Table of Content

10 November 2024, Volume 45 Issue 11

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Preface

蛋白质组学专刊

龙亿涛, 乔亮, 万晶晶

2024, 45(11):  1-3. 

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Content

Cover and Content of Chemical Journal of Chinese Universities Vol.45 No.11(2024)

2024, 45(11):  1-6. 

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Review

Advances and Challenges of Exosome Metabolomics in Body Fluids

CAO Yiqing, HOU Jingxin, LIU Jianye, LI Yan

2024, 45(11):  20240324.  doi:10.7503/cjcu20240324

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Exosomes， ubiquitously present in body fluids， serve as non-invasive biomarkers for disease diagnosis， monitoring， and treatment. As intercellular messengers， exosomes encapsulate a rich array of proteins， nucleic acids， and metabolites， although most studies have primarily focused on proteins and RNA. Recently， exosome metabolomics has demonstrated clinical value and potential advantages in disease detection and pathophysiology， despite significant challenges， particularly in exosome isolation and metabolite detection. This review discusses the significant technical challenges in exosome isolation and metabolite detection， highlighting the advancements in these areas that support the clinical application of exosome metabolomics， and illustrates the potential of exosomal metabolites from various body fluids as biomarkers for early disease diagnosis and treatment.



Advances in Single-cell Multi-omics Analysis Based on Mass Spectrometry

HUO Zhiyuan, ZHOU Jinping, MA Xiumin, ZHOU Yan, HUANG Lin

2024, 45(11):  20240389.  doi:10.7503/cjcu20240389

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With the rapid development of mass spectrometry technology in recent years， single-cell mass spectrometry has played an increasingly vital role in the field of life sciences. Mass spectrometry has been widely applied to detect the metabolome and proteome of cell populations. The multi-omics technology of single-cell mass spectrometry combines microscopic biology with cutting-edge mass spectrometry analysis， providing a powerful tool for in-depth study at the single-cell level and further clarifying the micro-level complexity of cells. Especially in biomedical applications， single-cell mass spectrometry multi-omics helps to build cell atlas， explore the molecular mechanisms of biological phenomena， and promote the development of precision medicine. Therefore， this article systematically summarizes and reviews the challenges and breakthroughs in the development of mass spectrometry-based single-cell metabolism， proteomics， and multi-omics technologies in recent years， and provides an outlook on its future development trends.



Research Progress in Exosome Isolation and Proteomics Analysis

JIN Ying, ZHANG Junjie, ZHANG Yixin, YUAN Yue, HAN Zhenzhen

2024, 45(11):  20240305.  doi:10.7503/cjcu20240305

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Exosomes are extracellular vesicles released via the fusion of multivesicular bodies with the cell plasma membrane， containing proteins， lipids， nucleic acids， etc. They transport cargo in the form of extracellular vesicles and participate in various cancer processes such as invasion and metastasis. As emerging targets for liquid biopsy， exosomes play crucial roles in cell communication， signal transduction， and immune response. Mass spectrometry has become an indispensable part of the field of proteomics research， and the proteomic analysis of exosomes is a promising method for discovering potential cancer biomarkers. Recent advances in high‐resolution separations， high‐performance mass spectrometry and comprehensive proteome databases have all contributed to the successful analysis of exosomes from patient samples. Here， this article reviews the isolation methods of exosomes， proteomics analysis techniques， and the application research of proteomics analysis based on exosomes in clinical disease diagnosis. Finally， the challenges faced by exosome isolation and proteomics， as well as the prospects for their clinical applications， are discussed and outlooked.



New Technologies for Mass Spectrometry-based Single-cell Resolved Spatial Proteomics Research

SHEN Fenglin, FENG Zhaoying, FANG Jing, ZHANG Lei, LIU Xiaohui, ZHOU Xinwen

2024, 45(11):  20240299.  doi:10.7503/cjcu20240299

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Cell-to-cell heterogeneity prevails in biological systems. Single-cell and single-cell resolved spatial proteomics can help human gain better understanding of the mechanism of any disease. Limited to the extremely low protein content in a single cell and sensitivity of detection technology， the development of the single-cell proteomic technology faces major technical challenges. Over the last few decades， the sensitivity， resolution and scanning speed of mass spectrometry have improved dramatically. Recently， new MS-based single cell resolved spatial proteomics methods have been developed for quantifying thousands of proteins in individual human cells. These spatial proteomics analyses at the single-cell level provide high-resolution data for disease， environment and biochemistry research. This review mainly focuses on the mass spectrometry-based approaches for the study of single-cell resolved spatial proteomics.



