Protein Adsorption onto Modified Porous Silica by Single and Binary Human Serum Protein Solutions

Typical porous silica (SBA-15) has been modified with pore expander agent (1,3,5-trimethylbenzene) and fluoride-species to diminish the length of the channels to obtain materials with different textural properties, varying the Si/Zr molar ratio between 20 and 5. These porous materials were characterized by X-ray Diffraction (XRD), N2 adsorption/desorption isotherms at −196 °C and X-ray Photoelectron Spectroscopy (XPS), obtaining adsorbent with a surface area between 420–337 m2 g−1 and an average pore diameter with a maximum between 20–25 nm. These materials were studied in the adsorption of human blood serum proteins (human serum albumin—HSA and immunoglobulin G—IgG). Generally, the incorporation of small proportions was favorable for proteins adsorption. The adsorption data revealed that the maximum adsorption capacity was reached close to the pI. The batch purification experiments in binary human serum solutions showed that Si sample has considerable adsorption for IgG while HSA adsorption is relatively low, so it is possible its separation.

Analysis of human serum protein extracts by matrix assisted laser desorption ionization and electrospray ionization quadrupole-quadrupole time-of-flight tandem mass spectrometry

Serum is a complex heterogeneous matrix contianing endogenous proteins with potential diagnostic value. No previously reported methods have allowed the detection of all its constituent proteins from a single sample. In the work described in this thesis, normal human serum was separated by reversed-phase or dye affinity chromatography, ultrafiltration and by extraction under various pH and salt concentrations in aqueous and organic solutions. The serum extracts were analyzed by SDS-PAGE, MALDI and LC-ESI-Qq-TOF MS/MS without prior enzymatic digestion. Peptides observed as multiple isotopic peaks in MS mode were correlated with known serum proteins by ProteinPilot and X!Tandem analysis of their fragmentation spectra using the no enzyme [XX] condition. Fragment ion masses within 0.04 Da of those predicted yielded high confidence identifications from both algorithms. Angiotensin 1 and [G1u1] fibrinopeptide B were clearly detected in serum spiked with as little as 250 and 100 femtomoles respectively. Post-translational modifications of serum peptides were identified using ProteinPilot.

Analysis of human serum protein extracts by matrix assisted laser desorption ionization and electrospray ionization quadrupole-quadrupole time-of-flight tandem mass spectrometry

Serum is a complex heterogeneous matrix contianing endogenous proteins with potential diagnostic value. No previously reported methods have allowed the detection of all its constituent proteins from a single sample. In the work described in this thesis, normal human serum was separated by reversed-phase or dye affinity chromatography, ultrafiltration and by extraction under various pH and salt concentrations in aqueous and organic solutions. The serum extracts were analyzed by SDS-PAGE, MALDI and LC-ESI-Qq-TOF MS/MS without prior enzymatic digestion. Peptides observed as multiple isotopic peaks in MS mode were correlated with known serum proteins by ProteinPilot and X!Tandem analysis of their fragmentation spectra using the no enzyme [XX] condition. Fragment ion masses within 0.04 Da of those predicted yielded high confidence identifications from both algorithms. Angiotensin 1 and [G1u1] fibrinopeptide B were clearly detected in serum spiked with as little as 250 and 100 femtomoles respectively. Post-translational modifications of serum peptides were identified using ProteinPilot.

A study of the binding between radicicol and four proteins by means of spectroscopy and molecular docking

The interactions between radicicol and four proteins (catalase, trypsin, pepsin, and human serum protein) are investigated by spectroscopic techniques and molecular docking. A static quenching process is confirmed. The binding constant value between radicicol and human serum protein is the largest among the four proteins. Results reveal changes in the micro-environment of the protein by the addition of radicicol. It is found that radicicol shows an inhibitory effect on the activity of proteins (catalase, trypsin, and pepsin). Molecular docking results are consistent with the thermodynamic experimental results. This work provides clues to the elucidation of the mechanisms of the interactions between radicicol and proteins.