MS data contain very rich information. Due to the large sizes and complex structures of the data-sets, performing comprehensive data analysis is very challenging. We have developed effective approaches of using multivariate analysis (e.g., Multivariate Curve Resolution (MCR)) and Machine Learning (ML) to efficiently extract the metabolomics information from metabolomic information from MS images. Similarly, ML based methods have used to analyze SCMS data to extract essential biological information. In addition, we use image fusion technique to integrate optical image (e.g. fluorescence microscopy image) with MS image, allowing for (1) significantly improved spatial resolution and (2) correlation between histological hallmarks (e.g., stained proteins) and metabolomic profiles.
(2) Mass Spectrometry Imaging (MSI)
Mass spectrometry imaging (MSI) allows for mapping the spatial distribution of a broad range of molecules on surfaces. MSI combines MS with other technologies in microscopy, motion control instrumentation, and software engineering. In general, a MSI measurement is conducted by simultaneously recording the MS data with the physical locations in spot-to-spot scanning way.
The Single-probe MSI setup
(3) Mass Spectrometry Analysis of Multicellular Spheroids
Spheroids are important for studies of diseases such as cancers. Compared with traditional 2D-culture cells, 3D-culture multicellular spheroids are better models for tumors.
• Extracellular molecules in spheroids. We produce micro-scale device, the Micro-funnel, that can be implanted into live spheroids to collect metabolites secreted from cancer cells. The collected extracellular metabolites can be analyzed by the Single-probe MS technique (Sun et al. Anal. Chem. 2017, 89, 9069.)
• Quantification of drug uptake in single spheroids. Among all drug resistance mechanisms, reduced drug uptake in tumors is regarded as an important pathway acquired by drug-resistant cancer cells. Chemoresistant cancer cells in ovarian cancer patients can develop into spheroids, which can spread and induce metastasis. These spheroids are highly heterogeneous with different shapes, sizes, and compositions of cell types. Studying drug uptake in single spheroids is necessary for understanding chemosensitivity and chemoresistance. We have developed LC/MS method to quantify anticancer drug uptake in single spheroids (Peng et al. ACS Pharmacol. Transl. Sci. 2024, ).
Research in the Yang laboratory centers on mass spectrometry. We are interested in a variety of areas.
(1) Single Cell Mass Spectrometry (SCMS) Analysis
Traditional cell bioanalysis is performed on samples prepared from large numbers of cells (e.g. cell lysate), such that the information of individual cells is concealed by the averaged results. Single cell mass spectrometry (SCMS) is an emerging field that could potentially revolutionize studies in basic scientific and biomedical research. We have developed multiple ambient SCMS techniques.
The Single-probe MSI results
- Single-probe
SCMS metabolomics. The Single-probe is a miniaturized sampling and ionization device that can be coupled to mass spectrometer for live single cell MS analysis in real-time under ambient conditions. The Single-probe, with a sampling tip smaller than eukaryotic cells (<10 μm), can be inserted into individual living cells to sample the intracellular compounds for immediate MS analysis.(Pan et al., Anal. Chem. 2014, 86, 9376).
Quantitative SCMS. The Single-probe technique can be used to quantitative SCMS analysis such as quantify drug uptake in single cells (adherent and suspension). (Pan et al. Anal. Chem. 2019, 91, 9018; Bensen et al. ACS Pharmacol. Transl. Sci., 2021, 4, 96).
Integrated fluorescence microscopy & SCMS. The Single-probe SCMS technique can be integrated with fluorescence microscopy to study cell-cell interactions. An application of this method is to study the influence of drug-resistant cells on drug-sensitive cells (Chen et al. Chem. Sci. 2022, 13, 6687).
Videos of the Single-probe fabrication and applications of live single cell analysis can be found from: Wei Rao, Ning Pan, Zhibo Yang*, Journal of Visualized Experiments 2016, 112, e53911, (doi:10.3791/53911).
