Nanoprobe‐based immobilized metal affinity chromatography for sensitive and complementary enrichment of multiply phosphorylated peptides

Abstract

Magnetic nanoparticles (MNP, <100 nm) have rapidly evolved as sensitive affinity probes for phosphopeptide enrichment. By taking advantage of the easy magnetic separation and flexible surface modification of the MNP, we developed a surface-blocked, nanoprobe-based immobilized metal ion affinity chromatography (NB-IMAC) method for the enhanced purification of multiply phosphorylated peptides. The NB-IMAC method allowed rapid and specific one-step enrichment by blocking the surface of titanium (IV) ion-charged nitrilotriacetic acid-conjugated MNP (Ti⁴-NTA-PEG@MNP) with low molecular weight polyethylene glycol. The MNP demonstrated highly sensitive and unbiased extraction of both mono- and multiply phosphorylated peptides from diluted β-casein (2 × 10⁻¹⁰ M). Without chemical derivation or fractionation, 1283 phosphopeptides were identified from 400 μg of Raji B cells with 80% purification specificity. We also showed the first systematic comparison on the particle size effect between nano-sclae IMAC and micro-scale IMAC. Inductively coupled plasma-mass spectrometry (ICP-MS) analysis revealed that MNP had a 4.6-fold higher capacity for metal ions per unit weight than did the magnetic micro-sized particle (MMP, 2-10 μm), resulting in the identification of more phosphopeptides as well as a higher percentage of multiply phosphorylated peptides (31%) at the proteome scale. Furthermore, NB-IMAC complements chromatography-based IMAC and TiO₂ methods because <13% of mono- and 12% of multiply phosphorylated peptide identifications overlapped among the 2700 phosphopeptides identified by the three methods. Notably, the number of multiply phosphorylated peptides was enriched twofold and threefold by NB-IMAC relative to micro-scale IMAC and TiO₂, respectively. NB-IMAC is an innovative material for increasing the identification coverage in phosphoproteomics.

Recent advances in phosphoproteomics and application to neurological diseases

“…The cations are immobilized on metal ion chelation groups, such as nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), or phosphate polymer, which are on the surface of agarose-, silica-, or magnetic-based solid supports. 39–41 MOAC, similar to IMAC, uses the affinity of metals in metal oxide particles (TiO 2 , ZrO 2 , Fe 3 O 4 , etc.) to retain the anionic phosphoryl groups on the matrixes.…”

On-target and nanoparticle-facilitated selective enrichment of peptides and proteins for analysis by MALDI-MS

“…NPs employed for selective enrichment of phosphopeptides by IEX have included unmodified Fe 3 O 4 mNPs and Fe 3 O 4 mNPs modified with PEI . NPs for selective enrichment of phosphopeptides by IMAC have been prepared by immobilization of N ′, N ″‐ bis (carboxymethyl)‐ l ‐lysine (NTA) on Fe 3 O 4 /SiO 2 core/shell mNPs modified with either 3‐(2‐aminoethyl)aminopropyltrimethoxysilane or 3‐aminopropyltriethoxysilane, respectively . Magnetic NPs for MOA have been prepared almost exclusively from Fe 3 O 4 /SiO 2 core/shell (silica encapsulated Fe 3 O 4 ) mNPs and include (i) Fe 3 O 4 /TiO 2 core/shell mNPs , (ii) Fe 3 O 4 /carbon/TiO 2 (carbon encapsulated Fe 3 O 4 ) core/shell mNPs , (iii) Fe 3 O 4 /SiO 2 /ZrO 2 core/shell mNPs , (iv) Fe 3 O 4 /SiO 2 core/shell mNPs coated with aluminum oxide , (v) Fe 3 O 4 /SiO 2 core/shell mNPs coated with zirconium phosphonate , and (vi) Fe 3 O 4 /SiO 2 core/shell mNPs‐coated tantalum oxide .…”

A poly(ethylene glycol)-brush decorated magnetic polymer for highly specific enrichment of phosphopeptides

“…23 The double phosphopeptides accounted for about 10% of the total identified phosphopeptides and the rest of the phosphopeptides identified by using the three materials were in highly phosphorylation. Although the distributions of phosphopeptides according to the number of phosphorylation sites are similar for the three materials, it was extensively reported that different materials have complementary performance towards phosphopeptides enrichment, 14,23,37 thus, it is of great interest to investigate the overlap of the identified phosphopeptides by the three materials. As predicted in Fig.…”

“…Despite the advance of these mentioned IMAC materials, some difficulties are still encountered. Due to the different surface properties and metal ligand complexes of IMAC materials, many acidic nonphosphopeptides are strongly bound to the magnetic supports 37,39 and the binding capacity of phosphopeptides still remains limited, which would result in low sensitivity and specificity. On the other hand, these reports highlighted that suitable surface modification of the magnetic supports is of great research demand to enhance the efficiency of phosphopeptide capture.…”