title: Phosphopeptide enrichment: Development of magnetic solid phase extraction method based on polydopamine coating and Ti(4+)-IMAC.
abstract: Protein post translational modifications currently represent one of the main challenges with proteomic analysis, due to the important biological role they play within cells. Protein phosphorylation is one of the most important, with several approaches developed for phosphopeptides enrichment and analysis, essential for comprehensive phosphoproteomic analysis. However, the development of new materials for phosphopeptides enrichment may overcome previous drawbacks and improve enrichment of such peptides. In this regard, new magnetic stationary phases based on polydopamine coating and Ti(4+) immobilization exploit the potential of IMAC enrichment and couple it with the versatility of magnetic solid phase extraction. In this work the use of such stationary phase was extended from the MALDI proof of concept stage with the development of an optimized method for phosphopeptides enrichment compatible with typical shotgun proteomics experimental workflows. Different loading and elution buffers were tested to improve phosphopeptides recovery and enrichment selectivity. Finally, the analysis of isolated peptides pointed out that polydopamine alone is an ideal support matrix for polar post translational modifications because it enables to reduce unspecific binding and preferentially binds hydrophilic peptides.
authors: Dipartimento di Chimica, Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Rome, Italy. Electronic address: susy.piovesana@uniroma1.it.
authors: Susy
authors: S
authors: Piovesana
authors: Dipartimento di Chimica, Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Rome, Italy. Electronic address: annalaura.capriotti@uniroma1.it.
authors: Anna Laura
authors: AL
authors: Capriotti
authors: Dipartimento di Chimica, Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Rome, Italy. Electronic address: chiara.cavaliere@uniroma1.it.
authors: Chiara
authors: C
authors: Cavaliere
authors: Dipartimento di Chimica, Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Rome, Italy. Electronic address: francesca.ferraris@uniroma1.it.
authors: Francesca
authors: F
authors: Ferraris
authors: Dipartimento di Chimica, Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Rome, Italy. Electronic address: roberto.samperi@uniroma1.it.
authors: Roberto
authors: R
authors: Samperi
authors: Dipartimento di Chimica, Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Rome, Italy. Electronic address: salvatore.ventura@uniroma1.it.
authors: Salvatore
authors: S
authors: Ventura
authors: Dipartimento di Chimica, Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Rome, Italy. Electronic address: aldo.lagana@uniroma1.it.
authors: Aldo
authors: A
authors: Laganà
mesh_terms: D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D007211:Indoles / Q000737:chemistry*; D060526:Magnetic Fields; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D010748:Phosphopeptides / Q000302:isolation & purification*; D011108:Polymers / Q000737:chemistry*; D052616:Solid Phase Extraction / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014025:Titanium / Q000737:chemistry*
chemical_list: D007211:Indoles; D058185:Magnetite Nanoparticles; D010748:Phosphopeptides; D011108:Polymers; C568283:polydopamine; D014025:Titanium
keywords: Magnetic solid phase extraction; Phosphoproteomics; Polydopamine; Sample preparation; Shotgun proteomics; Ti(4+)-immobilized metal-ion affinity chromatography