Preparation and loading buffer study of polyvinyl alcohol-based immobilized Ti⁴⁺ affinity chromatography for phosphopeptide enrichment

Abstract

Despite recent advances in phosphoproteomics, an efficient and simple enrichment protocol is still a challenge and of high demand aiming at large-scale plant phosphoproteomics studies. Here, we developed a novel loading buffer system for synthesized immobilized metal affinity chromatography material targeting plant samples, which was prepared by a simple one-step esterification between polyvinyl alcohol and phosphoric acid and then was subjected to immobilize Ti(4+). SEM and Fourier transform IR spectroscopy were used to assure the synthesis protocol of the polyvinyl alcohol-based Ti(4+) immobilized material, and the specific surface areas and pore volumes of the polymers were measured. The selectivity for phosphopeptide enrichment from α-casein was improved by optimizing the pH and components of the loading buffer. By using potassium hydrogen phthalate/hydrochloric acid with pH at 2.50 as the loading buffer, 19 phosphopeptides with high intensity were identified. The final optimized protocol was adapted to salt-stressed maize leaves for phosphoproteome analysis. A total of 57 phosphopeptides containing 59 phosphorylated sites from 50 phosphoproteins were identified in salt-stressed maize leaf. The research was meaningful to obtain much more information about phosphoproteins leading to the comprehension of salt resistance and salt-inducible phosphorylated processes of maize leaves.

Gadolinium oxide: Exclusive selectivity and sensitivity in the enrichment of phosphorylated biomolecules

“…Information about amino acid sequence, m/z values, amino acid position number is given in Table . Previously we have reported one phosphopeptide at m/z 1634 (ED S PEVIESPPEIN) detected from κ‐casein . Here, with Gd 2 O 3 NPs, two phosphopeptides from κ‐casein at m/z 1634 and 1241 (TIA S GEPTSTPT) are enriched.…”

“…In phosphoproteomics, selectivity is the ability of material to distinguish phosphorylated from nonphosphorylated molecules . The affinity probes are based on the high‐valence metal oxides like TiO 2 , ZrO 2 , La 2 O 3 , HfO 2 , SnO 2 , Ta 2 O 5 , CeO 2 , and the metal oxide composites with strong Lewis acidity. The incorporation of multiple metal ions into the enrichment material affects the phosphate affinity by changing the distributions of binding sites or changing the Lewis acidity of material .…”

Design of a ruthenium(III) immobilized affinity material based on a β‐cyclodextrin‐functionalized poly(glycidyl methacrylate‐ethylene dimethacrylate) monolith for the enrichment of hippuric acid

“…The affinity separation is primarily based on the interaction between the analytes and a chelated transition metal ion to a matrix immobilized ligand . IMAC has several advantages such as easy preparation, high adsorption capacity, and stability , and therefore it has attracted much attention in the past years. The immobilized metal ions are found to be crucial in IMAC since different metal ions have diverse‐binding affinities to the targets.…”

Efficient fabrication of high‐capacity immobilized metal ion affinity chromatographic media: The role of the dextran‐grafting process and its manipulation

“…Purification procedures based on the use of affinity tags are well developed to enable different proteins to be purified using some common methods. Among those affinity tags, short histidine stretches or His‐tags typically fused to the C‐ or N‐terminus, enables the purification of these recombinant proteins from the crude extracts of the host cells by a single step, i.e., immobilized metal ion affinity chromatography . More than 50% of the recombinant proteins expressed in prokaryotic host cell are purified by immobilized metal ion affinity chromatography and its future outlook is very bright indeed for many purification processes such as interferon, nucleoside diphosphatase, trypsin inhibitors, glycogen phosphorylase and lactate dehydrogenase (LDH) .…”