Robust dimethyl-based multiplex-DIA workflow doubles single-cell proteome depth via a reference channel

Abstract

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, a challenge that 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. In single runs of mammalian cells, a three-plex analysis of tryptic peptides quantified 7,700 proteins per channel. The Lys-N enzyme enables five-plex quantification at MS1 and MS2 level. Because the multiplex 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 feature and confidently quantifies close to 4,000 proteins in single cells with excellent reproducibility, while our workflow currently allows routine analysis of 80 single cells per day. The concept of stable proteome vs. stochastic transcriptome still holds at this deeper proteome coverage.

µPhos: a scalable and sensitive platform for functional phosphoproteomics

“…We observed a 4-fold increase in raw MS intensity compared to the previous generation protocol. However, single-cell proteomics workflows routinely process samples in 5 µL or less 51,52 , suggesting further sensitivity gains may be achievable through continued miniaturization. One practical limitation is the need for sufficient volumes to collect all cells while ensuring efficient lysis and mixing during enrichment.…”

Spatial single-cell mass spectrometry defines zonation of the hepatocyte proteome

“…To determine the quantities of the precursors in the DIA-NN report.tsv file, we utilized the Pythonbased RefQuant algorithm 11 . In brief, RefQuant determines the ratio between target-and reference channel for each individual fragment ion and MS1 peak that is available.…”

“…Encouraged by these spatial results, we next asked if single shapes alone could produce deep and interpretable proteomic results. To improve sensitivity further we adopted and optimized elements of our scProteomics workflow 11 . These include addition of the surfactant n-Dodecyl-β-D-maltoside (DDM) to maximize peptide recovery 12 , lowering the chromatographic flow rate to 100 nL/min for increased ionization efficiency 2 ('Whisper gradients' on the Evosep system) and achieving higher chromatographic resolution with zero dead volume columns (IonOpticks) 13 .…”

“…Peptides were prepared semi-automated on a Bravo pipetting robot (Agilent), similar as previously described 11 . For this, plates were removed from the freezer and centrifuged.…”

“…The timsTOF SCP was operated with an optimal dia-PASEF method generated with our Python tool py_diAID 24 . The method contained 8 dia-PASEF scans with variable width and 2 ion mobility windows per dia-PASEF scan, covering an m/z from 300 to 1200 and an ion mobility range from 0.7 to 1.3 Vs cm -2 , as previously used on the same gradient and similar input material amount 11 . The mass spectrometer was operated in high sensitivity mode, with an accumulation and ramp time at 100 ms, capillary voltage set to 1400 V and the collision energy as a linear ramp from 20 eV at 1/K0 = 0.6 Vs cm -2 to 59 eV at 1/K0 = 1.6 Vs cm -2 .…”