• The Single-probe is a multifunctional device that can also be used for MSI studies to reconstruct the spatial distribution of biomolecules from sections of animal tissues under ambient conditions (i.e. in atmosphere and under room temperature).
• We have obtained high spatial resolution (8.5 μm) on biological tissues, which is among the highest resolution that can be obtained using ambient MSI methods (Rao et al. JASMS, 2015, 26, 986).
Photo of mouse kidney slice
MSI of mouse kidney slice
(Spatial resolution 8.5 μm)
Research Funding
Active External Funding
(1) NSF, 8/2021–7/2025, Yihan Shao (PI), Zhibo Yang (Co-PI), total award: $444,855 ($125,000 to Yang).
(2) DoD, 4/2023–3/2027, Doris Benbrook and Anthony Burgett (Co-PI, OUHSC), Zhibo Yang (Co-I), $1,093,200 ($166,680 to Yang).
(3) NSF, 7/2023–6/2026, Zhibo Yang (PI), Atkinson, Linda (OU K20, co-PI), total award: $410,000 ($365,210 to Yang)).
(4) NIH-RO1, 7/2023–6/2028, NIH (1R01AI177469), Laura-Isobel (Multi PI), Zhibo Yang (Multi PI), total award: $2,607,600 ($1,447,000 to Yang).
(5) Chan Zuckerberg Initiative (CZI), 12/2023–11/2025, Laura-Isobel (Co-PI), Zhibo Yang (Co-PI), total award: $500,000 ($240,000 to Yang).
Past External Funding
(1) NIH-RO1, 8/2015–7/2022, Zhibo Yang (PI), Chuanbin Mao (co-I), Anthony Burgett (collaborator), total awarded: $1,530,000.
(2) NIH-R21, 6/2016–5/2019, Anthony Burgett (PI), Zhibo Yang (co-I), total awarded: $588,000.
(3) NSF, 8/2016-7/2022, Zhibo Yang (PI), Boris Wawrik (formal PI), total awarded: $537,000.
(4) NSF-MRI, 9/2016–8/2019, Robert Cichewicz (PI), Laura Bartley (co-PI), Marc Libault (co-PI), Zhibo Yang (co-PI), Si Wu (co-PI), total awarded: $416,500.
(5) NCI P30 Cancer Center Support Grant (CCSG), 5/2022–4/2023, Anthony Burgett (PI), Zhibo Yang (co-PI); total award: $25,000.
(6) NIH DSP (Diversity Supplements Program), 9/2017–8/2019, Zhibo Yang (PI), total awarded: $44,000
(7) ASMS (American Society for Mass Spectrometry) Research Award, 6/2014–5/2015, Zhibo Yang (PI), total awarded: $35,000
(8) OCAST (Oklahoma Center for the Advancement of Science and Technology) Health Research Program, 9/2014–8/2017, Zhibo Yang (PI), total awarded: $135,000
(9) NSF EPSCoR REU, 5/2016–8/2016, Zhibo Yang (co-PI, 50%), Marc Libault (co-PI, 50%), total awarded: $5,000
Internal Grants:
(1) VPRP, Bridge Funding Investment Program (BFIP), 4/2023-9/2023, PI (Yang), $50,000
(2) Faculty Investment Program (FIP), OU, 2013 – 2015, 2020, 2023 (PI: Zhibo Yang; awarded: $60,000)
(3) Honors Research Assistant Program (HRAP) for undergraduate student research, OU, 2014 – 2018, 2023 (PI: Zhibo Yang; total awarded: $6,000)
(4) BFIP (Bridge Funding Investment Program), OU, 4/2023-9/2023 (PI: Zhibo Yang; total award: $50,000)
(5) SEIP (Strategic Equipment Investment Program), OU, 6/2023-12/2023 (PI: Zhibo Yang; total award: $25,500)
(6) SFSF (Senior Faculty Summer Fellowship), PI (Yang), 2022, $8,000
(7) Junior Faculty Fellowship, OU, 2013 – 2014 (total awarded: $14,000)
(8) Faculty Enrichment Grant, OU, 2013 (total awarded: $1,200)
(9) Travel Assistance Program, OU, 2013 – 2016 (total awarded: $4,800)
(10) Research Publication Support, VRP and Research Council, OU, 2016 (total awarded: $2,000)
(11) VPR Supplemental Equipment Funding, 2013, OU (PI: Zhibo Yang; awarded: $139,400)
Single cells
SCMS analysis of HeLa cells using the Single-probe
(Click to watch the video)
Single-probe tip
Research Highlighted in the Media
(1) Awarded for Chan Zuckerberg Initiative with Laura-Isobel McCall for Bystander Metabolic Effects of Infection Across Scales, 2023.
