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Leaf1
Team TFS
Team TFS

A new single-cell proteomics pre-print from Juerg Straubhaar and Bogdan Budnik et al., Bogdan along with Nikolai Slavov, developed the SCoPE-MS approach for single-cell proteomics that is most commonly used at the moment on Orbitrap mass spectrometers. In this pre-print Bogdan highlights a new platform called SCREEN (Single Cell pRotEomE aNalysis) that does number of improvements from sample preparation to data acquisition.

 

One of the big issues with the original SCoPE-MS method was the number of cells that could be used in the carrier channel, thus, limiting the number of proteins that could be identified and quantified. The number of cells that could be used is around 100 cells. Increasing the number of cells in the carrier channel above 200 cells results in loss of peptide identifications mainly because of the high peaks of TMT label from the carrier channel. In order to overcome this what the authors in this paper did was to move the carrier channel from 131 position to 126 position. They then removed the 126 peak from the MS2 detection to improve spectra quality which enabled the identification of far more MS2 spectra. They did this by acquiring their MS2 spectra starting from a fixed mass of 126.5 m/z thereby, enabling the detection of all channels except the carrier channel. Since the authors can still detect the 127N and 127C channels in their MS2 spectra, they still know how much carrier channel isotopic impurity has leaked into 127C channel and they still have 127N channel as their internal “true empty” channel to detect the noise level of each LC-MS run. This new acquisition approach uses HCD since all experiments here were done on a Q Exactive HF-X. They refer to this new SCOPE-MS method as SCoPE X. Because they can increase the number of cells in the carrier channel, the number of peptides identified is boosted by 40% compared to the original SCoPE-MS. The final addition to the SCREEN method is acquiring data in a targeted manner. Initially, a library is created on a much larger cell sample using the identical gradient that a single-cell sample would be run on. The final targeted library will contain a list total of 15 to 30 thousand unique peptides for targeted masses for single cell analysis runs. They tested the method on Jurkat cells, which were the same sample as in the original SCOPE-MS paper. On average, they are identifying 2900 proteins. They do point out that if they could do this experiment with CID possibly on Orbitrap Eclipse, the numbers could be in the 5000 range.

 

The SCREEN platform was used in this paper to perform single cell proteomics experiment of separating Jurkat cells from U937 human blood cancer cells. It was also used to look at Jurkat cells treated with PMAi to show that SCREEN allows not only a clear distinction between treated and untreated cell populations based on their proteome shift. but also displays a digital response. SCREEN was able to observe a difference between drug-susceptible leukemia cancer cells and resistant cells from the same leukemia cell line.

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Last update:
‎08-09-2022 09:55 AM
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