Characterization of post-translationally modified peptides by hydrophilic interaction and reverse phase liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry
This work explores the use of both hydrophilic interaction liquid chromatography (HILIC) and reverse phase liquid chromatography (RPLC) for the separation and subsequent characterization of bovine caseinomacropeptide (CMP) phosphopeptides and O-glycopeptides using a quadrupole-time-of-flight (QTOF) mass spectrometer with electrospray ionization. Two neutral, ethylene bridged hybrid (BEH) amide and polyhydroxyethyl aspartamide (PHEA), and a zwitterionic, sulfobetaine (ZIC), stationary phases were used for the HILIC mode, whilst an octadecylsilane (C) stationary phase was employed for the RPLC separation. Overall, developed HILIC-QTOF method using the ZIC or BEH amide stationary phases resulted to be the most efficient methods to separate and characterize post-translationally modified (PTM) peptides without the need of any previous fractionation or derivatization step. The separation of phosphopeptides and differently sialylated O-glycopeptides in the ZIC stationary phase was dominated by an electrostatic repulsion interaction mechanism between the negatively charged phosphate groups or sialic acid moieties and the negatively charged terminal sulfonate group of the stationary phase, whereas the separation of either non-modified peptides or neutral O-glycopeptides both free of basic amino acids was based on a partitioning mechanism. In neutral amide columns, the separation was mainly dominated by hydrophilic partitioning, leading to a higher retention of the post-translationally modified peptides than the unmodified counterparts due to the hydrophilicity provided by the phosphate groups and/or O-glycans. As a consequence, HILIC-ESI-QTOF MS operating in the positive ion mode is a powerful tool for the characterization of underivatized O-glycopeptides and phosphopeptides.
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Elsevier
2016
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Subjects: | Tandem mass spectrometrya, HILIC, Post-translational modifications, Caseinomacropeptide, O-Glycopeptides, Phosphopeptides, |
Online Access: | http://hdl.handle.net/10261/133830 http://dx.doi.org/10.13039/501100004837 |
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dig-cial-es-10261-1338302020-06-01T12:37:25Z Characterization of post-translationally modified peptides by hydrophilic interaction and reverse phase liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry Hernández-Hernández, Oswaldo Quintanilla-López, Jesús Eduardo Lebrón-Aguilar, Rosa Sanz, M. Luz Moreno, F. Javier Ministerio de Ciencia e Innovación (España) Tandem mass spectrometrya HILIC Post-translational modifications Caseinomacropeptide O-Glycopeptides Phosphopeptides This work explores the use of both hydrophilic interaction liquid chromatography (HILIC) and reverse phase liquid chromatography (RPLC) for the separation and subsequent characterization of bovine caseinomacropeptide (CMP) phosphopeptides and O-glycopeptides using a quadrupole-time-of-flight (QTOF) mass spectrometer with electrospray ionization. Two neutral, ethylene bridged hybrid (BEH) amide and polyhydroxyethyl aspartamide (PHEA), and a zwitterionic, sulfobetaine (ZIC), stationary phases were used for the HILIC mode, whilst an octadecylsilane (C) stationary phase was employed for the RPLC separation. Overall, developed HILIC-QTOF method using the ZIC or BEH amide stationary phases resulted to be the most efficient methods to separate and characterize post-translationally modified (PTM) peptides without the need of any previous fractionation or derivatization step. The separation of phosphopeptides and differently sialylated O-glycopeptides in the ZIC stationary phase was dominated by an electrostatic repulsion interaction mechanism between the negatively charged phosphate groups or sialic acid moieties and the negatively charged terminal sulfonate group of the stationary phase, whereas the separation of either non-modified peptides or neutral O-glycopeptides both free of basic amino acids was based on a partitioning mechanism. In neutral amide columns, the separation was mainly dominated by hydrophilic partitioning, leading to a higher retention of the post-translationally modified peptides than the unmodified counterparts due to the hydrophilicity provided by the phosphate groups and/or O-glycans. As a consequence, HILIC-ESI-QTOF MS operating in the positive ion mode is a powerful tool for the characterization of underivatized O-glycopeptides and phosphopeptides. This work has been financed by project AGL2011-27884 fromthe Spanish Ministry of Science and Innovation (MICINN). We thank Laurie Davis from Davisco Foods International, Inc for kindly providing us with CMP. Peer Reviewed 2016-06-21T10:43:18Z 2016-06-21T10:43:18Z 2016 2016-06-21T10:43:19Z artículo http://purl.org/coar/resource_type/c_6501 doi: 10.1016/j.chroma.2015.07.096 issn: 0021-9673 e-issn: 1873-3778 Journal of Chromatography A 1428: 202-211 (2016) http://hdl.handle.net/10261/133830 10.1016/j.chroma.2015.07.096 http://dx.doi.org/10.13039/501100004837 http://dx.doi.org/10.1016/j.chroma.2015.07.096 none Elsevier |