(2) Interviewed with OCAST radio show, Oklahoma Innovations, for single cell mass spectrometry research,
2015.
(3) Single cell mass spectrometry studies has been reported by scientific magazines (Chemistry World and
C&E News) and websites (Proteo Monitor, Genomeweb, and Bioanalysis Zone), 2014.
(4) ASMS (American Society of Mass Spectrometry) Research Award, 2014.
(5) Astrochemistry research reported by NCSA (National Center for Supercomputing Applications) and published in
ACCESS magazine (2011, 24, 10-11) and on the iSGTW (International Science Grid This Week) website.
MS analysis of extracellular metabolites in live spheroids
MSI data analysis using MCR and ML
Single-probe SCMS metabolomics
The T-probe
- T-probe.
The T-probe is a miniaturized sampling and ionization device for live single cell analysis (Liu et al., Anal. Chem. 2018, 90, 11078).
A T-probe is fabricated by using two polycarbonate slides to sandwich three capillaries. The self-aspiration produced by the nanoESI provides a driving force to draw cellular contents and mix them with solvent for MS analysis.
Quantitative SCMS
- Micropipette needle.
The Micropipette Needle is a multifunctional device that can be used reactive SCMS studies.
The micropipette Needle integrates single cell selection, chemical reactions, and nanoESI for MS analysis.
Combined with the PB (Paternò-Buchi) reaction, this technique has been used to determine C=C bond locations in unsaturated lipids in single cells (Zhu et al., Anal. Chem. 2020, 92, 11380).
The Micropipette Needle
Quantification of anticancer drug uptake in single spheroids
Proteins act as machines underlying basic cell functions. Different groups of functional proteins are involved in cellular homeostasis, metabolic pathways, drug delivery, and chemoresistance. We are interested in using bottom-up proteomics methods to study biological processes and human diseases.
Cell-cell interaction. Cell–cell interactions are critical for the growth, health, and functions of cells. Understanding cell-cell interactions is needed for better understanding of diseases such as the development of chemoresistance. For example, drug-resistant cells can interact with drug-sensitive cells to elevate their drug resistance level. We performed direct coculture and indirect coculture of drug-resistant and drug-sensitive cell lines, aiming to investigate intracellular proteins responsible for cell communication.
we performed direct coculture and indirect coculture of drug-resistant and drug-sensitive cell lines, aiming to investigate intracellular proteins responsible for cell communication.
MS data contain very rich information. Due to the large sizes and complex structures of the data-sets, performing comprehensive data analysis is very challenging. We have developed effective approaches of using multivariate analysis (e.g., Multivariate Curve Resolution (MCR)) and Machine Learning (ML) to efficiently extract the metabolomics information from metabolomic information from MS images. Similarly, ML based methods have used to analyze SCMS data to extract essential biological information. In addition, we use image fusion technique to integrate optical image (e.g. fluorescence microscopy image) with MS image, allowing for (1) significantly improved spatial resolution and (2) correlation between histological hallmarks (e.g., stained proteins) and metabolomic profiles.
50 μm
Integrated fluorescence microscopy & SCMS
Proteomics of cell-cell interactions
