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Title: Making broad proteome protein measurements in 1-5 min using high-speed RPLC separations and high-accuracy mass measurments

Abstract

The throughput for proteomics measurements that provide broad protein coverage is limited by the quality and speed of both the separations and the subsequent mass analysis; present analysis times can range anywhere from hours to days (or longer). We have explored the basis for ultrahigh-throughput proteomics measurements using high-speed reversed-phase liquid chromatography (RPLC) combined with high accuracy mass spectrometric measurements. Time-of-flight (TOF) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometers were evaluated in conjunction with 0.8-µm porous C18 particle-packed RPLC using 50 µm i.d. capillary columns for identifying peptides using the Accurate Mass and Time (AMT) tag approach. Peptide RPLC relative retention (elution) times could be correlated to within 5% to elution times that differed by at least 25-fold in speed, which allowed peptides to be identified using AMT tags identified from much slower RPLC-MS/MS analyses. When coupled with RPLC, the mass spectrometers operated at fast spectrum acquisition speeds (e.g., 0.2 sec for TOF and either 0.3 or 0.6 sec for FTICR), and peptide mass measurement accuracies of better than ±15 ppm were obtained. Ion population control during fast separations limited the mass accuracies obtained with FTICR, but the use of fast regulation of ion populations using automated gainmore » control improved the mass accuracies. The detection of low abundance species was somewhat suppressed for fast analyses. The proteome coverage obtained using AMT tags was limited by the separation peak capacity, the sensitivity of the MS, and the accuracy of both the mass measurements and the relative RPLC peptide elution times. Experimental results demonstrated that accuracies of 5% for the RPLC relative elution times and better than ±15 ppm for mass measurements were sufficient for confident identification of >2800 peptides and >760 proteins from >13,000 different detected species from a Shewanella oneidensis tryptic digest.. The TOF instrumentation was found to be preferable for faster separations (of <120 sec), while FTICR MS was more effective for analysis times of >150 sec due to the improved mass accuracies achievable with longer spectrum acquisition times. The present work demonstrates the feasibility of very high throughput proteomics measurements and indicates additional significant improvements in throughput are achievable by further increasing the speed of high peak capacity separations, as well by increasing the measurement sensitivity and the accuracy of mass measurements.« less

Authors:
; ; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
876944
Report Number(s):
PNNL-SA-45881
400412000
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Journal Name:
Analytical Chemistry, 77(23):7763-7773
Additional Journal Information:
Journal Volume: 77; Journal Issue: 23
Country of Publication:
United States
Language:
English

Citation Formats

Shen, Yufeng, Strittmatter, Eric F., Zhang, Rui, Metz, Thomas O., Moore, Ronald J., Li, Fumin, Udseth, Harold R., Smith, Richard D., Unger, Klaus K., Kumar, Dhirendra, and Lubda, Dieter. Making broad proteome protein measurements in 1-5 min using high-speed RPLC separations and high-accuracy mass measurments. United States: N. p., 2005. Web. doi:10.1021/ac051257o.
Shen, Yufeng, Strittmatter, Eric F., Zhang, Rui, Metz, Thomas O., Moore, Ronald J., Li, Fumin, Udseth, Harold R., Smith, Richard D., Unger, Klaus K., Kumar, Dhirendra, & Lubda, Dieter. Making broad proteome protein measurements in 1-5 min using high-speed RPLC separations and high-accuracy mass measurments. United States. https://doi.org/10.1021/ac051257o
Shen, Yufeng, Strittmatter, Eric F., Zhang, Rui, Metz, Thomas O., Moore, Ronald J., Li, Fumin, Udseth, Harold R., Smith, Richard D., Unger, Klaus K., Kumar, Dhirendra, and Lubda, Dieter. Thu . "Making broad proteome protein measurements in 1-5 min using high-speed RPLC separations and high-accuracy mass measurments". United States. https://doi.org/10.1021/ac051257o.
@article{osti_876944,
title = {Making broad proteome protein measurements in 1-5 min using high-speed RPLC separations and high-accuracy mass measurments},
author = {Shen, Yufeng and Strittmatter, Eric F. and Zhang, Rui and Metz, Thomas O. and Moore, Ronald J. and Li, Fumin and Udseth, Harold R. and Smith, Richard D. and Unger, Klaus K. and Kumar, Dhirendra and Lubda, Dieter},
abstractNote = {The throughput for proteomics measurements that provide broad protein coverage is limited by the quality and speed of both the separations and the subsequent mass analysis; present analysis times can range anywhere from hours to days (or longer). We have explored the basis for ultrahigh-throughput proteomics measurements using high-speed reversed-phase liquid chromatography (RPLC) combined with high accuracy mass spectrometric measurements. Time-of-flight (TOF) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometers were evaluated in conjunction with 0.8-µm porous C18 particle-packed RPLC using 50 µm i.d. capillary columns for identifying peptides using the Accurate Mass and Time (AMT) tag approach. Peptide RPLC relative retention (elution) times could be correlated to within 5% to elution times that differed by at least 25-fold in speed, which allowed peptides to be identified using AMT tags identified from much slower RPLC-MS/MS analyses. When coupled with RPLC, the mass spectrometers operated at fast spectrum acquisition speeds (e.g., 0.2 sec for TOF and either 0.3 or 0.6 sec for FTICR), and peptide mass measurement accuracies of better than ±15 ppm were obtained. Ion population control during fast separations limited the mass accuracies obtained with FTICR, but the use of fast regulation of ion populations using automated gain control improved the mass accuracies. The detection of low abundance species was somewhat suppressed for fast analyses. The proteome coverage obtained using AMT tags was limited by the separation peak capacity, the sensitivity of the MS, and the accuracy of both the mass measurements and the relative RPLC peptide elution times. Experimental results demonstrated that accuracies of 5% for the RPLC relative elution times and better than ±15 ppm for mass measurements were sufficient for confident identification of >2800 peptides and >760 proteins from >13,000 different detected species from a Shewanella oneidensis tryptic digest.. The TOF instrumentation was found to be preferable for faster separations (of <120 sec), while FTICR MS was more effective for analysis times of >150 sec due to the improved mass accuracies achievable with longer spectrum acquisition times. The present work demonstrates the feasibility of very high throughput proteomics measurements and indicates additional significant improvements in throughput are achievable by further increasing the speed of high peak capacity separations, as well by increasing the measurement sensitivity and the accuracy of mass measurements.},
doi = {10.1021/ac051257o},
url = {https://www.osti.gov/biblio/876944}, journal = {Analytical Chemistry, 77(23):7763-7773},
number = 23,
volume = 77,
place = {United States},
year = {2005},
month = {12}
}