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Title: Subnanogram proteomics: Impact of LC column selection, MS instrumentation and data analysis strategy on proteome coverage for trace samples

Abstract

One of the greatest challenges for mass spectrometry (MS)-based proteomics is the limited ability to analyze small samples. Here we investigate the relative contributions of liquid chromatography (LC), MS instrumentation and data analysis methods with the aim of improving proteome coverage for sample sizes ranging from 0.5 ng to 50 ng. We show that the LC separations utilizing 30-µm-i.d. columns increase signal intensity by >3-fold relative to those using 75-µm-i.d. columns, leading to 32% increase in peptide identifications. The Orbitrap Fusion Lumos mass spectrometer significantly boosted both sensitivity and sequencing speed relative to earlier generation Orbitraps (e.g., LTQ-Orbitrap), leading to a ~3× increase in peptide identifications and 1.7× increase in identified protein groups for 2 ng tryptic digests of bacterial lysate. The Match Between Runs algorithm of open-source MaxQuant software further increased proteome coverage by ~ 95% for 0.5 ng samples and by ~42% for 2 ng samples. The present platform is capable of identifying >3000 protein groups from tryptic digestion of cell lysates equivalent to 50 HeLa cells and 100 THP-1 cells (~10 ng total proteins), respectively, and >950 proteins from subnanogram bacterial and archaeal cell lysates. The present ultrasensitive LC-MS platform is expected to enable deep proteome coveragemore » for subnanogram samples, including single mammalian cells.« less

Authors:
; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
1430424
Report Number(s):
PNNL-SA-125645
Journal ID: ISSN 1387-3806; 49001; KP1704020
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Journal Name:
International Journal of Mass Spectrometry
Additional Journal Information:
Journal Volume: 427; Journal Issue: C; Journal ID: ISSN 1387-3806
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
Environmental Molecular Sciences Laboratory

Citation Formats

Zhu, Ying, Zhao, Rui, Piehowski, Paul D., Moore, Ronald J., Lim, Sujung, Orphan, Victoria J., Paša-Tolić, Ljiljana, Qian, Wei-Jun, Smith, Richard D., and Kelly, Ryan T.. Subnanogram proteomics: Impact of LC column selection, MS instrumentation and data analysis strategy on proteome coverage for trace samples. United States: N. p., 2018. Web. doi:10.1016/j.ijms.2017.08.016.
Zhu, Ying, Zhao, Rui, Piehowski, Paul D., Moore, Ronald J., Lim, Sujung, Orphan, Victoria J., Paša-Tolić, Ljiljana, Qian, Wei-Jun, Smith, Richard D., & Kelly, Ryan T.. Subnanogram proteomics: Impact of LC column selection, MS instrumentation and data analysis strategy on proteome coverage for trace samples. United States. doi:10.1016/j.ijms.2017.08.016.
Zhu, Ying, Zhao, Rui, Piehowski, Paul D., Moore, Ronald J., Lim, Sujung, Orphan, Victoria J., Paša-Tolić, Ljiljana, Qian, Wei-Jun, Smith, Richard D., and Kelly, Ryan T.. Sun . "Subnanogram proteomics: Impact of LC column selection, MS instrumentation and data analysis strategy on proteome coverage for trace samples". United States. doi:10.1016/j.ijms.2017.08.016.
@article{osti_1430424,
title = {Subnanogram proteomics: Impact of LC column selection, MS instrumentation and data analysis strategy on proteome coverage for trace samples},
author = {Zhu, Ying and Zhao, Rui and Piehowski, Paul D. and Moore, Ronald J. and Lim, Sujung and Orphan, Victoria J. and Paša-Tolić, Ljiljana and Qian, Wei-Jun and Smith, Richard D. and Kelly, Ryan T.},
abstractNote = {One of the greatest challenges for mass spectrometry (MS)-based proteomics is the limited ability to analyze small samples. Here we investigate the relative contributions of liquid chromatography (LC), MS instrumentation and data analysis methods with the aim of improving proteome coverage for sample sizes ranging from 0.5 ng to 50 ng. We show that the LC separations utilizing 30-µm-i.d. columns increase signal intensity by >3-fold relative to those using 75-µm-i.d. columns, leading to 32% increase in peptide identifications. The Orbitrap Fusion Lumos mass spectrometer significantly boosted both sensitivity and sequencing speed relative to earlier generation Orbitraps (e.g., LTQ-Orbitrap), leading to a ~3× increase in peptide identifications and 1.7× increase in identified protein groups for 2 ng tryptic digests of bacterial lysate. The Match Between Runs algorithm of open-source MaxQuant software further increased proteome coverage by ~ 95% for 0.5 ng samples and by ~42% for 2 ng samples. The present platform is capable of identifying >3000 protein groups from tryptic digestion of cell lysates equivalent to 50 HeLa cells and 100 THP-1 cells (~10 ng total proteins), respectively, and >950 proteins from subnanogram bacterial and archaeal cell lysates. The present ultrasensitive LC-MS platform is expected to enable deep proteome coverage for subnanogram samples, including single mammalian cells.},
doi = {10.1016/j.ijms.2017.08.016},
journal = {International Journal of Mass Spectrometry},
issn = {1387-3806},
number = C,
volume = 427,
place = {United States},
year = {2018},
month = {4}
}