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Tandem mass spectrometrya HILIC Post-translational modifications Caseinomacropeptide O-Glycopeptides Phosphopeptides Tandem mass spectrometrya HILIC Post-translational modifications Caseinomacropeptide O-Glycopeptides Phosphopeptides |
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Tandem mass spectrometrya HILIC Post-translational modifications Caseinomacropeptide O-Glycopeptides Phosphopeptides Tandem mass spectrometrya HILIC Post-translational modifications Caseinomacropeptide O-Glycopeptides Phosphopeptides Hernández-Hernández, Oswaldo Quintanilla-López, Jesús Eduardo Lebrón-Aguilar, Rosa Sanz, M. Luz Moreno, F. Javier Characterization of post-translationally modified peptides by hydrophilic interaction and reverse phase liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry |
description |
This work explores the use of both hydrophilic interaction liquid chromatography (HILIC) and reverse phase liquid chromatography (RPLC) for the separation and subsequent characterization of bovine caseinomacropeptide (CMP) phosphopeptides and O-glycopeptides using a quadrupole-time-of-flight (QTOF) mass spectrometer with electrospray ionization. Two neutral, ethylene bridged hybrid (BEH) amide and polyhydroxyethyl aspartamide (PHEA), and a zwitterionic, sulfobetaine (ZIC), stationary phases were used for the HILIC mode, whilst an octadecylsilane (C) stationary phase was employed for the RPLC separation. Overall, developed HILIC-QTOF method using the ZIC or BEH amide stationary phases resulted to be the most efficient methods to separate and characterize post-translationally modified (PTM) peptides without the need of any previous fractionation or derivatization step. The separation of phosphopeptides and differently sialylated O-glycopeptides in the ZIC stationary phase was dominated by an electrostatic repulsion interaction mechanism between the negatively charged phosphate groups or sialic acid moieties and the negatively charged terminal sulfonate group of the stationary phase, whereas the separation of either non-modified peptides or neutral O-glycopeptides both free of basic amino acids was based on a partitioning mechanism. In neutral amide columns, the separation was mainly dominated by hydrophilic partitioning, leading to a higher retention of the post-translationally modified peptides than the unmodified counterparts due to the hydrophilicity provided by the phosphate groups and/or O-glycans. As a consequence, HILIC-ESI-QTOF MS operating in the positive ion mode is a powerful tool for the characterization of underivatized O-glycopeptides and phosphopeptides. |
author2 |
Ministerio de Ciencia e Innovación (España) |
author_facet |
Ministerio de Ciencia e Innovación (España) Hernández-Hernández, Oswaldo Quintanilla-López, Jesús Eduardo Lebrón-Aguilar, Rosa Sanz, M. Luz Moreno, F. Javier |
format |
artículo |
topic_facet |
Tandem mass spectrometrya HILIC Post-translational modifications Caseinomacropeptide O-Glycopeptides Phosphopeptides |
author |
Hernández-Hernández, Oswaldo Quintanilla-López, Jesús Eduardo Lebrón-Aguilar, Rosa Sanz, M. Luz Moreno, F. Javier |
author_sort |
Hernández-Hernández, Oswaldo |
title |
Characterization of post-translationally modified peptides by hydrophilic interaction and reverse phase liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry |
title_short |
Characterization of post-translationally modified peptides by hydrophilic interaction and reverse phase liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry |
title_full |
Characterization of post-translationally modified peptides by hydrophilic interaction and reverse phase liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry |
title_fullStr |
Characterization of post-translationally modified peptides by hydrophilic interaction and reverse phase liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry |
title_full_unstemmed |
Characterization of post-translationally modified peptides by hydrophilic interaction and reverse phase liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry |
title_sort |
characterization of post-translationally modified peptides by hydrophilic interaction and reverse phase liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry |
publisher |
Elsevier |
publishDate |
2016 |
url |
http://hdl.handle.net/10261/133830 http://dx.doi.org/10.13039/501100004837 |
work_keys_str_mv |
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