumes, preventing sample loss, and avoiding contamination during sample preparation. Innovative methods and improvements, such as the utilization of capillaries for sample processing and microchips as platforms to minimize the contact area of the droplets, have been proposed. The integration of these techniques with HPLC-MS shows some progress. In summary, this article focuses on the recent advances in HPLC-MS technology for proteomics analysis and provides a comprehensive reference for future research in the field of proteomics.

Picoliter Single-Cell Reactor for Proteome Profiling by In Situ Cell Lysis, Protein Immobilization, Digestion, and Droplet Transfer.

Global profiling of single-cell proteomes can reveal cellular heterogeneity, thus benefiting precision medicine. However, current mass spectrometry (MS)-based single-cell proteomic sample processing still faces technical challenges associated with processing efficiency and protein recovery. Herein, we present an innovative sample processing platform based on a picoliter single-cell reactor (picoSCR) for single-cell proteome profiling, which involves in situ protein immobilization and sample transfer. PicoSCR helped minimize surface adsorptive losses by downscaling the processing volume to 400 pL with a contact area of less than 0.4 mm2. Besides, picoSCR reached highly efficient cell lysis and digestion within 30 min, benefiting from optimal reagent and high reactant concentrations. Using the picoSCR-nanoLC-MS system, over 1400 proteins were identified from an individual HeLa cell using data-dependent acquisition mode. Proteins with copy number below 1000 were identified, demonstrating this system with a detection limit of 1.7 zmol. Furthermore, we profiled the proteome of circulating tumor cells (CTCs). Data are available via ProteomeXchange with the identifier PXD051468. Proteins associated with epithelial-mesenchymal transition and neutrophil extracellular traps formation (which are both related to tumor metastasis) were observed in all CTCs. The cellular heterogeneity was revealed by differences in signaling pathways within individual cells. These results highlighted the potential of the picoSCR platform to help discover new biomarkers and explore differences in biological processes between cells.

Water-stable hydrophilic metal organic framework composite for the recognition of N-glycopeptides during diabetes progression by mass spectrometry.

A hydrophilic Al-MOFs composite was prepared using cheap and available reagents in water via a suitable large-scale production, an economical and environment-friendly method for capturing N-glycopeptides. The prepared Al-MOFs composite with high hydrolytically stable and hydrophilic 1D channels exhibits an ultralow detection limit (0.5 fmol/µL), and excellent reusability (at least 10 cycles) in the capture of N-glycopeptides from standard bio-samples. Interestingly, the Al-MOFs composite also shows remarkable performance in practical applications, where 300 N-glycopeptides ascribed to 124 glycoproteins were identified in 1 µL human serum and were successfully applied in profiling the differences of N-glycopeptides during diabetes progression. Moreover, 12 specific glycoproteins used as biomarkers to accurately distinguish the progression of diabetes are identified. The present work provides a potential commercial method for large-scale glycoproteomics research in complex clinical samples while offering new guidance for the precise diagnosis of diabetes progression.

Facile preparation of a novel chitosan-derived porous graphitized carbon biomaterial for highly efficient capture of N-glycans.

The comprehensive characterization of N-glycans is of significant importance for the discovery of potential biomarkers and the diagnosis and therapy of diseases. Herein, we designed and fabricated a porous graphitized carbon biomaterial (CS-900-1C) using cheap and available chitosan as the carbon source via a facile pyrolysis process and a post-oxidation strategy for the effective capture of N-glycans. Thanks to its large surface area (2846 m2 g-1), high graphitization degree, suitable oxidation degree and unique porous structure, the CS-900-1C biomaterial exhibits an ultralow detection limit (1 ng µL-1), an excellent size-exclusion effect (OVA digest : BSA protein : OVA protein, 1 : 1000 : 1000, w/w/w) and satisfactory reusability (at least 8 cycles) in the capture of standard N-glycans. Moreover, CS-900-1C has successfully been applied in profiling the difference of N-glycans during diabetes progression (obesity, impaired glucose tolerance, diabetes patients and healthy control) where we discovered that the expression levels of five N-glycans show a gradually increasing trend as diabetes progresses. Remarkably, the five specific N-glycans could be considered as biomarkers to accurately diagnose the progression of diabetes. Our work not only developed a novel porous graphitized carbon biomaterial for the large-scale characterization of N-glycans but also provided new guidance for the precise therapy of diabetes.

Emerging role of ferroptosis in glioblastoma: Therapeutic opportunities and challenges.

Glioblastoma (GBM) is the most common malignant craniocerebral tumor. The treatment of this cancer is difficult due to its high heterogeneity and immunosuppressive microenvironment. Ferroptosis is a newly found non-apoptotic regulatory cell death process that plays a vital role in a variety of brain diseases, including cerebral hemorrhage, neurodegenerative diseases, and primary or metastatic brain tumors. Recent studies have shown that targeting ferroptosis can be an effective strategy to overcome resistance to tumor therapy and immune escape mechanisms. This suggests that combining ferroptosis-based therapies with other treatments may be an effective strategy to improve the treatment of GBM. Here, we critically reviewed existing studies on the effect of ferroptosis on GBM therapies such as chemotherapy, radiotherapy, immunotherapy, and targeted therapy. In particular, this review discussed the potential of ferroptosis inducers to reverse drug resistance and enhance the sensitivity of conventional cancer therapy in combination with ferroptosis. Finally, we highlighted the therapeutic opportunities and challenges facing the clinical application of ferroptosis-based therapies in GBM. The data generated here provide new insights and directions for future research on the significance of ferroptosis-based therapies in GBM.

Counting Protein Number in a Single Cell by a Picoliter Liquid Operating Technology.

Ultra-low-copy number proteins play a crucial role in exploring cellular heterogeneity and the insight of protein biomarkers in a single cell. However, counting ultra-low-copy number target proteins in a single cell remains a grand challenge. Herein, we developed a so-called single-cell picoliter liquid operating technology for counting target proteins in a single cell. An ingenious volume-controllable sampling technique was employed to capture a single cell for subsequent analysis. Remarkably, 50 pL of sample volume was employed for sample preparation, single-cell capture, in-droplet lysis, and target protein immobilization on a functionalized coverslip in a monolayer. Then, target protein antibodies coupled with quantum dots were added and incubated to label those immobilized proteins. After clean-up, a single-view image under 100× objective was taken, and the 80 × 80 µm2 view image was then applied to count the precise copy number of the target proteins in the single cell. Furthermore, good linearity and repeatability were achieved for ultra-low-copy number proteins, ranging from 1 to 1500. Finally, the expression level of human epidermal growth factor receptor 2 in single cells from both MCF-7 and MDA-MB-231 cell lines was also analyzed. In a word, this work stimulated the development of capillary-based single-cell analysis and updated the connotation of counting ultra-low-copy number proteins.