Single Cell Proteomic Analysis by Mass Spectrometry

FAN Zhirui, FANG Qun, YANG Yi

2024, 45(11):  20240294.  doi:10.7503/cjcu20240294

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Single-cell proteomics allows revealing precisely the differences of proteins between individual cells， which has become a research hotspot showing indispensable application value in many important fields. Its difficulties lie in the fact that the proteins in a single cell are of extremely low abundance， which calls for ingenious solutions to the problems of sample loss during preparation， low sensitivity of chromatography-mass spectrometry detection， and insufficient analysis of spectral data with low signal intensities. This review summarizes the current research progress of mass spectrometry-based single-cell proteomic analysis， including single-cell sorting， sample preparation， chromatography-mass spectrometry acquisition， and data analysis， as well as its applications in biomedical fields. Its potential future development is also discussed.



Advances in MALDI MS Matrices for the Detection of Small Molecules

XU Hongmei, WANG Liangchen, MIN Qianhao

2024, 45(11):  20240285.  doi:10.7503/cjcu20240285

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The analysis of small molecules is of great interest to profile the distribution of small pharmaceutical drugs rapidly， or to trace and understand metabolic pathways. Matrix-assisted laser desorption/ionization mass spectrometry（MALDI MS） plays a central role in the analysis of biological molecules. However， its application for analysis of low molecular weight compounds is restricted by conventional organic matrices interferences in the low m/z region. In addition， inhomogeneity of matrix and sample crystals also impedes the detection repeatability and imaging spatial resolution. Several strategies regarding matrices preparation and functionalization have been investigated to overcome this problem. This review gives an overview on the rational design strategies used to develop matrix systems for the analysis of small molecules， focusing on the exploration of deprotonating matrices， modified conventional matrices， high molecular weight organic matrices， reactive matrices， and nanomaterial-based matrices. In final， the design and application of the new matrices for mass spectrometry analysis of small molecules are summarized and prospected.



Research Progress of Allergen Detection in Aquatic Products

ZUO Chunqian, XU Ruirui, BI Hongyan

2024, 45(11):  20240073.  doi:10.7503/cjcu20240073

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Food allergy is an abnormal reaction of the body’s immune system to certain foods. It is a health problem that has gained widespread attention around the world. Food allergies commonly occur as a result of the immune system’s response to specific foods， often mediated by immunoglobulin E or immune cells. Allergens play a key role in triggering these reactions， underscoring the importance of detecting and identifying allergens to maintain food safety. As an important source of protein， aquatic products are an indispensable part of people’s daily diet， the detection of allergens in aquatic products is highly necessary. This review briefly describes the research progress of allergen detection in aquatic products such as fish and shellfish in recent years， and summarizes common allergens， detection methods and technologies， as well as challenges in current research and future development trends. At present， the detection methods for common food allergens currently face challenges， including limitations associated with low detection efficiency， reduced accuracy and high overall costs. These constraints underscore the need for ongoing research and innovation in this area to address these shortcomings. Combining different technical methods to establish efficient， accurate and low-cost allergen detection methods is still an important aspect in the development of this research field.



Article

Mass Spectrometry-based Deep Coverage Proteome： Evaluation of Cellular Protein Extraction Methods

XU Xia, QIN Weida, LI Ruomeng, WANG Qianqian, LIU Ning, LI Gongyu

2024, 45(11):  20240344.  doi:10.7503/cjcu20240344

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The current study comprehensively evaluates four different protein extraction methods based on urea， sodium dodecyl sulfate（SDS）， anionic surfactants（BT）， and total RNA extractor（Trizol）， aiming to optimize the sample preparation workflow for mass spectrometry-based proteomics. Using HeLa cells as an example， we found that the method employing the mass spectrometry-compatible surfactant BT reagent significantly reduces the total time consumed for protein extraction and minimizes protein losses during the sample preparation process. Further integrating the four protein extraction methods， we identified over 7000 proteins from HeLa cells without relying on pre-fractionation techniques， and 2990 of them were quantified using label-free quantification. It is worth noting that the BT and SDS methods demonstrate higher efficiency in extracting membrane proteins， while the Urea and Trizol methods are more effective in extracting proteins from nuclear and cytoplasmic fractions. In summary， this study provides a novel solution for deep proteome coverage， particularly in the context of cellular protein extraction， by integrating mass spectrometry-compatible surfactants with traditional extraction methods to effectively enhance protein identification numbers.



