RESUMEN
Phosphorylation events are important during cellular function. Analysis of phosphorylation in complex samples has been extensively studied using large-scale phosphopeptide enrichment methods. Quantitative analysis of the enriched phosphopeptides is subsequently performed using label-based methodologies (e.g., SILAC, iTRAQ, and others). Here we describe the protocol for the quantitative analysis of phosphopeptides, enriched with titanium dioxide micro-column, using an intensity-based label-free quantitation.
Asunto(s)
Cromatografía de Afinidad , Fosfoproteínas/metabolismo , Proteómica/métodos , Titanio , Cromatografía de Afinidad/métodos , Análisis de Datos , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Fosfoproteínas/química , Fosforilación , Titanio/químicaRESUMEN
Titanium dioxide (TiO2) has very high affinity for phosphopeptides and in recent years it has become one of the most popular methods for phosphopeptide enrichment from complex biological samples. Peptide loading onto TiO2 resin in a highly acidic environment in the presence of 2,5-dihydroxybenzoic acid (DHB), phthalic acid, lactic acid, or glycolic acid has been shown to improve selectivity significantly by reducing unspecific binding of non-phosphorylated peptides. The phosphopeptides bound to the TiO2 are subsequently eluted from the chromatographic material using an alkaline buffer. TiO2 chromatography is extremely tolerant towards most buffers used in biological experiments, highly robust and as such it has become the method of choice in large-scale phosphoproteomics. Here we describe a batch mode protocol for phosphopeptide enrichment using TiO2 chromatographic material followed by desalting and concentration of the sample by reversed phase micro-columns prior to downstream MS and LC-MS/MS analysis.
Asunto(s)
Cromatografía de Afinidad , Fosfopéptidos/agonistas , Proteómica/métodos , Titanio/química , Adsorción , Animales , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , Biología Computacional , Bases de Datos de Proteínas , Células HeLa , Humanos , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Propiedades de Superficie , Espectrometría de Masas en Tándem , Titanio/metabolismo , Flujo de TrabajoRESUMEN
Phosphoproteomics relies on methods for efficient purification and sequencing of phosphopeptides from highly complex biological systems, especially when using low amounts of starting material. Current methods for phosphopeptide enrichment, e.g., Immobilized Metal ion Affinity Chromatography and titanium dioxide chromatography provide varying degrees of selectivity and specificity for phosphopeptide enrichment. The number of multi-phosphorylated peptides identified in most published studies is rather low. Here we describe a protocol for a strategy that separates mono-phosphorylated peptides from multiply phosphorylated peptides using Sequential elution from Immobilized Metal ion Affinity Chromatography. The method relies on the initial enrichment and separation of mono- and multi-phosphorylated peptides using Immobilized Metal ion Affinity Chromatography and a subsequent enrichment of the mono-phosphorylated peptides using titanium dioxide chromatography. The two separate phosphopeptide fractions are then subsequently analyzed by mass spectrometric methods optimized for mono-phosphorylated and multi-phosphorylated peptides, respectively, resulting in improved identification of especially multi-phosphorylated peptides from a minimum amount of starting material.