An Automated Nanowell-Array Workflow for Quantitative Multiplexed Single-Cell Proteomics Sample Preparation at High Sensitivity

Abstract

Multiplexed and label-free mass spectrometry-based approaches with single-cell resolution have attributed surprising heterogeneity to presumed homogenous cell populations. Even though specialized experimental designs and instrumentation have demonstrated remarkable advances, the efficient sample preparation of single cells still lags. Here, we introduce the proteoCHIP, a universal option for single-cell proteomics sample preparation including multiplexed labeling up to 16-plex with high sensitivity and throughput. The automated processing using a commercial system combining single-cell isolation and picoliter dispensing, the cellenONE, reduces final sample volumes to low nanoliters submerged in a hexadecane layer simultaneously eliminating error-prone manual sample handling and overcoming evaporation. The specialized proteoCHIP design allows direct injection of single cells via a standard autosampler resulting in around 1500 protein groups per TMT10-plex with reduced or eliminated need for a carrier proteome. We evaluated the effect of wider precursor isolation windows at single-cell input levels and found that using 2 Da isolation windows increased overall sensitivity without significantly impacting interference. Using the dedicated mass spectrometry acquisition strategies detailed here, we identified on average close to 2000 proteins per TMT10-plex across 170 multiplexed single cells that readily distinguished human cell types. Overall, our workflow combines highly efficient sample preparation, chromatographic and ion mobility-based filtering, rapid wide-window data-dependent acquisition analysis, and intelligent data analysis for optimal multiplexed single-cell proteomics. This versatile and automated proteoCHIP-based sample preparation approach is sufficiently sensitive to drive biological applications of single-cell proteomics and can be readily adopted by proteomics laboratories.

Keywords: automated sample preparation; high field asymmetric waveform ion mobility spectrometry; isobaric peptide labeling; proteoCHIP; single-cell proteomics.

Graphical abstract

Fig. 1

The proteoCHIP multiplexing workflow for TMT10 and TMTpro.A, up to 16 nanowells/single cells per TMT set are prepared inside the cellenONE, (B) automatically combined via benchtop centrifugation, and (C) directly interfaced with a standard autosampler. D, identified proteins, peptides, PSMs, and ID-rate and (E) log₁₀ reporter ion S/N of single cells labeled with TMT10 (red) or TMTpro (green) at 20× or no-carrier. A total of 250 single cells were processed for TMT10 no-carrier, while 56 were included for TMT10 20× carrier. For TMTpro with no-carrier, 80 single cells were processed, while for TMTpro 20× carrier, 196 single cells were used. PSM, peptide spectral match; RI, reporter ion; S/N, signal to noise; TMT, tandem mass tag.

Fig. 2

Data completeness and reproducibility of multiplexed single-cell proteomes with TMT10 and TMTpro reagents at different sorted carrier ratios. Log₂ reporter ion S/N correlation of two single-cell samples within one TMTplex for (A) TMT10 with a 20× carrier channel or (B) no-carrier, (C) TMTpro with a 20× carrier channel, or (D) no-carrier; r = Pearson correlation estimate. Cumulative missing reporter ions per quantified protein across three analytical runs for (E) TMT10 with a 20× carrier channel or (F) no-carrier, (G) TMTpro with a 20× carrier channel, or (H) no-carrier samples across three injections per condition. RI, reporter ion; S/N, signal to noise; TMT, tandem mass tag.

Fig. 3

Large isolation windows do not result in frequent chimeric spectra at single-cell peptide input with FAIMS-based ion mobility filtering.A, protein identifications assigned per species and isolation windows (0.4, 0.7, 1, 1.5, 2, or 3 m/z) across 1∗10, 10, or 100 ng total sample input. B, % PSMs identified from chimeric MS/MS scans (>2 peptides identified per MS/MS scan) per isolation window (0.4, 0.7, 1, 1.5, 2, or 3 m/z) across 1, 10, or 100 ng total sample input. C, % isolation interference and (D) % matched fragment ions per PSM. Colors indicate isolation window (0.4, 0.7, 1, 1.5, 2, or 3 m/z) across 1, 10, or 100 ng total sample input. Cell type–specific separation by PCA on the peptide level using a 0.4 m/z isolation window with (E) 1, (F) 10, or (G) 100 ng total sample input. PCA of two-proteome mix isolated with 2 m/z at (H) 1, (I) 10, or (J) 100 ng peptide input. Colors indicate different species; each data point represents one sample. FAIMS, field-asymmetric ion mobility spectrometry; MS/MS, tandem mass spectrometry; PCA, principal component analysis; PSM, peptide spectral match.

Fig. 4

Comparison of HeLa and HEK-293 single-cell proteomes.A, protein groups, peptides, PSMs, MS/MS scans, ID-rate and (B) reporter ion S/N of HeLa/HEK-293 samples with 20× sorted and no-carrier. C, distinct peptide sequence overlap across three analytical runs of HeLa/HEK-293 no-carrier samples. Fifty single-cell (D) PCA with Kernel density estimates and (E) differential protein abundance or 170 single-cell (F) PCA with Kernel density estimates and (G) distinct proteins of HeLa (pink) and HEK-293 (blue). For volcano plots, log₂ fold change and −log₁₀p-value is shown. MS/MS, tandem mass spectrometry; PCA, principal component analysis; PSM, peptide spectral match; RI, reporter ion; S/N, signal to noise.