High-throughput Analysis of Tyrosinase Activity and Inhibitors Based on Matrix-assisted Laser Desorption/ionization Mass Spectrometry

SHI Qian, LIU Dongmei, FANG Xiaoni, LIU Baohong

2024, 45(11):  20240330.  doi:10.7503/cjcu20240330

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The efficient and convenient separation capability and strong ultraviolet absorption of magnetic graphene nanomaterials（Fe₃O₄@G） were applied in the analysis of tyrosinase activity and inhibitors using matrix-assisted laser desorption/ionization mass spectrometry（MALDI-MS）. Compared to other methods， this approach reduces sample loss and improves detection sensitivity by avoiding cumbersome sample pretreatment processes. Additionally， the combination of MALDI-MS enables high-throughput sample analysis. The development of this research is of significant importance for gaining a deeper understanding of melanin synthesis processes， studying related diseases， and developing effective drugs and cosmetic products.



Untargeted Lipidomics Reveals Lipid Metabolism Dysfunction During Macrophage Foaming

WANG Zengyu, LIU Baohong, QIAO Liang, LIN Ling

2024, 45(11):  20240053.  doi:10.7503/cjcu20240053

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Atherosclerosis is a multifactorial chronic complex disease characterized by the accumulation of lipids， inflammatory responses， and ultimately fibrous plaque formation within arterial walls. Plaque formation begins with the abnormal accumulation of lipids engulfed by macrophages within arterial walls， forming so-called foam cells. Despite the pivotal role of foam cell formation in the pathophysiological remodeling process of blood vessels， in-depth research into lipid metabolism disturbances during macrophage foam cell formation is still relatively lacking. In this study， we constructed and optimized a lipidomics analysis workflow， applying it to analyzing metabolic reprogramming during macrophage foam cell formation. A total of 645 lipid molecules belonging to 16 lipid subclasses were identified. Principal component analysis， time-series pattern analysis， and volcano plot analysis revealed significant differences in lipid levels at different stages of foam cell formation. As incubation time with oxidized low-density lipoprotein increased， the degree of lipid metabolism dysfunction in foam cells increased. Triglycerides， hemolytic phospholipids， and ether phospholipids were upregulated， while phosphatidylserine was downregulated. The significant accumulation of triglycerides enhanced the inflammatory response of macrophages， promoting foam cell formation by further engulfing oxidized low-density lipoprotein. Meanwhile， the synthesis of phosphatidylserine and hemolytic phosphatidylcholine increased significantly in the late stages of foam cell formation， indicating a positive correlation between foam cell formation and cell apoptosis. These lipid molecules may serve as signaling molecules to attract macrophages for the clearance of apoptotic cells. This study not only reveals the significant upregulation of inflammatory responses during foam cell formation but also elucidates the close connection between lipid metabolism disturbances and cell apoptosis signaling.



Plasmonic Composites Aid Semi-quantitative Analysis and Identification of Low-molecular-weight Metabolites by Mass Spectrometry

CAO Ting, SHU Weikang, WAN Jingjing

2024, 45(11):  20240325.  doi:10.7503/cjcu20240325

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DMSN@Ag/Au composites， with DMSN（dendritic mesoporous silica nanosphere）， as the support and Au/Ag nanoparticles homogeneously loading on the surface， were employed as MALDI（matrix-assisted laser desorption/ionization） matrices. These composites enable highly sensitive MALDI metabolic analysis with a detection limit of 0.005 mg/mL. Moreover， these composites demonstrated good reproducibility in detection and provided an intrinsic Au⁺ signal as a reference to enhance the accuracy of quantitative analysis. Additionally， DMSN@Ag/Au facilitated Ag⁺ addition to the analyte and assisted in metabolite identification process. The composites are expected to serve as a new generation of multifunctional MALDI matrix and provide new ideas for clinical mass spectrometry analysis.



