Precursor carboxy-silica for functionalization with interactive ligands. IV. Carbodiimide assisted preparation of immobilized antibody stationary phases for high performance immuno-affinity chromatography of human serum.

In this Part IV of the article series dealing with the functionalization of the precursor carboxy silica with various chromatographic ligands, immuno affinity (IA) columns were prepared with immobilized anti-apolipoprotein B (AAP B) and anti-haptoglobin (AHP) antibodies for use in immuno affinity chromatography (IAC) in the aim of selectivily capturing their corresponding antigens from healthy and cancer human sera. Diseased human serum with adenocarcinoma cancer was selected as a typical diseased biological fluid. Besides preferentially capturing their corresponding antigens, the AAP B column captured from disease-free and cancer sera, 34 proteins and 33 proteins, respectively, while the AHP column enriched 38 and 47 proteins, respectively. This nonspecific binding can be attributed to the many proteins human serum have, which could mediate protein-protein interactions thus leading to the so-called "sponge effect". This kind of behavior can be exploited positively in the determination of differentially expressed proteins (DEPs) for diseased serum with respect to healthy serum and in turn allow the identification of an array of potential biomarkers for cancer. In fact, For AHP column, 13 upregulated and 22 downregulated proteins were identified whereas for AAP B column the numbers were 23 and 10, respectively. The DEPs identified with both columns match those reported in the literature for other types of cancers. The different expression of proteins in each IAC column can be related to the variability of protein-protein interactions. In addition, an array of a few biomarkers is more indicative of a certain disease than a single biomarker.

Precursor carboxy-silica for functionalization with interactive ligands. III. Carbodiimide assisted preparation of immobilized lectin stationary phases for high performance lectin affinity chromatography of sub-glycoproteomics from cancer and disease free human sera.

In this study, a precursor carboxy-silica support was demonstrated in the immobilization of two different lectins, namely concanavalin A (Con A) and wheat germ agglutinin (WGA) for use in high performance lectin affinity chromatography (LAC) for the selective capturing and enrichment of glycoproteins from healthy/disease free and cancer human sera. The lectin columns thus obtained (i.e., Con A- and WGA-columns) showed no nonspecific interactions toward some chosen standard glycoproteins and non-glycoproteins. Both columns were shown in sub-glycoproteomics enrichment from human sera including disease free and adenocarcinoma cancer sera. The collected fractions were subjected to LC-MS/MS for identification of the captured glycoproteins, whereby the total number of identified proteins using Con A column from disease-free and cancer sera were 164 and 188, respectively while 133 and 103 proteins were identified in the fractions captured by the WGA column from disease-free and cancer sera samples, respectively. Differentially expressed proteins (DEPs) between the disease free and cancer sera in both the Con A and WGA column fractions were identified via the plot of the abundance vs. the protein ratio whereby the binary logarithm of average intensities of cancer and disease free sera were plotted against the binary logarithm of cancer/disease free sera ratios. The proteins that exhibit log 2 (cancer/healthy) ratio values greater than +2 and less than -2 in both categories are considered as DEPs. Furthermore, for visualization of the data arrangement, Q-Q scatterplot were also used whereby the binary logarithm of cancer serum was plotted against the binary logarithm of disease-free serum for both Con A and WGA. For Con A column, 28 up-regulated and 10 down regulated proteins were identified with a total of 38 DEPs while only two being non-glycoproteins. Furthermore, the up-regulated, and down regulated proteins recorded for WGA column are 14 and 6, respectively, totaling 20 proteins including 3 non-glycoproteins. Some of the non-specific binding to lectin are most likely due to protein-protein interactions.

Precursor carboxy-silica for functionalization with interactive ligands. I. Carbodiimide-assisted preparation of silica-bonded stationary phases with octadecyl, naphthyl, and anthracenyl ligands: Comparison of their selectivity and retentivity.

A precursor carboxy-silica support was introduced for grafting retentive ligands for use in high-performance liquid chromatography. This support was prepared by sequentially reacting 5 µm silica particles with vinyltrimethoxysilane and then thioglycolic acid. The carboxy-silica thus obtained was subsequently functionalized with octadecylamine, 2-naphthylamine, or 2-aminoanthracene by on-column reactions via a carbodiimide conjugation reaction. The carbodiimide with its zero-length carboxyl-to-amine coupling ability works by activating the surface carboxyl groups of the precursor support for direct reaction with the primary amines of octadecylamine, 2-naphthylamine, or 2-aminoanthracene via amide bond formation. These reactions series, which are applied for the first time in high-performance liquid chromatography column fabrication, yielded the octadecyl-, naphthyl-, and anthracenyl-silica columns. The three columns were evaluated for their reversed-phase chromatography retention properties with alkylbenzenes, alkylphenyl ketones, nitroalkanes, benzene and toluene derivatives, polyaromatic hydrocarbons, and nitro-substituted amino acids. The naphthyl- and anthracenyl-silica exhibited a good selectivity and efficiency toward most of the aromatic analytes when compared to the octadecyl-silica. Nitro-substituted amino acids containing electron withdrawing groups showed greater selectivity than other analytes on the aromatic-based columns than the C18 column. This is because of the ability of the π electron system of the analyte to accept electrons from the aromatic-based stationary phase (a Lewis base).