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A simple approach for accurate peptide quantification in MS-based proteomics

View ORCID ProfileTeresa Mendes Maia, View ORCID ProfileAn Staes, Kim Plasman, View ORCID ProfileJarne Pauwels, Katie Boucher, View ORCID ProfileAndrea Argentini, View ORCID ProfileLennart Martens, Tony Montoye, View ORCID ProfileKris Gevaert, View ORCID ProfileFrancis Impens
doi: https://doi.org/10.1101/703397
Teresa Mendes Maia
VIB-UGent Center for Medical Biotechnology, Albert Baertsoenkaai 3, 9000 Ghent, BelgiumDepartment of Biomolecular Medicine, Ghent University, Albert Baertsoenkaai 3, 9000 Ghent, BelgiumVIB Proteomics Core, Albert Baertsoenkaai 3, 9000 Ghent, Belgium
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  • ORCID record for Teresa Mendes Maia
An Staes
VIB-UGent Center for Medical Biotechnology, Albert Baertsoenkaai 3, 9000 Ghent, BelgiumDepartment of Biomolecular Medicine, Ghent University, Albert Baertsoenkaai 3, 9000 Ghent, BelgiumVIB Proteomics Core, Albert Baertsoenkaai 3, 9000 Ghent, Belgium
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Kim Plasman
Alzheimer Research Foundation, Kalkhoevestraat 1, 8790 Waregem, Belgium
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Jarne Pauwels
VIB-UGent Center for Medical Biotechnology, Albert Baertsoenkaai 3, 9000 Ghent, BelgiumDepartment of Biomolecular Medicine, Ghent University, Albert Baertsoenkaai 3, 9000 Ghent, BelgiumVIB Proteomics Core, Albert Baertsoenkaai 3, 9000 Ghent, Belgium
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Katie Boucher
VIB-UGent Center for Medical Biotechnology, Albert Baertsoenkaai 3, 9000 Ghent, BelgiumDepartment of Biomolecular Medicine, Ghent University, Albert Baertsoenkaai 3, 9000 Ghent, BelgiumVIB Proteomics Core, Albert Baertsoenkaai 3, 9000 Ghent, Belgium
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Andrea Argentini
VIB-UGent Center for Medical Biotechnology, Albert Baertsoenkaai 3, 9000 Ghent, BelgiumDepartment of Biomolecular Medicine, Ghent University, Albert Baertsoenkaai 3, 9000 Ghent, BelgiumBioinformatics Institute Ghent, Ghent University, Ghent, Belgium.
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Lennart Martens
VIB-UGent Center for Medical Biotechnology, Albert Baertsoenkaai 3, 9000 Ghent, BelgiumDepartment of Biomolecular Medicine, Ghent University, Albert Baertsoenkaai 3, 9000 Ghent, BelgiumBioinformatics Institute Ghent, Ghent University, Ghent, Belgium.
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Tony Montoye
Business Development Management, VIB, 9000 Ghent, Belgium
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Kris Gevaert
VIB-UGent Center for Medical Biotechnology, Albert Baertsoenkaai 3, 9000 Ghent, BelgiumDepartment of Biomolecular Medicine, Ghent University, Albert Baertsoenkaai 3, 9000 Ghent, BelgiumVIB-UGent Center for Medical Biotechnology, Albert Baertsoenkaai 3, 9000 Ghent, Belgium
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  • For correspondence: francis.impens@vib-ugent.be kris.gevaert@vib-ugent.be
Francis Impens
VIB-UGent Center for Medical Biotechnology, Albert Baertsoenkaai 3, 9000 Ghent, BelgiumDepartment of Biomolecular Medicine, Ghent University, Albert Baertsoenkaai 3, 9000 Ghent, BelgiumVIB Proteomics Core, Albert Baertsoenkaai 3, 9000 Ghent, BelgiumVIB-UGent Center for Medical Biotechnology, Albert Baertsoenkaai 3, 9000 Ghent, Belgium
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  • ORCID record for Francis Impens
  • For correspondence: francis.impens@vib-ugent.be kris.gevaert@vib-ugent.be
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ABSTRACT

Despite its growing popularity and use, bottom-up proteomics remains a complex analytical methodology. Its general workflow consists of three main steps: sample preparation, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and computational data analysis. Quality assessment of the different steps and components of this workflow is instrumental to identify technical flaws and to avoid loss of precious measurement time and sample material. However, assessment of the extent of sample losses along the sample preparation protocol, in particular after proteolytic digestion, is not yet routinely implemented because of the lack of an accurate and straightforward method to quantify peptides. Here, we report on the use of a microfluidic UV/visible spectrophotometer to quantify MS-ready peptides directly in MS loading solvent, consuming only 2 μl of sample. We determined the optimal peptide amount for LC-MS/MS analysis on a Q Exactive HF mass spectrometer using a dilution series of a commercial K562 cell digest. Careful evaluation of selected LC and MS parameters allowed us to define 3 μg as an optimal peptide amount to be injected on this particular LC-MS/MS system. Finally, using tryptic digests from human HEK293T cells, we showed that injecting equal peptide amounts, rather than approximated ones, results into less variable LC-MS/MS and protein quantification data. The obtained quality improvement together with easy implementation of the approach makes it possible to routinely quantify MS-ready peptides as a next step in daily proteomics quality control.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted July 16, 2019.
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A simple approach for accurate peptide quantification in MS-based proteomics
Teresa Mendes Maia, An Staes, Kim Plasman, Jarne Pauwels, Katie Boucher, Andrea Argentini, Lennart Martens, Tony Montoye, Kris Gevaert, Francis Impens
bioRxiv 703397; doi: https://doi.org/10.1101/703397
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A simple approach for accurate peptide quantification in MS-based proteomics
Teresa Mendes Maia, An Staes, Kim Plasman, Jarne Pauwels, Katie Boucher, Andrea Argentini, Lennart Martens, Tony Montoye, Kris Gevaert, Francis Impens
bioRxiv 703397; doi: https://doi.org/10.1101/703397

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