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 (C18) 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.
Instituto de Química Orgánica General, Consejo Superior de Investigaciones Científicas (CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain. Electronic address: ohernandez@natac.es.
Oswaldo
O
Hernandez-Hernandez
Instituto de Química-Física "Rocasolano", Consejo Superior de Investigaciones Científicas (CSIC), C/Serrano 119, 28006 Madrid, Spain. Electronic address: je.quintanilla@iqfr.csic.es.
Jesus Eduardo
JE
Quintanilla-Lopez
Instituto de Química-Física "Rocasolano", Consejo Superior de Investigaciones Científicas (CSIC), C/Serrano 119, 28006 Madrid, Spain. Electronic address: rlebron@iqfr.csic.es.
Rosa
R
Lebron-Aguilar
Instituto de Química Orgánica General, Consejo Superior de Investigaciones Científicas (CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain. Electronic address: mlsanz@iqog.csic.es.
Maria Luz
ML
Sanz
Instituto de Investigación en Ciencias de la Alimentación (CIAL), Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), C/Nicolás Cabrera 9, Campus de Cantoblanco, 28049 Madrid, Spain. Electronic address: javier.moreno@csic.es.
F Javier
FJ
Moreno
D000818:Animals; D002417:Cattle; D002623:Chemistry Techniques, Analytical / Q000295:instrumentation / Q000379:methods*; D002853:Chromatography, Liquid; D056148:Chromatography, Reverse-Phase; D006020:Glycopeptides / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry*; D011499:Protein Processing, Post-Translational
D006020:Glycopeptides; D010455:Peptides
Caseinomacropeptide; HILIC; O-Glycopeptides; Phosphopeptides; Post-translational modifications; Tandem mass spectrometry