A High-Sensitivity and High-Throughput LC-MS Workflow for Low-Input and Single-Cell Proteomics
- 1Kazusa DNA Research Institute
Abstract
Single-cell proteomics (SCP) requires sample preparation that minimizes sample loss and highly sensitive LC-MS analysis because the amount of protein available from a single cell is extremely limited. Here, we developed an integrated workflow for low-input and single-cell proteomics based on single-cell proteomics by LMNG-assisted sample preparation (scpLASP), which enables microliter-scale sample preparation using standard laboratory consumables. Systematic optimization of peptide reconstitution conditions identified 0.01% n-dodecyl-{beta}-D-maltoside (DDM) combined with 0.01% lauryl maltose neopentyl glycol (LMNG) as the condition yielding the highest number of identified peptides among those tested for low-input samples. Optimization of the LC mobile-phase composition identified 0.5% acetic acid with 1% dimethyl sulfoxide (DMSO) as the optimal condition, increasing the mean number of identified protein groups from 6,767 with 0.1% formic acid to 7,409 for 250 pg HeLa digests analyzed using an Orbitrap Astral Zoom. Shortening the LC gradient from 11 to 5.5 min enabled higher analytical throughput while maintaining mean identification counts of 6,558 and 7,088 protein groups from 100 and 250 pg HeLa digests, respectively, with median protein-level coefficients of variation of 9.20% and 6.99%. When the optimized workflow was applied to HEK293-derived Expi293F single cells using the same 5.5-min LC gradient, an effective analytical throughput of 135 samples per day was achieved with an injection volume of 4.5 {micro}L, accounting for sample-loading time. This workflow identified a median of 5,655 protein groups per cell using an empirical spectral library generated from 20-cell samples. Overall, this study establishes a sensitive and high-throughput workflow for low-input and single-cell proteomics that combines low-loss microliter-scale sample preparation with optimized LC-MS analysis, achieving high proteome depth and analytical throughput without requiring specialized nanoliter-scale sample-preparation devices.
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BibTeX
@misc{okuda2026highsensitivity,
title = {{A High-Sensitivity and High-Throughput LC-MS Workflow for Low-Input and Single-Cell Proteomics}},
author = {Y. Okuda and S. Watanabe and Y. Endo and R. Konno and H. Mitsui and D. Nakajima and Y. Kodera and O. Ohara and D. Higo and Y. Kawashima},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.26.762077},
url = {https://www.biorxiv.org/content/10.64898/2026.10.26.762077v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - A High-Sensitivity and High-Throughput LC-MS Workflow for Low-Input and Single-Cell Proteomics
AU - Y. Okuda
AU - S. Watanabe
AU - Y. Endo
AU - R. Konno
AU - H. Mitsui
AU - D. Nakajima
AU - Y. Kodera
AU - O. Ohara
AU - D. Higo
AU - Y. Kawashima
PY - 2026
DA - 2026/10/26
PB - bioRxiv
DO - 10.64898/2026.10.26.762077
UR - https://www.biorxiv.org/content/10.64898/2026.10.26.762077v1
AB - Single-cell proteomics (SCP) requires sample preparation that minimizes sample loss and highly sensitive LC-MS analysis because the amount of protein available from a single cell is extremely limited. Here, we developed an integrated workflow for low-input and single-cell proteomics based on single-cell proteomics by LMNG-assisted sample preparation (scpLASP), which enables microliter-scale sample preparation using standard laboratory consumables. Systematic optimization of peptide reconstitution conditions identified 0.01% n-dodecyl-{beta}-D-maltoside (DDM) combined with 0.01% lauryl maltose neopentyl glycol (LMNG) as the condition yielding the highest number of identified peptides among those tested for low-input samples. Optimization of the LC mobile-phase composition identified 0.5% acetic acid with 1% dimethyl sulfoxide (DMSO) as the optimal condition, increasing the mean number of identified protein groups from 6,767 with 0.1% formic acid to 7,409 for 250 pg HeLa digests analyzed using an Orbitrap Astral Zoom. Shortening the LC gradient from 11 to 5.5 min enabled higher analytical throughput while maintaining mean identification counts of 6,558 and 7,088 protein groups from 100 and 250 pg HeLa digests, respectively, with median protein-level coefficients of variation of 9.20% and 6.99%. When the optimized workflow was applied to HEK293-derived Expi293F single cells using the same 5.5-min LC gradient, an effective analytical throughput of 135 samples per day was achieved with an injection volume of 4.5 {micro}L, accounting for sample-loading time. This workflow identified a median of 5,655 protein groups per cell using an empirical spectral library generated from 20-cell samples. Overall, this study establishes a sensitive and high-throughput workflow for low-input and single-cell proteomics that combines low-loss microliter-scale sample preparation with optimized LC-MS analysis, achieving high proteome depth and analytical throughput without requiring specialized nanoliter-scale sample-preparation devices.
ER -