Development of Affinity Chromatographic Tools for Evaluation of Proteins/Peptides from Biological Samples

Affinity chromatography has been a method of choice in last decade due to its higher selectivity and versatility for extraction and purification of target analytes. Enrichment of target proteins and peptides through affinity materials is one of the primary focused area of separation science. Field of proteomics has coupled the affinity chromatography to modern mass spectrometry techniques. A large variety of affinity materials for the selective capturing of phosphorylated biomolecules have been developed with varying efficiency. Carbon based nanomaterials have been used as affinity sorbents in phosphoproteomics with different modifications. Diamond nanopowder from carbon family is known for its biocompatibility and inertness towards biomolecules. It is therefore commonly used in the field of separation science and life science. In this work, the diamond nanopowder is modified to design affinity sorbent for phosphopeptides enrichment. Diamond nanopowder is functionalized as IMAC (Immobilized Metal Affinity Chromatography) material loaded with Fe3+ and La3+ ions and MOAC (Metal Oxide Affinity Chromatography) material in the form of composites with lanthanum oxide and samarium oxide. The characterization is carried out by Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM) and energy dispersive X-ray (EDX) spectroscopy. Diamond-IMAC material is synthesized for phosphopeptides enrichment and diamond reversed phase (C-18) media for desalting of smaples and profiling of human serum. Optimization of phosphopeptides enrichment to diamond-IMAC material loaded with Fe3+ and La3+ ions is carried out by standard protein digest (β-casein) followed by the real biological samples. Material shows excellent selectivity for phosphopeptides from complex samples such as non-fat milk and human serum. Furthermore, diamond-IMAC is also applied to the serum samples from gall bladder carcinoma and it shows excellent specificity towards phosphopeptides. MOAC materials, i.e. diamond-lanthanum oxide and diamond-samarium oxide composites show selectivity upto 1:3000 and 1:1500 (β-casein: BSA ratio) while sensitivity down to 1 fM and 20 fM levels, respectively. Enrichment efficiency is evaluated for non-fat milk as semi-complex sample and 18 phosphopeptides are enriched from the milk digest. Human serum, without any pre-treatment is also applied and nanocomposites capture characteristic xix | P a g e phosphopeptides from human serum. The materials show promise towards identifying the phosphopeptides biomarkers from body fluids. Apart from diamond nanopowder, Titania grafted magnetic sporopollenin is also synthesized by the liquid phase deposition (LPD) technique as an affinity material for phosphopeptides enrichment. The material exhibits good selectivity, sensitivity and capacity for phosphopeptides from standard samples (β-casein and BSA mixture). Enrichment of phosphorylated peptides is carried out from milk digest, diluted human serum and rat brain cell lysate. The material is further applied to tryptic digest of rat brain cell lysate for phosphopeptides enrichment where 2718 phosphopeptides are identified by using LC-MS/MS. TiO2 coated magnetic material shows better enrichment efficiency in comparison to the commercial TiO2. The magnetic property of Titania coated magnetic sporopollenin facilitates the fast and effective isolation of phosphopeptides from complex mixtures through external magnet.Similarly, an anion exchange affinity using polyethylenimine modified chitosan nanomagnets is developed for the enrichment of phosphopeptides from biological samples. A two step strategy is used to synthesize the affinity sorbent. The potential of anion exchange affinity material is evaluated through β-casein digest. Enrichment parameters such as pH of loading (pH:2) and elution buffer (pH:11), loading time (5 minutes), selectivity (β-casein and BSA ratio 1:1000), sensitivity (1 fmol) and reproducibility have been optimized before applying it to real biological fluids such as egg yolk digest, non-fat milk digest and diluted human serum. The material shows excellent enrichment efficiency from complex biological fluids. Simple and low cast synthesis, fast enrichment due to magnetic nature have been some of the attributes of this anion exchange material. All these affinity materials show excellent results in terms of phosphopeptides enrichment and can be applied to cancer protein profiling for the diagnosis and biomarker identification after further optimizations.