Prioritized single-cell proteomics reveals molecular and functional polarization across primary macrophages

Abstract

Major aims of single-cell proteomics include increasing the consistency, sensitivity, and depth of protein quantification, especially for proteins and modifications of biological interest. To simultaneously advance all of these aims, we developed prioritized Single Cell ProtEomics (pSCoPE). pSCoPE ensures duty-cycle time for analyzing prioritized peptides across all single cells (thus increasing data consistency) while analyzing identifiable peptides at full duty-cycle, thus increasing proteome depth. These strategies increased the quantified data points for challenging peptides and the overall proteome coverage about 2-fold. pSCoPE enabled quantifying proteome polarization in primary mouse macrophages and linking it to phenotypic variability in endocytic activity. Proteins annotated to phagosome maturation and proton transport showed concerted variation for both untreated and lipopolysaccharide-treated macrophages, indicating a conserved axis of polarization. pSCoPE further quantified proteolytic products, suggesting a gradient of cathepsin activities within a treatment condition. pSCoPE is easily accessible and likely to benefit many applications, especially mechanistic analysis seeking to focus on proteins of interest without sacrificing proteome coverage.

Exploring functional protein covariation across single cells using nPOP

“…2a) and 3) 15 sets lost because of LC malfunctions. To increase the depth and consistency of proteome coverage, the single-cell samples were analyzed by prioritized Single Cell ProtEomics (pSCoPE) introduced by Huffman et al 20 , following the guidelines of the SCoPE2 protocol 13,18 .…”

“…Before injecting samples for analysis, wells can be rehydrated with 0.1 % formic acid (which may contain isobaric carrier diluted to the desired concentration), resealed with foil, and placed into a compatible autosampler for automated sample injection and MS analysis, Taking advantage of the flexibility afforded by nPOP, we quantified proteins with two different methods, one performing quantification at MS1 level and the other at MS2 level. Specifically, using nPOP we prepared multiplexed single-cell samples for data independent acquisition by plex-DIA 24,26 and for prioritized data acquisition by pSCoPE 27 , Fig. 2.…”

“…A spectral library was built from two injections of a 10x concentrated aliquot of combined carrier and reference sample analyzed by DIA instrument methods 1 and 2, as well as an injection of a 5x concentrated aliquot of combined carrier and reference sample analyzed by DIA method 1. Both of these instrument methods are detailed in the methods section of Huffman, et al [20]. A subsequent injection of a 1x concentrated aliquot of carrier and reference sample was analyzed by DIA instrument method 1 to serve as a retention-time-calibration run.…”

“…A prioritized analysis workflow [27] was used to increase consistency of identification and depth of coverage for the nPOP prepared single-cell data shown in Fig. 2, Fig.…”

Increasing the throughput of sensitive proteomics by plexDIA

“…The parallel sample and peptide analysis by plexDIA becomes increasingly important for lowly abundant samples since they require long ion accumulation times that undermine the throughput of serial acquisition methods, such as TMT-DDA, even when the vast majority of MS2 scans result in confident peptide identifications 7, 52 . Thus, plexDIA is particularly attractive for the analysis of nanogram samples as it may afford accurate and deep proteome quantification without using 2-dimensional peptide separation (offline fractionation).…”