Optimized data-independent acquisition approach for proteomic analysis at single-cell level

Abstract

Background Single-cell proteomic analysis provides valuable insights into cellular heterogeneity allowing the characterization of the cellular microenvironment which is difficult to accomplish in bulk proteomic analysis. Currently, single-cell proteomic studies utilize data-dependent acquisition (DDA) mass spectrometry (MS) coupled with a TMT labelled carrier channel. Due to the extremely imbalanced MS signals among the carrier channel and other TMT reporter ions, the quantification is compromised. Thus, data-independent acquisition (DIA)-MS should be considered as an alternative approach towards single-cell proteomic study since it generates reproducible quantitative data. However, there are limited reports on the optimal workflow for DIA-MS-based single-cell analysis. Methods We report an optimized DIA workflow for single-cell proteomics using Orbitrap Lumos Tribrid instrument. We utilized a breast cancer cell line (MDA-MB-231) and induced drug resistant polyaneuploid cancer cells (PACCs) to evaluate our established workflow. Results We found that a short LC gradient was preferable for peptides extracted from single cell level with less than 2 ng sample amount. The total number of co-searching peptide precursors was also critical for protein and peptide identifications at nano- and sub-nano-gram levels. Post-translationally modified peptides could be identified from a nano-gram level of peptides. Using the optimized workflow, up to 1500 protein groups were identified from a single PACC corresponding to 0.2 ng of peptides. Furthermore, about 200 peptides with phosphorylation, acetylation, and ubiquitination were identified from global DIA analysis of 100 cisplatin resistant PACCs (20 ng). Finally, we used this optimized DIA approach to compare the whole proteome of MDA-MB-231 parental cells and induced PACCs at a single-cell level. We found the single-cell level comparison could reflect real protein expression changes and identify the protein copy number. Conclusions Our results demonstrate that the optimized DIA pipeline can serve as a reliable quantitative tool for single-cell as well as sub-nano-gram proteomic analysis.

High Sensitivity Limited Material Proteomics Empowered by Data-Independent Acquisition on Linear Ion Traps

“…Finally, as we are using very short gradients in this work (31 and 58 min methods run-to-run), peptide and protein identifications can be significantly increased by lengthening the runtime as previously tested by others. 16,17 Similarly, we opted not to include library-based searches or cosearching strategies with higher-load samples, not to confound our technical results by those enhancements obtained by search strategies offering ID transfer.…”

“…Brunner et al included a similar platform during their recent demonstration of advances in instrument development to analyze single-cell proteomes on a trapped ion mobility mass spectrometer with diaPASEF . Currently, most single-cell proteomics and low-input proteomics studies were performed on an orbitrap mass analyzer instrument ,− or a time-of-flight instrument. ,, Linear ion traps stand as an attractive alternative mass analyzer to orbitraps for low-input applications in mass spectrometry-based proteomics thanks to their increased sensitivity and efficient scanning speed. To enhance the results of data acquisition for low-input quantitative proteomics experiments, data-independent acquisition (DIA) is an attractive approach, as precursor ions are fragmented and acquired independently from their intensity, which makes this acquisition method less biased and reduces missing values compared to data-dependent acquisition (DDA)…”

High sensitivity limited material proteomics empowered by data-independent acquisition on linear ion traps

“…For the quantification in figure 2, samples were diluted in buffer A so it is possible that we were integrating background noise because the background is dropping at the same rate as the signal. Finally, as we are using very short gradients in this work (31min and 58min methods run-to-run), peptide and protein identifications can be significantly increased by lengthening the runtime as previously tested by others 16,17 . Similarly, we opted not to include library-based searches or co-searching strategies with higher-load samples, not to confound our technical results by those enhancements obtained by search strategies offering ID transfer.…”

“…Brunner et al 5 included a similar platform during their recent demonstration of advances in instrument development to analyze single-cell proteomes on a trapped ion mobility mass spectrometer with diaPASEF 12 . Currently, most single-cell proteomics and low-input proteomics studies were performed on an orbitrap mass analyser instrument 4,[13][14][15][16][17] or a time-of-flight instrument 5,15,18 . Linear ion traps stand as an attractive alternative mass analyzer to orbitraps for low-input applications in mass spectrometry-based proteomics 19 thanks to their increased sensitivity and efficient scanning speed.…”

Recent advances in microfluidics for single-cell functional proteomics

“…Multiple studies have demonstrated that optimized acquisition methods (such as DIA) led to increased ion utilization which further resulted in enhanced coverage for SCP. 178,179 Together, these improvements drastically enhanced sensitivity resulting in increased protein identification and quantification from single-cell samples.…”