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Spectral counting robust on high mass accuracy mass spectrometers
Wolfgang Hoehenwarter1, Stefanie Wienkoop
1Department of Molecular Systems Biology, Faculty of Life Sciences, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria. wolfgang.hoehenwarter@univie.ac.at
Rapid Communications in Mass Spectrometry : RCM
|November 26, 2010
Summary
Optimizing mass spectrometry settings enhances shotgun proteomics by increasing quantified low-abundance proteins. Spectral counting remains reliable for protein abundance, even with highly abundant proteins present.
Area of Science:
- Proteomics
- Mass Spectrometry
- Biochemistry
Background:
- Shotgun proteomics aims to quantify the entire protein complement of biological samples.
- Modern mass spectrometers offer high accuracy, resolution, and speed, aiding proteomic quantification.
- Spectral counting (SC) is a reliable method for estimating peptide and protein relative abundance.
Purpose of the Study:
- To optimize mass spectrometry (MS) configurations for enhanced quantification of low-abundance proteins in shotgun proteomics.
- To evaluate the impact of dynamic exclusion parameters on protein quantification in complex samples.
- To confirm the robustness of spectral counting for protein abundance estimation in the presence of highly abundant proteins.
Main Methods:
- Liquid chromatography-tandem mass spectrometry (LC/MS/MS) was performed on an LTQ-OrbiTrap mass spectrometer.
- MS/MS acquisition was tuned to minimize redundancy and maximize proteome resolution.
- Dynamic exclusion parameters, including exclusion duration and width, were systematically varied and tested.
Main Results:
- Tuning the mass spectrometer significantly increased the number of quantified low-abundance proteins.
- An exclusion duration of 90 seconds and an exclusion width of 10 ppm were found to be optimal.
- Protein spectral counts were highly reproducible, and abundance ratios were unaffected by parameter changes.
Conclusions:
- Spectral counting is a robust measure of protein abundance on high-accuracy mass spectrometry instruments, even in complex samples.
- Optimizing dynamic exclusion parameters is crucial for improving the depth of proteome coverage in shotgun proteomics.
- This study demonstrates a method to enhance the quantification of low-abundance proteins, improving comprehensive proteomic analysis.
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