Improved Single-Cell Proteome Coverage Using Narrow-Bore Packed NanoLC Columns and Ultrasensitive Mass Spectrometry

Journal Article · Tue Feb 04 00:00:00 EST 2020 · Analytical Chemistry

DOI:https://doi.org/10.1021/acs.analchem.9b04631· OSTI ID:1600520

Cong, Yongzheng  ^[1]; Liang, Yiran ^[1]; Motamedchaboki, Khatereh ^[2]; Huguet, Romain ^[2]; Truong, Thy ^[3]; Zhao, Rui ^[1]; Shen, Yufeng ^[4]; Lopez-Ferrer, Daniel ^[5]; Zhu, Ying  ^[1]; Kelly, Ryan T.  ^[6]

---

1. BATTELLE (PACIFIC NW LAB)
2. Thermo Fisher Scientific
3. Brigham Young University
4. UNKNOWN
5. VISITORS
6. BRIGHAM YOUNG UNIVERSITY

Single-cell proteomics can provide unique insights into biological processes by resolving heterogeneity that is obscured by bulk measurements. Gains in the overall sensitivity and proteome coverage through improvements in sample processing and analysis increase the information content obtained from each cell, particularly for less abundant proteins. Here we report on improved single-cell proteome coverage through the combination of the previously developed Nanodroplet Processing in One Pot for Trace Samples (nanoPOTS) platform with further miniaturization of liquid chromatography (LC) separations and implementation of an ultrasensitive latest-generation mass spectrometry (MS) instrument. Following nanoPOTS sample preparation, protein digest from single cells were separated using a 20-µm-i.d. in-house-packed nanoLC column. Separated peptides were ionized using an etched fused silica emitter capable of stable ionization at the ~20 nL/min flow rate provided by the LC separation. Ultrasensitive LC-MS analysis was achieved using the Orbitrap Eclipse Tribrid mass spectrometer. An average of 362 protein groups were identified by MS/MS from single HeLa cells, and 874 protein groups were identified using the Match Between Runs (MBR) feature of MaxQuant. This represents a >70% increase in label-free proteome coverage for single cells relative to previous efforts using larger-bore (30-µm-i.d.) LC columns coupled to a previous-generation MS (Orbitrap Fusion Lumos).

• Research Organization: Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
• Sponsoring Organization: USDOE
• DOE Contract Number: AC05-76RL01830
• OSTI ID: 1600520
• Report Number(s): PNNL-SA-148385
• Journal Information: Analytical Chemistry, Vol. 92, Issue 3
• Country of Publication: United States
• Language: English