Newly Developed Poly(Allyl Glycidyl Ether/Divinyl Benzene) Polymer for Phosphopeptides Enrichment and Desalting of Biofluids

Abstract

The polymeric materials have contributed significantly in the area of bioanalytical science. The functionalization of polymeric backbone after its development brings unique selectivity towards the target biomolecules. In present work, the functionalities of choice have been introduced through the ring-opening of allyl glycidyl ether. The utility of polymer is widened through derivatizations to immobilized metal ion affinity chromatographic (IMAC) material for the phosphopeptides enrichment and Reversed Phase (C-18) for the desalting prior to MALDI-MS analysis. The polymer-IMAC in addition to Fe(3+) is also immobilized with lanthanide ions like La(3+), Eu(3+), and Er(3+). The amount of Fe(3+) immobilized is determined as 0.7928 mg/g. Spherical morphology with narrow particle size dispersion is revealed by scanning electron microscopy (SEM). The surface area, pore volume and size distribution is determined by nitrogen adsorption porosimetery. The elemental composition and purity level is confirmed by energy dispersive X-ray spectroscopy (EDX) data. The derivatization to IMAC and RP is evaluated by Fourier transform infrared (FT-IR) spectroscopy. The polymer enables the efficient phosphopeptide enrichment to equal degree from casein variants, non-fat milk, egg yolk, human serum, and HeLa cell extract. The identification of phosphorylation sites can lead to the phosphorylation pathways to understand the post-translational modifications. The identification with their sequence coverage is made using Mascot and Phosphosite Plus. It is sensitive to enrich the phosphopeptides down to 2 femtomoles with very high selectivity of 1:2000 with BSA background. These attributes are linked to the higher surface area (173.1554 m(2)/g) of the designed polymer. The non-specific bindings, particularly the Fe(3+) linked acidic residues are also avoided. Four characteristic phosphopeptides (fibrinopeptide A and their hydrolytic products) from fibrinogen α-chain are identified from the human serum after the enrichment, which have link to the hepatocellular carcinoma (HCC). The proportions of fibrinogen and their phosphorylation products enriched by poly(AGE/DVB)-IMAC open new horizons in the biomarker discovery.

Preparation of on-plate immobilized metal ion affinity chromatography platform via dopamine chemistry for highly selective isolation of phosphopeptides with matrix assisted laser desorption/ionization mass spectrometry analysis

“…In order to demonstrate the feasibility of our method to complex samples, we carried out the onplate enrichment of phosphopeptide from human serum, which is expected containing four endogenous phosphopeptides with molecular masses of 1389, 1460, 1545 and 1616 Da coming from fibrinogen α-chain [28][29][30][31]. Because a large amount of nonphosphopeptides, abundant proteins and inorganic salts existing in the serum increases the complexity of condition largely, the detection of endogenous phosphopeptides contained in human serum will be suppressed, which results in almost no peptides found in the spectrum before enrichment (Fig.…”

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

“…S6). The importance and cancer relevance of FPA phosphopeptides has been previously discussed [36,37]. Another phosphopeptide at Ser 609 derived from fibrinogen alpha chain is identified at m/z 1952 (MADEAGSEADHEGTHSTK, 603-620, Supporting Information Fig.…”

“…Four phosphopeptides above 3500 Da are shown as inset. Non-phosphopeptides (NPP) at 1267(YLGYLEQLLR, ␣ S1 , 106-115) and 1758 (HQGLPQEVLNENLLR, ␣ S2 , [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] are difficult to remove. They bind with Gd 2 O 3 at applied pH because of the presence of glutamic acid (E).…”

Preparation of Ti4+-immobilized modified silica capillary trapping column for on-line selective enrichment of phosphopeptides

“…Ti 4 þ -IMAC is the most commonly used to isolate phosphopeptides for its highly specific interaction with the phosphate group on phosphopeptides [12]. The metal ions are immobilized on different matrix (polymer beads [23], porous materials [20], nanocomposites [21], graphene [24] or magnetic core-shell architectures [15,25]), using various bridging molecules, such as acidic chelating ligands of phosphate groups, iminodiacetic acid (IDA) [17] or nitrilotriacetic acid (NTA) [16]. Recently, hydrophilic polydopamine (PDA) has successfully been developed [26][27][28] as a novel chelating ligand for indirectly immobilize metal ions on various substrates modified for enrichment of phosphopeptides [15,29,30].…”