Exploring functional protein covariation across single cells using nPOP
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Abstract
Background
Many biological processes, such as cell division cycle and drug resistance, are reflected in protein covariation across single cells. This covariation can be quantified and interpreted by single-cell mass spectrometry with sufficiently high throughput and accuracy.
Results
Here, we describe nPOP, a method that enables simultaneous sample preparation of thousands of single cells, including lysing, digesting, and labeling individual cells in volumes of 8–20 nl. nPOP uses piezo acoustic dispensing to isolate individual cells in 300 pl volumes and performs all subsequent sample preparation steps in small droplets on a fluorocarbon-coated glass slide. Protein covariation analysis identifies cell cycle dynamics that are similar and dynamics that differ between cell types, even within subpopulations of melanoma cells delineated by markers for drug resistance priming. Melanoma cells expressing these markers accumulate in the G1 phase of the cell cycle, display distinct protein covariation across the cell cycle, accumulate glycogen, and have lower abundance of glycolytic enzymes. The non-primed melanoma cells exhibit gradients of protein abundance, suggesting transition states. Within this subpopulation, proteins functioning in oxidative phosphorylation covary with each other and inversely with proteins functioning in glycolysis. This protein covariation suggests divergent reliance on energy sources and its association with other biological functions. These results are validated by different mass spectrometry methods.
Conclusions
nPOP enables flexible, automated, and highly parallelized sample preparation for single-cell proteomics. This allows for quantifying protein covariation across thousands of single cells and revealing functionally concerted biological differences between closely related cell states. Support for nPOP is available at https://scp.slavovlab.net/nPOP .
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This review reflects comments and contributions by Sónia Gomes Pereira, Julia Grzymkowski and Wasim Sayyad.
The authors reported an improved method called nano-PrOteomics sample Preparation (nPOP) to quantify single-cell proteomics and its use in cell cycle analysis. The high throughput sample preparation method uses CellenOne cell sorting and liquid handling system which overcome the limitations of their previously proposed method Minimal ProteOmics sample Preparation (mPOP method) by reducing the sample volume 100-fold to a few nanoliters. The nPOP Workflow is explained very clearly. The Cell Cycle Analysis data presented in Figure 4 is an interesting validation of the technique, which seems really useful for single cell-omics studies.
The text in the paper could be tidied up slightly, especially surrounding Figure 1. Some minor …
This review reflects comments and contributions by Sónia Gomes Pereira, Julia Grzymkowski and Wasim Sayyad.
The authors reported an improved method called nano-PrOteomics sample Preparation (nPOP) to quantify single-cell proteomics and its use in cell cycle analysis. The high throughput sample preparation method uses CellenOne cell sorting and liquid handling system which overcome the limitations of their previously proposed method Minimal ProteOmics sample Preparation (mPOP method) by reducing the sample volume 100-fold to a few nanoliters. The nPOP Workflow is explained very clearly. The Cell Cycle Analysis data presented in Figure 4 is an interesting validation of the technique, which seems really useful for single cell-omics studies.
The text in the paper could be tidied up slightly, especially surrounding Figure 1. Some minor issues which can be easily addressed:
- Please provide the full forms of some of the abbreviations used (e.g. SILAC, SP3, iST, RI, or TMT).
- There are some missing labels of subpanels in the Figures or the text. E.g.
a) Subpanel Fig. 1c and 1d mentioned in the text are missing in Fig. 1 as well as in the legend.
b) On page 5 and line 7 (PDF file), Fig. 2b should be mentioned at the end of the sentence “After 20 minutes of cell lysis, …”.
c) On page 7 (PDF file), ‘The relatively low CV values indicate that protein quantification derived from different peptides is internally consistent, Fig. 3c.’ - Fig. 3c should read Fig. 3b.
‘The samples were then diluted and combined for digestion. These results suggest that DMSO allows for efficient cell lysis without detectable biases against proteins originating from different cell compartments, Fig. 1a’ - There may be a sentence missing here to explain the graph in Fig. 1a. The first sentence describes the last part of the method and the next sentence starts with "These results suggest…", though no results were discussed. Perhaps the reference to Fig. 1a should be updated to refer to other figure panels.