Protein-Small Molecule Interaction Electrospray Ionization Mass Spectrometry Study of the Ubiquitin/Adenosine Triphoshate Couple over Temperature Variation

LIU Siying, SU Wen, ZHOU Zhongyan, YANG Zhiyu, PEI Huafu, HE Zhiru, WANG Na, YUE Lei

2024, 45(11):  20240382.  doi:10.7503/cjcu20240382

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In this paper， protein-small molecule interaction electrospray ionization mass spectrometry（PSMI-ESI-MS） was used to study a model protein， ubiquitin（Ubi）， and one of the most important bioactive small molecules， adenosine triphosphate（ATP） system. At room temperature， the electrospray mass spectra of the Ubi/ATP couple at the concentration ratio of 1 μmol/L∶50 μmol/L mainly showed charge states at +5， +6， and +7， corresponding well with the charge of Ubi in native state. ATP mainly formed 1∶1 and 1∶2（molar ratio） complexes with +5 and +6 ubiquitin while complexes with +7 was much less abundant， indicating that ubiquitin in lower charge state has a stronger binding affinity for ATP. Analysis of Ubi/ATP mass spectra at different ratios showed that there was no significant difference in the binding state at the concentration dimension. However， there was a significant difference in the charge distribution of Ubi and Ubi-ATP complexes at temperature variation. The calculated binding affinity increased with increasing temperature， indicating that the interaction between Ubi and ATP was enhanced after unfolding. Furthermore， Gibbs free energy of folded and unfolded Ubi indicated that the presence of ATP increased the energy required for unfolding， thereby enhancing the stability of ubiquitin. In this study， multi-dimensional information such as stoichiometric ratio， affinity， and Gibbs free energy were obtained based on electrospray mass spectrometry analysis of ubiquitin and ATP in the temperature dimension. It provides a general strategy for subsequent studies on protein-small molecule interactions.



Tip Fe₂O₃ Nanorods Driven High-performance Mass Spectrometry Analysis for Constructing Metabolic Fingerprint of PM_2.5-exposed Mice

ZHANG Yihan, HUA Tianyu, HOU Shijiao, ZHANG Yangyang, YIN Dan, JI Xiangbo, ZHANG Yanhao, PEI Congcong, ZHANG Shusheng

2024, 45(11):  20240376.  doi:10.7503/cjcu20240376

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The performance of laser desorption/ionization mass spectrometry（LDI MS） is greatly limited by the choice and design of the matrix. Structural design of the matrix can further improve the detection performance. The sharper corner can effectively enhance charge transfer and photothermal conversion efficiency， thereby improving the ionization and desorption ability in LDI MS. Therefore， we constructed an LDI MS platform based on tip Fe₂O₃ nanorods（Nr-Fe₂O₃）. The nanoscale surface roughness， strong light absorption， enhanced ionization ability， and photothermal conversion properties of Nr-Fe₂O₃ have improved the selectivity and sensitivity of LDI-MS for metabolite detection［3—10 fold signal enhancement compared with Fe₂O₃ nanoparticles（Np-Fe₂O₃）， 10—15 fold signal enhanced compared to commercial organic matrices］. In the serum samples， the Nr-Fe₂O₃ was used to collect serum metabolic fingerprints of fine particulate matter（PM_2.5）-exposed mice， and screen the fluctuating features by T-test. Our approach guides us in matrix design for LDI MS metabolic analysis and provides the theoretical support for subsequent PM_2.5 exposure toxicology research.



Cell Map of Mouse Peripheral Blood Mononuclear Cells with a Label-free Single-cell Proteomics Method

HUANG Yuying, YU Chengkun, LIU Siqi, REN Yan

2024, 45(11):  20240355.  doi:10.7503/cjcu20240355

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Fluorescently labeled antibodies were used to isolate T cells， B cells， natural killer cells， and dendritic cells. Single cells were then sorted using the CellenONE single-cell sorting system. During the sorting process， an approach of mass-adaptive coating-assisted single-cell proteomics（Mad-CASP） was pre-added to the wells of the sorting plate to reduce subsequent protein adsorption to the plate and chromatography column. The single-cell proteins were extracted and digested， and the resulting peptides were analyzed using a liquid chromatography-tandem mass spectrometry（LC-MS/MS）. Protein identification was conducted using the Maxquant software’s spectral library function in combination with the "match-between-runs" feature. Venn diagrams and UMAP（Uniform manifold approximation and projection） were employed to analyze the protein expression differences among the four cell types. The specific proteins of each cell type were subjected to molecular signature database mouse immune pathway enrichment， with the top two pathways selected for further analysis. Simultaneously， fuzzy C-means clustering and KEGG（Kyoto encyclopedia of genes and genomes） pathway enrichment were used to analyze the quantitative changes of shared proteins among immune cells. Finally， a single-cell proteomics map specifically for mouse peripheral blood mononuclear cells was generated， providing significant insights into understanding the functional characteristics of immune cells and identifying key protein markers related to diseases.



