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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 in this paper, 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 MS significantly boosted both sensitivity and sequencing speed relative to earlier generation Orbitraps (e.g., LTQ-Orbitrap), leading to a ~3-fold increase in peptide identifications and 1.7-fold increase in identified protein groups for 2 ng tryptic digests of the bacterium S. oneidensis. 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. Using the best combination of the above variables, we were able to identify >3000 proteins from 10 ng tryptic digests from both HeLa and THP-1 mammalian cell lines. We also identified >950 proteins from subnanogram archaeal/bacterial cocultures. The present ultrasensitive LC–MS platform achieves a level of proteome coverage notmore » previously realized for ultra-small sample loadings, and is expected to facilitate the analysis of subnanogram samples, including single mammalian cells.« less

Authors:
 [1];  [1];  [2];  [2];  [3];  [3];  [1];  [2];  [2];  [1]
  1. Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
  2. Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Biological Sciences Division
  3. California Inst. of Technology (CalTech), Pasadena, CA (United States). Division of Geological and Planetary Sciences
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); National Institutes of Health (NIH)
OSTI Identifier:
1395289
Alternate Identifier(s):
OSTI ID: 1379954; OSTI ID: 1413502
Report Number(s):
PNNL-SA-125645
Journal ID: ISSN 1387-3806; PII: S1387380617302130
Grant/Contract Number:  
AC02-05CH11231; AC05-76RL01830; P41 GM103493; R21 EB020976-01A1; 1R21EB020976-01A1
Resource Type:
Accepted Manuscript
Journal Name:
International Journal of Mass Spectrometry
Additional Journal Information:
Journal Volume: 427; Journal ID: ISSN 1387-3806
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Ultrasensitive; NanoLC; Orbitrap Fusion Lumos; Match between runs; Subnanogram proteomics; Small cell populations; 97 MATHEMATICS AND COMPUTING; ultrasensitive; nanoLC; match between runs; subnanogram proteomics; small cell populations

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., 2017. 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. Fri . "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. https://www.osti.gov/servlets/purl/1395289.
@article{osti_1395289,
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 in this paper, 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 MS significantly boosted both sensitivity and sequencing speed relative to earlier generation Orbitraps (e.g., LTQ-Orbitrap), leading to a ~3-fold increase in peptide identifications and 1.7-fold increase in identified protein groups for 2 ng tryptic digests of the bacterium S. oneidensis. 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. Using the best combination of the above variables, we were able to identify >3000 proteins from 10 ng tryptic digests from both HeLa and THP-1 mammalian cell lines. We also identified >950 proteins from subnanogram archaeal/bacterial cocultures. The present ultrasensitive LC–MS platform achieves a level of proteome coverage not previously realized for ultra-small sample loadings, and is expected to facilitate the analysis of subnanogram samples, including single mammalian cells.},
doi = {10.1016/J.IJMS.2017.08.016},
journal = {International Journal of Mass Spectrometry},
number = ,
volume = 427,
place = {United States},
year = {2017},
month = {9}
}

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Figures / Tables:

Fig. 1 Fig. 1: Comparison of LC column size on the performance of proteomic analysis. (a1–a2) Base peak chromatograms of 10-ng tryptic digest of Shewanella oneidensis with (a1)75-μm and (a2) 30-μm i.d. LC columns and a LTQ-Orbitrap MS. Y axis was fixed at 8E6 to show the signal gain for the 30-μmmore » LC. (b) Peptide intensity ratio between 30-μm and 75-μm i.d. LC. Each point in the chart corresponds to a peptide identified from both LC columns. (c) The number of unique peptides and protein groups identified with 75-μm and 30-μm LC columns. In each condition, two replicates were analyzed and averaged to generate results. LC conditions: 30-μm i.d. column operated at 60 nL/min;75-μm i.d. column operated at 350 nL/min; a 150-min gradient from 5% to 28% Buffer B was used.« less

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