New cellulose–silica composite IMAC/C18 for the selective enrichment of phosphorylated molecules and the improved recovery of hydrophilic species

Abstract

Cellulose and silica are the traditional quality sorbents with range of reported applications, particularly for the hydrophilic biomolecules. Considering those characteristics, cellulose and silica are brought into a composite form to take the benefits on a single platform. Choice of cellulose and silica is made to obtain complete hydrophilic composite, followed by its derivatization for the selective enrichment of phosphopeptides and as desalting material prior to the mass spectrometric analysis. The composite is characterized with FT-IR, EDX and SEM. As immobilized metal ion affinity chromatographic (IMAC) material, the cellulose-silica enriches phosphopeptides from complex mixture in which β-casein is spiked in de-phosphorylated HeLa cell extract. The inherent hydrophilic nature of composite gives higher selectivity up to 2000 times complexity level and sensitivity down to 1 femtomole for phosphopeptides. Other than the immobilized metal ions onto the IMAC composite, the role of base materials in the composite, i.e. cellulose and silica is also tested in the enrichment which helps thereof in optimizing the best sample preparation protocol. With the introduction of successive elution conditions, phospholipids and phosphopeptides are enriched and identified from egg yolk digest using single batch extraction method. The phosphopeptides are analyzed with MALDI-MS whereas newly designed gold nanoparticles and carbon based materials are used to analyze low molecular weight phospholipids through LDI-MS.Phosphopeptides are also enriched from complex serum digest and discussed in relevance to the prostate cancer. As RP material, it provides combination of hydrophilic composite and hydrophobic C18 chain. Efficient desalting of higher salt concentrations, improved recovery of hydrophilic peptides and the high sequence coverage in comparison to Development of enrichment strategy relies on the reproducibility of material and method independent of time and space. Figure S9 is representative of the reproducible results using serum digest. The intensity difference can be attributed to sample preparation, cocrystallization procedure and instrumental factors as laser power and laser intensity. For cellulose-silica IMAC-Sm 3+ , statistical analysis is applied on the reproducibility study.The highest standard deviation (SD) is found 0.379 and the lowest 0.150 (Table S2). Cellulose-Silica-IMAC-Sm 3+ for Phosphoproteins/PhosphopeptidesThe developed enrichment strategy for cellulose-silica IMAC-Sm 3+ is tested on protein mixture containing phosphoproteins like α-, and β-casein with non-phosphoproteins as lysozyme, myoglobin, cytochrome c and BSA. The non-phosphoproteins are selected considering the range from hydrophobic to hydrophilic. Phosphoproteins α-, and β-casein having mass in the range of 24 kDa to 26 kDa are successfully enriched from the protein mixture with no trace of non-phosphoproteins ( Figure S10). Both α-casein variants, αS1 and αS2 including the β-casein, are detected.The complexity level is further enhanced and a ...

Enrichment of HDL proteome and phospholipidome from human serum via IMAC/MOAC affinity

“…Cellulose–SiO 2 –IMAC–Sm 3+ (Najam‐ul‐Haq, Jabeen, Shafiq, Sajid, & Saba, ), SiO 2 –La 2 O 3 composite (Jabeen et al, ) and Fe 3 O 4 @Sm 2 O 3 core–shell nanocomposites (Jabeen et al, ) were synthesized using the reported protocols.…”

In-Tip Lanthanum Oxide Monolith for the Enrichment of Phosphorylated Biomolecules

“…The tips were washed with 80% ACN in 0.2% TFA followed by DHB buffer (110 mg of DHB in 0.5% ACN) and deionized water. The elution was performed using 1.5% aqueous ammonium hydroxide solution for phosphopeptides and 1% ammonium hydroxide in chloroform for phospholipids . The eluted fractions were subjected to MALDI-MS analysis.…”

“…The elution was performed using 1.5% aqueous ammonium hydroxide solution for phosphopeptides and 1% ammonium hydroxide in chloroform for phospholipids. 12 The eluted fractions were subjected to MALDI-MS analysis.…”

“…Reported strategies include precipitation, chemical derivatization, hydrophilic interaction chromatography (HILIC), and ion exchange chromatography . Common techniques are immobilized metal ion affinity chromatography (IMAC; Fe 3+ , Ti 4+ , Zr 4+ , Sm 3+ , etc.) and metal oxide affinity chromatography (MOAC; Fe 3 O 4 , TiO 2 , ZrO 2 , CeO 2 , La 2 O 3 , etc.)…”

Liquid‐phase based separation systems for depletion, prefractionation, and enrichment of proteins in biological fluids and matrices for in‐depth proteomics analysis—An update covering the period 2014–2016

“…A new cellulose–silica composite IMAC/C18 sorbent that was derivatized for the selective enrichment of phosphopeptides has been reported . The IMAC/C18 sorbent was utilized to enrich phosphopeptides from a complex sample in which β‐casein is spiked in dephosphorylated HeLa cell extract.…”