Bacterial Protein Profiling

JIANG Yan, CHEN Yanlin, SONG Gaoyu, CHEN Yanyan, BAI Jing, ZHU Yingdi, LI Juan

2024, 45(11):  20240345.  doi:10.7503/cjcu20240345

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Profiling the protein composition of bacteria is essential for understanding their biology， physiology and interaction with environment. Mass spectrometry has become a pivotal tool for protein analysis， facilitating the examination of expression levels， molecular masses and structural modifications. In this study， we compared the performance of three widely-used mass spectrometry methods， i.e.， matrix-assisted laser desorption/ionization （MALDI） protein fingerprinting， top-down proteomics and bottom-up proteomics， in the profiling of bacterial protein composition. It was revealed that bottom-up proteomics provided the highest protein coverage and exhibited the greatest protein profile overlap between bacterial species. In contrast， MALDI protein fingerprinting demonstrated superior detection reproducibility and effectiveness in distinguishing between bacterial species. Although top-down proteomics identified fewer proteins than bottom-up approach， it complemented MALDI fingerprinting in the discovery of bacterial protein markers， both favoring abundant， stable， and hydrophilic bacterial ribosomal proteins. This study represents the most systematic and comprehensive comparison of mass spectrometry-based protein profiling methodologies to date. It provides valuable guidelines for the selection of appropriate profiling strategies for specific analytical purposes. This will facilitate studies across various fields， including infection diagnosis， antimicrobial resistance detection and pharmaceutical target discovery.



Detection of Coffee Polyphenols in Nanoliter Cerebrospinal Fluid by Microextraction Coupled to Nanoelectrospray Ionization Mass Spectrometry

YAN Yongjie, GAO Wenbo, LU Chenhui, YANG Cheng, XU Shuting

2024, 45(11):  20240327.  doi:10.7503/cjcu20240327

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An analytical technique was designed and constructed for the in situ extraction and rapid qualitative and quantitative detection of polyphenols in nanoliter cerebrospinal fluid. By preparing polypyrrole microextraction probes with good biocompatibility and stability， the rapid and efficient enrichment of polyphenols in cerebrospinal fluid was achieved， and the matrix interference was reduced. Combined with nanoelectrospray ionization mass spectrometry（nESI-MS）， high-throughput rapid detection of polyphenols in micro-volume mice cerebrospinal fluid was realized. The method for simultaneous detection of four phenolic acids（caffeic acid， 3-hydroxyphenylacetic acid， homovanillic acid and ferulic acid） and two flavonoids（epicatechin and epigallocatechin gallate） was established based on microextraction coupled to nESI-MS. The linear ranges were 0.1/0.5/1—20 µg/mL（R²=0.994—0.999） with recovery rates in the cerebrospinal fluid matrix of 96.9%—108%， and the detection limits were 0.027—0.39 µg/mL（0.014—0.20 ng）， which took the advantages of low sample consumption， wide linear range， low detection limit and accurate quantification. In addition， the new method was utilized to analyze the content and dynamic changes of polyphenols in mice cerebrospinal fluid after polyphenol ingestion， which showed high potential application value in understanding the distribution and metabolism of polyphenols in cerebrospinal fluid and exploring their mechanism in neuroprotection.



Dynamic Proteome Profiling of Neonatal Hair Shaft Using DEEP SEQ Method

HU Yuhong, YU Xiangming, SONG Lili, XING Qinghe, ZHOU Feng

2024, 45(11):  20240326.  doi:10.7503/cjcu20240326

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This article utilizes the deep efficient peptide sequencing and quantification proteomics method in conjunction with isobaric tags for relative and absolute quantitation to perform differential proteomic analysis of proteins in naturally shed hair samples collected at 24， 48， 72 and 96 h after birth in newborns. Among the four hair samples collected within 96 h after birth， a total of 1735 proteins were identified， with a large number of proteins showing dynamic changes in expression， involving embryonic development， energy metabolism， and neural pathways. These findings indicate that hair samples， as a novel clinical specimen， can reflect the early and drastic physiological and molecular changes in newborns， providing new insights for non-invasive diagnosis and early disease screening in newborns.



