Highly selective enrichment of phosphorylated peptides using titanium dioxide

Abstract

The characterization of phosphorylated proteins is a challenging analytical task since many of the proteins targeted for phosphorylation are low in abundance and phosphorylation is typically substoichiometric. Highly efficient enrichment procedures are therefore required. Here we describe a protocol for selective phosphopeptide enrichment using titanium dioxide (TiO2) chromatography. The selectivity toward phosphopeptides is obtained by loading the sample in a 2,5-dihydroxybenzoic acid (DHB) or phthalic acid solution containing acetonitrile and trifluoroacetic acid (TFA) onto a TiO2 micro-column. Although phosphopeptide enrichment can be achieved by using TFA and acetonitrile alone, the selectivity is dramatically enhanced by adding DHB or phthalic acid since these compounds, in conjunction with the low pH caused by TFA, prevent binding of nonphosphorylated peptides to TiO2. Using an alkaline solution (pH > or = 10.5) both monophosphorylated and multiphosphorylated peptides are eluted from the TiO2 beads. This highly efficient method for purification of phosphopeptides is well suited for the characterization of phosphoproteins from both in vitro and in vivo studies in combination with mass spectrometry (MS). It is a very easy and fast method. The entire protocol requires less than 15 min per sample if the buffers have been prepared in advance (not including lyophilization).

Monoliths with immobilized zirconium ions for selective enrichment of phosphopeptides

“…To date, mass spectrometry (MS) analysis has become an effective and commonly used method for characterization of phosphoproteins [4]. To reduce the interference of non-phosphopeptides, selective enrichment strategies, such as immobilized metal ion affinity chromatography (IMAC) [5][6][7], metal oxide affinity chromatography (MOAC) [8,9], and ion exchange chromatography [10][11][12][13], are necessary before MS analysis.…”

“…To date, mass spectrometry (MS) analysis has become an effective and commonly used method for characterization of phosphoproteins [4]. To reduce the interference of non-phosphopeptides, selective enrichment strategies, such as immobilized metal ion affinity chromatography (IMAC) [5][6][7], metal oxide affinity chromatography (MOAC) [8,9], and ion exchange chromatography [10][11][12][13], are necessary before MS analysis.Recently, chip-based system has been introduced as a promising platform for phosphopeptide analysis, due to the benefits of low sample amount, high throughput, and easy integration [14]. Several microfluidic devices have been developed by integrating phosphopeptide enrichment with other functions, such as protein alkylation and reduction [15], protein digestion [16], peptide separation and on-line interface for MS instrument [17][18][19][20], which have shown great potentials in the field of phosphoproteome research.…”

Mass spectrometric analysis of mono- and multi-phosphopeptides by selective binding with NiZnFe2O4 magnetic nanoparticles

“…MALDI-MS (matrix assisted laser desorption ionization-mass spectrometry) was used for methodology demonstration because it minimizes losses of multi-phosphorylated peptides that are often confronted with liquid chromatography-mass spectrometry approaches 26 . Tryptic peptides of a-casein were loaded in 50% ACN (acetonitrile) containing 0.1% TFA (trifluoroacetic acid) and eluted with 1 M ammonium phosphate as described in Methods.…”

Mesoporous TiO2 aerogel for selective enrichment of phosphopeptides in rat liver mitochondria

“…Recently, metal oxide affinity chromatography (MOAC), has been developed for phosphoproteome to selectively enrich phosphorylated peptides from proteome digests with improved selectivity and stability. A variety of materials are employed, such as titanium dioxide (TiO 2 ) [12][13][14][15][16], zirconium dioxide (ZrO 2 ) [17,18], niobium oxide (Nb 2 O 5 ) [19] and tantalum oxide (Ta 2 O 5 ) [20]. To further enhance the phosphopeptide binding capacity of MOAC materials, considerable efforts have recently been devoted to explore mesoporous metal oxides with high specific surface area [21][22][23][24][25][26].…”