A sample of 50 million cells was used for the surfaceome, while the sample for the whole cell proteome was made up of 5 million cells. Nine GO terms were used to identify the cell surface proteins. GO terms are a classification system for categorizing protein function. All of the surface proteins identified fell into one or more of the 9 GO term categories. The results in Table 1 indicate that the surfaceome method led to finding significantly more proteins (3780) than the whole cell proteome method (2517). Almost 2000 of the proteins identified by the surfaceome were not in the whole cell proteome data. Most importantly, the surface protein IDs were doubled (1640 vs. 818). They made up a larger percentage of the proteins identified in the surfaceome (43%) than the whole cell proteome (32%). Therefore, my hypothesis was supported by the data.
This researcher performed all the procedures outlined above for the whole cell proteome data collection. However, since maintaining and processing the cells is necessary for the collection of the data below required more than the 3 days/week in the mentorship schedule, the raw data was collected by others and this researcher analyzed and presented the data herein.
Which extraction method captures the greatest number of cell surface proteins?
Because the surfaceome workflow is an enrichment method, I hypothesized that it will capture more protein IDs than the whole cell proteome method. My justification for this is that the surfaceome method requires more cells and it enriches the cell surface proteins, unlike the whole cell proteome method, which requires fewer cells and does not enrich for cell surface proteins.
Acute myeloid leukemia (AML) is a malignant disease of myeloid cells in the blood and bone marrow. Chemotherapy and immunotherapy have resulted in a survival rate of 65% in children and 25% in adults, leaving room for improvement. In the past 40 years, there have been incremental improvements in AML survival rates, exposing the need for new therapies (Elgarten et al, 2019). Drugs often target the surface proteins of cells as they are the easiest to reach, so identifying ones that are unique to leukemic cells will provide potential new AML drug targets (Yıldırım, M. et al, 2007). The surfaceome and whole cell proteome are two extraction methods that capture the proteins of a cell. Enrichment is a process which concentrates proteins for analysis. The whole cell proteome is not an enrichment method. For the surfaceome method, sucrose gradient ultracentrifugation is used for enrichment of cell surface proteins, but it requires more cells and time. Previous work developed the surfaceome workflow used in this study (Glisovic-Aplenc et al, 2017).
Protein Class | Surfaceome | Whole Cell Proteome |
Surface Proteins | 1640 | 818 |
Internal Proteins | 2140 | 1699 |
Total | 3780 | 2517 |
Further research involving these surface proteins can lead to new drug targets being discovered. It would be beneficial to run multiple samples. Replication of this experiment can validate the data. I am also interested in seeing how the results would turn out with a different cell line.
Table 1. Comparison of how many surface, other, and total protein IDs each method identified.
Figure 2. Comparison of how many proteins were identified by each method and how many both identified.
The current treatments used for AML leave many patients uncured. Through examining the surface proteins of leukemic cells, it is hoped that new drug targets will be discovered for more effective treatments. The goal of this experiment was to compare the cell surface proteome (surfaceome) and whole cell proteome extraction methods in combination with the S-trap processing protocol in capturing cell surface proteins. For this experiment we used the AML cell line, MOLM-14. The surfaceome involves sucrose gradient ultracentrifugation for enrichment of cell surface proteins. The whole cell proteome is not an enrichment method. Whole cell proteins are extracted using the Triple Prep Kit. Both the surfaceome and whole cell proteome are further processed using S-trap before MS proteomics analysis. The data revealed that the surfaceome workflow detected more total protein IDs and twice as many surface proteins compared to the whole cell proteome. It needs more cells and time to be completed, but it is more effective at capturing surface proteins. It would be advantageous to replicate this experiment for validity and work with other cell lines.
Discovering Novel Therapeutic Targets in AML
Abstract
Background Information
Analysis and Conclusion
Future Considerations
Question
Hypothesis
Materials
References
Procedures
Splitting MOLM-14 cells
Extracting proteins using the Triple Prep Kit
Loading S-Trap plate into Centrifuge for washes
The photos above were taken by my research mentor, showing me conducting the outlined procedures.
Figure 1. Photos for Whole Cell Proteome Method
Purpose
Although work has been done using the surfaceome and whole cell proteome methods to identify surface proteins, we wanted to compare the efficacy of the two methods in combination with the S-trap downstream processing protocol in identifying surface proteins. Therefore, the purpose of this investigation was to compare the efficacy of these two workflows and to determine which method identifies the most cell surface proteins.
Hypothesis
Whole Cell Proteome
Figure 3. Comparison of surface protein percentage between each methods.