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

Wang, Xuantang; Yan, Guoquan; Zheng, Haoyang; Gao, Mingxia; Zhang, Xiangmin

doi:10.1016/j.chroma.2021.462351

Cited by 13 publications

(4 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As a result, it involves some manual steps in operation and is limited in terms of analytical throughput and automation, thereby restricting its use for high-throughput cell analysis at present. We plan to implement strategies such as parallel column analysis and optimization of liquid chromatography–mass spectrometry methods to improve throughput in the future. − Although our method is not suitable for analyzing large numbers of cells, it demonstrates promising prospects for rare cell analysis. This capillary-based approach enables cell selection under a microscope, reducing sample requirements compared with instrument-based cell sorting methods.…”

Section: Discussionmentioning

confidence: 99%

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

Weng,

Yan,

Liu

et al. 2024

J. Proteome Res.

Self Cite

View full text Add to dashboard Cite

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 mm 2 . 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.

show abstract

Section: Discussionmentioning

confidence: 99%

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

Weng,

Yan,

Liu

et al. 2024

J. Proteome Res.

Self Cite

View full text Add to dashboard Cite

show abstract

“…The cofractionation/mass spectrometry (CoFrac-MS) approach can achieve large-scale protein complex identification, which combines native cofractionation protein complexes with MS for inferring protein complexes and PPI based on correlative analysis of chromatographic elution profiles. − The recently developed SEC-SWATH-MS method further extends CoFrac-MS and offers a feasible solution for quantifying protein complexes . However, they also showed that the SEC-SWATH-MS approach faces the limitation that its data analysis requires the corresponding SEC’s fraction and molecular weight information to verify the protein complex and highly depends on a database with known protein complex information as a library.…”

Section: Introductionmentioning

confidence: 99%

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

Liu,

Wang,

et al. 2024

Anal. Chem.

Self Cite

View full text Add to dashboard Cite

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 complexcentric 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.

show abstract

“…Phenolic acids in Salvia miltiorrhiza [11], ginsenosides in Panax ginseng [12] and Panax notoginseng [13], sphingolipids in Caenorhabditis elegans [14], and proteins in tissues [15] have been comprehensively characterized using 2D-HILIC × RPLC, which demonstrate its enhanced separation efficiency, reduced ion suppression for the subsequent MS detection, and powerful characterization ability. Other orthogonal separation systems, including 2D-NPLC × RPLC [16], 2D-SEC × RPLC [17][18][19], 2D-chiral × chiral [20], and 2D-achiral × chiral [21], temperatureresponsive LC × RPLC [22], countercurrent chromatography × RPLC [23], IEC × assay-based RPLC [24], SFC × RPLC [25], IPC × RPLC [26], and IEC × SEC [27] have also been developed and applied toward the analysis of biosamples. Moreover, advanced strategies [28][29][30] were proposed for the development of a more systematic, less tedious, and less reliant on user expertise 2D-LC method.…”

Section: Introductionmentioning

confidence: 99%

Comprehensive profiling of Sanguisorba officinalis using offline two‐dimensional mixed‐mode liquid chromatography × reversed‐phase liquid chromatography, tandem high‐resolution mass spectrometry, and molecular network

Dai

Zhang

Xiong

et al. 2022

J of Separation Science

View full text Add to dashboard Cite

The profiling of natural products is important in modern biological sciences and new drug development. However, the separation and characterization of complex herbal extracts are significantly challenging for researchers in the biochemical field. Herein, an offline two-dimensional mixed-mode liquid chromatography × reversed-phase liquid chromatography system is developed. Our system exhibits high orthogonality and is composed of a newly prepared stationary phase in the first dimension and a traditional C 18 phase in the second dimension, and is operated in combination with a high-resolution mass spectrometry and molecular network. Sanguisorba officinalis L. is studied using the proposed method owing to its bioactivity. With the aid of orthogonal separation, the ionization of the individual components is improved. The number of detected compounds and separated peaks are significantly increased when one-dimensional liquid chromatography is upgraded to two-dimensional liquid chromatography.In addition, 270 compounds (127 of which are tentatively characterized as new compounds, and further confirmation is needed) are successfully characterized based on their fragmentation patterns under the guidance of molecular network, while only 95 compounds are characterized using one-dimensional liquid chromatography and high-resolution mass spectrometry. The results indicate that the developed offline two-dimensional mixed-mode liquid chromatography ×

show abstract

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

Cited by 13 publications

References 43 publications

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

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

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

Comprehensive profiling of Sanguisorba officinalis using offline two‐dimensional mixed‐mode liquid chromatography × reversed‐phase liquid chromatography, tandem high‐resolution mass spectrometry, and molecular network

Contact Info

Product

Resources

About