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Proteomic characterisation of immune cells with a novel FACS-into-evotip workflow

Kry, Elsa LU (2025) KIMM05 20251
Department of Immunotechnology
Abstract (Swedish)
Proteomic analysis can reveal substantial information about cells and cell types, including biomarkers that can be used to distinguish subpopulations. Established protocols include manual pipetting steps of cells that are both time consuming and problematic in terms of samples losses. In this study, a new “FACS-into-evotip” protocol for proteomic analysis was evaluated with the goal to automate sample preparation by sorting cells directly into Evotips with FACS. Proteomic analysis of B-cells, CD4T-cells, CD8T-cells, regulatory T-cells, NK-cells, NKT-cells, cDC1, cDC2, pDC, Monocytes and Basophils was conducted with the new protocol and revealed 63 proteins uniquely expressed in single cell types and 172 proteins uniquely expressed in... (More)
Proteomic analysis can reveal substantial information about cells and cell types, including biomarkers that can be used to distinguish subpopulations. Established protocols include manual pipetting steps of cells that are both time consuming and problematic in terms of samples losses. In this study, a new “FACS-into-evotip” protocol for proteomic analysis was evaluated with the goal to automate sample preparation by sorting cells directly into Evotips with FACS. Proteomic analysis of B-cells, CD4T-cells, CD8T-cells, regulatory T-cells, NK-cells, NKT-cells, cDC1, cDC2, pDC, Monocytes and Basophils was conducted with the new protocol and revealed 63 proteins uniquely expressed in single cell types and 172 proteins uniquely expressed in groups of two or more cell types, which may serve as potential biomarkers for distinguishing immune cell populations. The protocol was also applied on single cell analysis to investigate intra-subset heterogeneity at the single cell level. The results from the single cell analysis yielded limited depth but several known biomarker proteins were found, showing potential for the protocol. Overall, the new “FACS-into-evotip” protocol was successfully established as an automated proteomics protocol with detection of several biomarkers and identification of a high number of uniquely detected and differentially expressed protein. However, for single cell analysis and intra-subset heterogeneity investigation the protocol needs further development to rise the number of detected proteins. (Less)
Popular Abstract (Swedish)
A novel method that maps the secrets of the immune cells

A new method has potential to let researchers map the proteome of miniscule amounts of immune cells in a matter of hours – a step towards new insights about the immune system. The immune system is the military defence of our body, protecting us from attacking diseases. Within the immune system there are different types of cells, playing different roles in the defence. To understand how our immune system works on a deeper level and to understand the different roles of the different cells better, we have done something called proteomic analysis. Most of the functions of a cell are performed by proteins and different cell types express different setups of proteins, making them who... (More)
A novel method that maps the secrets of the immune cells

A new method has potential to let researchers map the proteome of miniscule amounts of immune cells in a matter of hours – a step towards new insights about the immune system. The immune system is the military defence of our body, protecting us from attacking diseases. Within the immune system there are different types of cells, playing different roles in the defence. To understand how our immune system works on a deeper level and to understand the different roles of the different cells better, we have done something called proteomic analysis. Most of the functions of a cell are performed by proteins and different cell types express different setups of proteins, making them who they are. A complete setup of protein for a cell type is called the proteome, and that is what is investigated with proteomic analysis. The information you gain from proteomic analysis can help to both understand the immune system and the diseases on a deeper level which can be important while developing treatments to certain diseases, such as cancers.

With the new protocol we managed to identify several proteins specific for one cell type or a group of cell types, information that can be used to characterize differences and similarities between different immune cell types. The analyses were generally done with 500 cells of the same cell type per sample, but we also tried with only 1 cell per sample. When you only analyze one cell at the time it is called single cell analysis and it can give information about differences between cells that belong to the same cell type and help investigate sub groups of a cell type. Since one cell is a very small amount of sample, it requires high quality of both the method and instruments used. For our experiment, the results were limited, and a much lower number of proteins were detected compared to the samples with 500 cells, even though a more sensitive instrument was used. However, some interesting proteins were detected. Both some that were expected since they are commonly expressed for the cell type and some that were found specifically expressed for the cell type in the analysis with 500 cells.

The new protocol is a fast and automated protocol for proteomic analysis of cell types. To get the immune cells, you first need to isolate the cells from blood. Then you need to sort them depending on which cell type they belong to, and it is here we have made a difference. Instead of sorting the cells into vials and then transferring an amount of sample to the tip they are further processed in, we sort the cells directly into the tip, minimizing the sample losses and letting us know the exact number of cells we have in the tip. So even though the results for the single cell experiment were limited, our new protocol shows great potential for improving and streamlining proteomic analysis of immune cells and with further development it could be a promising method for deeper understanding of the immune system. (Less)
Please use this url to cite or link to this publication:
author
Kry, Elsa LU
supervisor
organization
course
KIMM05 20251
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
9200669
date added to LUP
2025-06-24 13:40:19
date last changed
2025-06-24 13:40:19
@misc{9200669,
  abstract     = {{Proteomic analysis can reveal substantial information about cells and cell types, including biomarkers that can be used to distinguish subpopulations. Established protocols include manual pipetting steps of cells that are both time consuming and problematic in terms of samples losses. In this study, a new “FACS-into-evotip” protocol for proteomic analysis was evaluated with the goal to automate sample preparation by sorting cells directly into Evotips with FACS. Proteomic analysis of B-cells, CD4T-cells, CD8T-cells, regulatory T-cells, NK-cells, NKT-cells, cDC1, cDC2, pDC, Monocytes and Basophils was conducted with the new protocol and revealed 63 proteins uniquely expressed in single cell types and 172 proteins uniquely expressed in groups of two or more cell types, which may serve as potential biomarkers for distinguishing immune cell populations. The protocol was also applied on single cell analysis to investigate intra-subset heterogeneity at the single cell level. The results from the single cell analysis yielded limited depth but several known biomarker proteins were found, showing potential for the protocol. Overall, the new “FACS-into-evotip” protocol was successfully established as an automated proteomics protocol with detection of several biomarkers and identification of a high number of uniquely detected and differentially expressed protein. However, for single cell analysis and intra-subset heterogeneity investigation the protocol needs further development to rise the number of detected proteins.}},
  author       = {{Kry, Elsa}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Proteomic characterisation of immune cells with a novel FACS-into-evotip workflow}},
  year         = {{2025}},
}