Quantitative Accuracy Evaluation of Mass Spectrometry Based Proteomics Methods Commonly Used in Biomarker Research

ZHANG Lei, SHEN Huali

2024, 45(11):  20240311.  doi:10.7503/cjcu20240311

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In the progression of diseases， changes in protein expression levels， modification states， or interaction patterns may reflect pathological conditions or disease advancement， making proteins commonly used as disease biomarkers. Mass spectrometry-based proteomics has identified numerous potential disease biomarkers. However， these biomarkers and their mass spectrometry detection methods need to have high quantitative accuracy and stability for further clinical application. This study synthesized 32 standard peptides and evaluated three quantitative methods data independent acquisition（DIA）， multiple reaction monitoring（MRM）， and parallel reaction monitoring（PRM）， for their potential in large-scale clinical applications in mass spectrometry. We simulated actual samples at four different concentrations to assess peptide discovery rates， standard curve linearity， inter-day and intra-day precision， and retention time shift using the aforementioned three mass spectrometry methods. The results indicate that MRM quantification is most suitable for clinical applications due to its good stability， sensitivity， and quantitative accuracy. PRM is suitable for targeted quantification in research settings， which can offer high sensitivity and stability. Finally， despite its high throughput nature， DIA demonstrates inferior quantitative accuracy for specific peptides compared to PRM and MRM.



Analysis of N-Terminal Fragment of β⁃ Amyloid Peptides Using an Aerolysin Nanopore

CHEN Tianze, HU Fangzhou, LIN Xubo, YING Yilun, ZOU Aihua

2024, 45(11):  20240192.  doi:10.7503/cjcu20240192

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Alzheimer’s disease（AD） is one of the most common diseases caused by multiple neurodegenerative protein misfolding and aggregation disorders. Abnormal deposition of amyloid protein caused by β-amyloid（Aβ） peptides has been suggested as a possible predisposing factor for Alzheimer’s disease. Unlike human Aβ peptide， rodent Aβ peptide rarely has these characteristic lesions. The difference between rodent Aβ peptide and human Aβ peptide is that the 5th， 10th and 13th amino acids（Arg， Tyr， His） are replaced by Gly， Phe and Arg， respectively. In this study， molecular dynamics simulation and nanopore-based single molecule detection technology were used to study the structural differences between human Aβ_1—15 and rodent Aβ_1—15. The experimental results show that rodent Aβ_1—15 has lower blocking frequency and energy barrier when passing through nanopore than human Aβ_1—15， which proves that aerolysin nanopore can distinguish Aβ_1—15 with small structural differences. Furthermore， the interaction between Aβ_1—15 and sulfate ion was studied by using sulfate K₂SO₄ as a simplified model of glycosaminoglycan （glycosaminoglycans， GAGs）. Statistical analysis showed that both peptides could bind to sulfate ions and reduce their capture frequency by aerolysin nanopore， reducing the capture frequency of human Aβ_1—15 by 25% and rodent Aβ_1—15 by 59%. However， after the addition of sulfate ion， there was a significant difference in the dwell time of the two peptides. Compared with the results in the absence of sulfate， the dwell time of human Aβ_1—15 increased by 14% and that of rodent Aβ_1—15 decreased by 7%. It is inferred from the experimental results that the different sequences and conformations of the two peptides lead to different binding ways and binding intensity to sulfate ions， which have different effects on the translocation behavior. This study is helpful to better screen small molecular inhibitors and further promote the diagnosis and treatment of Alzheimer’s disease.



A New Method for Large-scale Enrichment and Stepwise Identification of RNA-protein Complexes

DONG Peiying, LIU Tong, QIN Weijie

2024, 45(11):  20240091.  doi:10.7503/cjcu20240091

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RNA-protein complexes（RPC） regulate various physiological processes in organisms and play a crucial role in the synthesis and degradation of RNA. For deep RPC profiling in addition to develop RNA-binding proteins（RBP） enrichment methods， some researchers also turned their attention to RNA-associated proteins（RAP）， which interact with RBP via protein-protein interaction. However， previous methods often enrich all proteins in the RPC as a whole and cannot differentiate RBP from RAP. Therefore， we reported a new method using combination of 254 nm UV crosslinking of RBP and RNA and DSP crosslinking of RBP and RAP. Next， RBP and RAP can be eluted in a stepwise way and separately identified by mass spectrometry. A total of 2007 high confidence RBP and 927 high confidence RAP were identified in HeLa cells， of which 243 RAP were identified for the first time. This method aims to refine the RNA-protein interaction network and provide a favorable foundation for subsequent biological investigations.