Ti (IV) attached‐phosphonic acid functionalized capillary monolith as a stationary phase for in‐syringe‐type fast and robust enrichment of phosphopeptides

Abstract

In this study, poly(vinylphosphonic acid-co-ethylene dimethacrylate), poly(VPA-co-EDMA) capillary monolith was synthesized as a starting material for obtaining a stationary phase for microscale enrichment of phosphopeptides. The chelation of active phosphonate groups with Ti (IV) ions gave a macroporous monolithic column with a mean pore size of 5.4 μm. The phosphopeptides from different sources were enriched on Ti (IV)-attached poly(VPA-co-EDMA) monolith using a syringe-pump. The monolithic capillary columns exhibited highly sensitive/selective enrichment performance with phosphoprotein concentrations as low as 1.0 fmol/mL. Six different phosphopeptides were detected with high intensity by the treatment of β-casein digest with the concentration of 1.0 fmol/mL, using Ti (IV)@poly(VPA-co-EDMA) monolith. Highly selective enrichment of phosphopeptides was also successfully carried out even at trace amounts, in a complex mixture of digested proteins (molar ratio of β-casein to bovine serum albumin, 1:1500) and three phosphopeptides were successfully detected. Four highly intense signals of phosphopeptides in human serum were also observed with high signal-to-noise ratio and a clear background after enrichment with Ti (IV)@poly(VPA-co-EDMA) monolith. It was concluded that the capillary microextraction system enabled fast, efficient and robust enrichment of phosphopeptides from microscale complex samples. The whole enrichment process was completed within 20 min, which was shorter than in the previously reported studies.KEYWORDS capillary monolith, human serum, immobilized metal affinity chromatography, phosphopeptide enrichment, syringe type microextration, vinylphosphonic acid

Recent trends in sorbents for bioaffinity chromatography

“…The importance of glycosylation can be also understood in diseases characterized by the abnormal glycosylation of proteins [5]. Effective enrichments are necessary and imperative before determining glycosylation and phosphorylation, which define cellular behavior and functions, in complex samples by mass spectrometry (MS) technology due to their low abundance and poor ionization efficiency [6][7][8][9]. Bioaffinity chromatography is based on the principle that the affinity ligand immobilized on the solid support in a suitable solution selectively captures the desired target molecule by molecular recognition.…”

Preparation of zirconium arsenate‐modified monolithic column for selective enrichment of phosphopeptides

“…To date, a number of strategies, including immobilized metal ion affinity chromatography (IMAC) [7][8][9][10][11][12], metal oxide affinity chromatography (MOAC) [13][14][15][16][17][18], ion exchange chromatography [19][20][21], and molecularly imprinted polymers [22][23][24][25][26], have been developed for the selective enrichment of phosphopeptides. IMAC is one of the widely applied techniques for phosphopeptide enrichment.…”

“…In addition, the advantages of flexible synthesis, adjustable porosity, fast mass transport, and low column backpressure also make it an ideal substrate of IMAC adsorbent [39]. However, most of the reported polymer monolith-based IMAC materials required tedious and time-consuming post-modification approaches to introduce chelating groups into the monolithic column [40,41], and the employed chelators were mostly phosphate group [8,11,[41][42][43].…”

In situ synthesis of a novel metal oxide affinity chromatography affinity probe for the selective enrichment of low‐abundance phosphopeptides

“…9 However, when the biological sample is directly measured by mass spectrometry (MS), 10 the low ionization efficiency of phosphopeptides and the signal suppression of non-phosphopeptides seriously affect the detection of the phosphopeptides. 11 Thus, it is important to adopt effective enrichment methods to specifically select phosphopeptides from complex biological samples. 12 Among the enrichment strategies that have been developed so far, 13,14 metal oxide affinity chromatography (MOAC), 15,16 which is based on reversible Lewis acid-base interactions between the metal oxides and the negatively charged phosphate groups to achieve the specific selection of phosphopeptides, is one of the most widely used and the one with the most potential.…”

“…Related research shows that aberrant phosphoproteins with high clinical specificity and sensitivity have become the biomarkers of clinical diagnosis, which means that research into aberrant phosphoproteins will help study the pathogenesis of human diseases 9 . However, when the biological sample is directly measured by mass spectrometry (MS), 10 the low ionization efficiency of phosphopeptides and the signal suppression of non‐phosphopeptides seriously affect the detection of the phosphopeptides 11 . Thus, it is important to adopt effective enrichment methods to specifically select phosphopeptides from complex biological samples 12 …”