‘We lysed U-937 monocytes and Jurkat T-cells with both DMSO and urea and compared the protein ratios estimated from the cells lysed with DMSO and with urea, Fig. 1c. U-937 monocytes cultured in heavy SILAC media and Jurkat T-Cells cultured in standard media were combined in equal amounts and lysed with either 90 % DMSO or 6M urea as shown in Fig. 1c.’ - Consider condensing this fragment into a single sentence.
‘This gave us further confidence that DMSO lysis is well suited for miniaturizing sample preparation on an open surface without using MS-incompatible chemicals’ - The confidence in using DMSO for lysis and therefore avoiding the use of "MS-incompatible chemicals" is supported. However, based solely on the information given in the paragraph and in Figure 1a, it is unclear how conclusions can be made about "miniaturizing sample preparation on an open surface," because it looks like the method was done in centrifuge tubes. Can more detail be provided about steps taken to establish whether DMSO lysis is suited for this miniaturization?
‘The addition brings the total volume to 13.5 nl, Fig. 2a. Samples are digested by 75 ng/μl trypsin for 5 hours’ - In the Methods it is stated that the volume is 14 nl and the trypsin concentration is 100 ng/ul. Suggest checking these details for consistency and accuracy. As in Fig. 1a the trypsin is given in nl (10nl), maybe both values should be in the text (e.g. 10nl of 75 ng/ul).
‘room temperature’ - Room temperature can mean different values in different countries. Could the authors clarify or provide a range of temperatures? This might be particularly important when taking into consideration the drying of samples with such reduced volumes.
Figure 2
- ‘a) A schematic of the nPOP sample preparation method illustrates the steps of cell lysis, protein digestion, peptide labeling with TMT, and quenching with two additions of hydroxylamine’ - The drying of the droplets appears to be done without the use of the nozzle. If this is correct, suggest removing it from this particular stage on the scheme. Also, please note that each incubation with hydroxylamine was indicated as lasting 20min (so a total of 40min), but the scheme indicates 1h.
- A suggestion for the visualization of Figure 2a is to divide the sections using vertical dashed lines into the following sections: Cell isolation - Lysis – Digestion - Labeling – Quenching - Pooling
- ‘(c) Total ion current chromatograms from three runs demonstrate low contaminants and consistent chromatography’ - This could be discussed in the text.
‘To improve the recovery of labeled peptides, the footprint of each array is washed by 4 μl of acetonitrile, which is collected and added to the corresponding combined set. This wash is option and used to maximize the recovery of labeled peptides from the slide.’ - Suggest rephrasing this fragment to "To improve the recovery of labeled peptides, an optional step of washing the footprint of each array…"
Figure 3 ‘The consistency of protein quantification is estimated as the coefficient of variation (CV) of the relative levels of peptides originating from the same protein. The median CVs per cells are tightly distributed, suggesting high consistency of sample preparation’ - It is not immediately clear what the pink and black numbers of cells represent; this could be clarified in the legend or in the text.
‘Similar to previous analysis the protein ratios in bulk samples agreed well with ratios in the single cells single cells’ - ‘single cells’ is duplicated.
‘A Pearson correlation of 0.8 indicates that the single-cell protein quantification is consistent with the proteomic measurements of established bulk method’ - Fig. 3d should be mentioned at the end of this sentence.
Figure 4
- Figure 4b, 'The boxplots display distributions for correlations between the phase markers and proteins from the large ribosomal subunit assembly GO term'- In the image itself it's written "Microtubule-based process," so please clarify what is shown on the boxplots.
- Figure 4c - as in b, please clarify in the legend what the boxplots represent.
Methods ‘for a final concentration of 100 ng/μl of trypsin’ - as mentioned above, in the main text the value indicated for trypsin concentration is 75ng/ul, can this be clarified?
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