Robust dimethyl-based multiplex-DIA doubles single-cell proteome depth via a reference channel.
Marvin ThielertEricka Cm ItangConstantin AmmarFlorian A SchoberIsabell BludauLisa SchweizerThierry M NordmannPatricia SkowronekMaria WahleWen-Feng ZengXie-Xuan ZhouAndreas-David BrunnerSabrina RichterMitchell Paul LevesqueFabian Joachim TheisMartin StegerMatthias MannPublished in: Molecular systems biology (2023)
Single-cell proteomics aims to characterize biological function and heterogeneity at the level of proteins in an unbiased manner. It is currently limited in proteomic depth, throughput, and robustness, which we address here by a streamlined multiplexed workflow using data-independent acquisition (mDIA). We demonstrate automated and complete dimethyl labeling of bulk or single-cell samples, without losing proteomic depth. Lys-N digestion enables five-plex quantification at MS1 and MS2 level. Because the multiplexed channels are quantitatively isolated from each other, mDIA accommodates a reference channel that does not interfere with the target channels. Our algorithm RefQuant takes advantage of this and confidently quantifies twice as many proteins per single cell compared to our previous work (Brunner et al, PMID 35226415), while our workflow currently allows routine analysis of 80 single cells per day. Finally, we combined mDIA with spatial proteomics to increase the throughput of Deep Visual Proteomics seven-fold for microdissection and four-fold for MS analysis. Applying this to primary cutaneous melanoma, we discovered proteomic signatures of cells within distinct tumor microenvironments, showcasing its potential for precision oncology.
Keyphrases
- single cell
- mass spectrometry
- rna seq
- high throughput
- label free
- induced apoptosis
- multiple sclerosis
- ms ms
- cell cycle arrest
- optical coherence tomography
- machine learning
- electronic health record
- palliative care
- signaling pathway
- cell death
- genome wide
- endoplasmic reticulum stress
- clinical practice
- big data
- cell proliferation
- pi k akt