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Title: Automated Nanoflow Two-Dimensional Reversed-Phase Liquid Chromatography System Enables In-Depth Proteome and Phosphoproteome Profiling of Nanoscale Samples

Abstract

Two-dimensional reversed-phase liquid chromatography (2D RPLC) separations have enabled near-comprehensive proteome profiling of biological systems. However, large samples containing milligram amounts of protein are usually required due to the significant sample losses incurred during offline fractionation. The large sample requirements preclude the application of 2D RPLC to biological systems having sample availability in the nanogram to low-microgram range. To achieve in-depth proteomic studies of trace samples, we have developed an automated nanoflow 2D RPLC platform in which the first dimension high-pH fractionation was performed at 300 nL/min on a 75-µm i.d. capillary column.Each fraction was then fully transferred to a second-dimension nanoLC separation using an autosampler equipped with a custom-machined syringe. In addition to optimizing fractionation conditions, we found that the use of a plastic 96-well plate as collection device and the adding of n-Dodecyl ß-D-Maltoside (0.01%) in the collection buffer can significantly improve sample recovery. We have demonstrated the nanoflow 2D RPLC platform can achieve confident identifications of ~37,000–69,000 unique peptides, corresponding to ~5300–6900 protein groups using only 100 to 1000 ng of HeLa tryptic digest. Furthermore, by integrating phosphopeptide enrichment, the platform can identify as many as ~20,000 phosphopeptides from 100 µg of MCF7 protein digest. Because thismore » platform is assembled from commercially available components, our approach can be readily adapted in other proteomics laboratories.« less

Authors:
 [1];  [1]; ORCiD logo [1];  [1];  [1];  [1];  [1];  [2];  [3]; ORCiD logo [1]; ORCiD logo [1]; ORCiD logo [4];  [1]; ORCiD logo [1]
  1. BATTELLE (PACIFIC NW LAB)
  2. UNIVERSITY PROGRAMS
  3. Pacific Northwest National Laboratory
  4. BRIGHAM YOUNG UNIVERSITY
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1572492
Report Number(s):
PNNL-SA-141437
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Journal Name:
Analytical Chemistry
Additional Journal Information:
Journal Volume: 91; Journal Issue: 15
Country of Publication:
United States
Language:
English
Subject:
Mass spectrometry, multidimensional separations, nanoflow fractionation, proteomics, phosphoproteomics

Citation Formats

Dou, Maowei, Tsai, Chia-Feng, Piehowski, Paul D., Wang, Yang, Fillmore, Thomas L., Zhao, Rui, Moore, Ronald J., Zhang, Pengfei, Qian, Wei-Jun, Smith, Richard D., Liu, Tao, Kelly, Ryan T., Shi, Tujin, and Zhu, Ying. Automated Nanoflow Two-Dimensional Reversed-Phase Liquid Chromatography System Enables In-Depth Proteome and Phosphoproteome Profiling of Nanoscale Samples. United States: N. p., 2019. Web. doi:10.1021/acs.analchem.9b01248.
Dou, Maowei, Tsai, Chia-Feng, Piehowski, Paul D., Wang, Yang, Fillmore, Thomas L., Zhao, Rui, Moore, Ronald J., Zhang, Pengfei, Qian, Wei-Jun, Smith, Richard D., Liu, Tao, Kelly, Ryan T., Shi, Tujin, & Zhu, Ying. Automated Nanoflow Two-Dimensional Reversed-Phase Liquid Chromatography System Enables In-Depth Proteome and Phosphoproteome Profiling of Nanoscale Samples. United States. doi:10.1021/acs.analchem.9b01248.
Dou, Maowei, Tsai, Chia-Feng, Piehowski, Paul D., Wang, Yang, Fillmore, Thomas L., Zhao, Rui, Moore, Ronald J., Zhang, Pengfei, Qian, Wei-Jun, Smith, Richard D., Liu, Tao, Kelly, Ryan T., Shi, Tujin, and Zhu, Ying. Tue . "Automated Nanoflow Two-Dimensional Reversed-Phase Liquid Chromatography System Enables In-Depth Proteome and Phosphoproteome Profiling of Nanoscale Samples". United States. doi:10.1021/acs.analchem.9b01248.
@article{osti_1572492,
title = {Automated Nanoflow Two-Dimensional Reversed-Phase Liquid Chromatography System Enables In-Depth Proteome and Phosphoproteome Profiling of Nanoscale Samples},
author = {Dou, Maowei and Tsai, Chia-Feng and Piehowski, Paul D. and Wang, Yang and Fillmore, Thomas L. and Zhao, Rui and Moore, Ronald J. and Zhang, Pengfei and Qian, Wei-Jun and Smith, Richard D. and Liu, Tao and Kelly, Ryan T. and Shi, Tujin and Zhu, Ying},
abstractNote = {Two-dimensional reversed-phase liquid chromatography (2D RPLC) separations have enabled near-comprehensive proteome profiling of biological systems. However, large samples containing milligram amounts of protein are usually required due to the significant sample losses incurred during offline fractionation. The large sample requirements preclude the application of 2D RPLC to biological systems having sample availability in the nanogram to low-microgram range. To achieve in-depth proteomic studies of trace samples, we have developed an automated nanoflow 2D RPLC platform in which the first dimension high-pH fractionation was performed at 300 nL/min on a 75-µm i.d. capillary column.Each fraction was then fully transferred to a second-dimension nanoLC separation using an autosampler equipped with a custom-machined syringe. In addition to optimizing fractionation conditions, we found that the use of a plastic 96-well plate as collection device and the adding of n-Dodecyl ß-D-Maltoside (0.01%) in the collection buffer can significantly improve sample recovery. We have demonstrated the nanoflow 2D RPLC platform can achieve confident identifications of ~37,000–69,000 unique peptides, corresponding to ~5300–6900 protein groups using only 100 to 1000 ng of HeLa tryptic digest. Furthermore, by integrating phosphopeptide enrichment, the platform can identify as many as ~20,000 phosphopeptides from 100 µg of MCF7 protein digest. Because this platform is assembled from commercially available components, our approach can be readily adapted in other proteomics laboratories.},
doi = {10.1021/acs.analchem.9b01248},
journal = {Analytical Chemistry},
number = 15,
volume = 91,
place = {United States},
year = {2019},
month = {8}
}