Title: Label-Free Profiling of up to 200 Single-Cell Proteomes per Day Using a Dual-Column Nanoflow Liquid Chromatography Platform

Abstract

Single-cell proteomics (SCP) has great potential to advance biomedical research and personalized medicine. The sensitivity of such measurements increases with low-flow separations (<100 nL/min) due to improved ionization efficiency, but the time required for sample loading, column washing, and regeneration in these systems can lead to low measurement throughput and inefficient utilization of the mass spectrometer. Herein, we developed a two-column liquid chromatography (LC) system that dramatically increases the throughput of label-free SCP using two parallel subsystems to multiplex sample loading, online desalting, analysis, and column regeneration. The integration of MS1-based feature matching increased proteome coverage when short LC gradients were used. The high-throughput LC system was reproducible between the columns, with a 4% difference in median peptide abundance and a median CV of 18% across 100 replicate analyses of a single-cell-sized peptide standard. An average of 621, 774, 952, and 1622 protein groups were identified with total analysis times of 7, 10, 15, and 30 min, corresponding to a measurement throughput of 206, 144, 96, and 48 samples per day, respectively. When applied to single HeLa cells, we identified nearly 1000 protein groups per cell using 30 min cycles and 660 protein groups per cell for 15 min cycles.more » Finally, we explored the possibility of measuring cancer therapeutic targets with a pilot study comparing the K562 and Jurkat leukemia cell lines. This work demonstrates the feasibility of high-throughput label-free single-cell proteomics.« less

Authors:

Webber, Kei G. I. ^[1]; Truong, Thy ^[1]; Johnston, S. Madisyn ^[1]; Zapata, Sebastian E. ^[1]; Liang, Yiran ^[1]; Davis, Jacob M. ^[1]; Buttars, Alexander D. ^[1]; Smith, Fletcher B. ^[1]; Jones, Hailey E. ^[1]; Mahoney, Arianna C. ^[1]; Carson, Richard H. ^[1]; Nwosu, Andikan J. ^[1]; Heninger, Jacob L. ^[1]; Liyu, Andrey V. ^[2]; Nordin, Gregory P.  ^[1];

• Search DOE PAGES for author "Nordin, Gregory P."
• Search DOE PAGES for ORCID "0000-0001-7241-5764"

Zhu, Ying  ^[2];

• Search DOE PAGES for author "Zhu, Ying"
• Search DOE PAGES for ORCID "0000-0002-5416-0566"

Kelly, Ryan T.  ^[3]

• Search DOE PAGES for author "Kelly, Ryan T."
• Search DOE PAGES for ORCID "0000-0002-3339-4443"

---

+ Show Author Affiliations

1. Brigham Young Univ., Provo, UT (United States)
2. Pacific Northwest National Laboratory (PNNL), Richland, WA (United States). Environmental Molecular Sciences Laboratory (EMSL)
3. Brigham Young Univ., Provo, UT (United States); Pacific Northwest National Laboratory (PNNL), Richland, WA (United States). Environmental Molecular Sciences Laboratory (EMSL)

Publication Date:

Wed Apr 06 00:00:00 EDT 2022

Research Org.:

Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

Sponsoring Org.:

USDOE; National Institute of General Medical Sciences (NIGMS)

OSTI Identifier:

1983636

Report Number(s):

PNNL-SA-170317
Journal ID: ISSN 0003-2700

Grant/Contract Number:  

AC05-76RL01830; R01GM138931

Resource Type:

Accepted Manuscript

Journal Name:

Analytical Chemistry

Additional Journal Information:

Journal Volume: 94; Journal Issue: 15; Journal ID: ISSN 0003-2700

Publisher:

American Chemical Society (ACS)

Country of Publication:

United States

Language:

English

Subject:

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; proteomics, nanoPOTS; cells; liquid chromatography; mass spectrometry; peptides and proteins; protein identification