Chemical and Physical Characterization of Therapeutic Proteins in Solution and Amorphous Solids

The chemical and physical stability of proteins in solution and solids was addressed in this dissertation. Protein-excipient interactions in lyophilized solids were studied by hydrogen/deuterium exchange with mass spectrometry (chapter 3) while glycosylation quanitification (chapter 4) and deamidation (chapter 5) was characterized in antibodies in solution. LC/ESI-MS was the method of choice for all studies. Hydrogen/deuterium exchange study showed that the method can be used to obtain region specific information about protein-excipient interactions in solids. It was demonstrated that exchange protection did not occur uniformly along the backbone of the protein and was dependant on excipient type and protein structure. The glycosylation quanitification study demonstrated that the Fc/2 (limited proteolysis followed by reduction) method was relatively quick and accurate and showed comparable values to the standard sugar release assay. Antibody deamidation study demonstrated that secondary structure played a pivotal role in determination of the deamidation products in antibodies

Comparison of LC and LC/MS Methods for Quantifying N-Glycosylation in Recombinant IgGs

High-performance liquid chromatography (LC) and liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-MS) methods with various sample preparation schemes were compared for their ability to identify and quantify glycoforms in two different production lots of a recombinant monoclonal IgG1 antibody. IgG1s contain a conserved N-glycosylation site in the fragment crystallizable (Fc) subunit. Six methods were compared: (1) LC/ESI-MS analysis of intact IgG, (2) LC/ESI-MS analysis of the Fc fragment produced by limited proteolysis with Lys-C, (3) LC/ESI-MS analysis of the IgG heavy chain produced by reduction, (4) LC/ESI-MS analysis of Fc/2 fragment produced by limited proteolysis and reduction, (5) LC/MS analysis of the glycosylated tryptic fragment (293EEQYNSTYR301) using extracted ion chromatograms, and (6) normal phase HPLC analysis of N-glycans cleaved from the IgG using PNGase F. The results suggest that MS quantitation based on the analysis of Fc/2 (4) is accurate and gives results that are comparable to normal phase HPLC analysis of N-glycans (6)

Comprehensive analysis of temporal alterations in cellular proteome of bacillus subtilis under curcumin treatment

Curcumin is a natural dietary compound with antimicrobial activity against various gram positive and negative bacteria. This study aims to investigate the proteome level alterations in Bacillus subtilis due to curcumin treatment and identification of its molecular/cellular targets to understand the mechanism of action. We have performed a comprehensive proteomic analysis of B. subtilis AH75 strain at different time intervals of curcumin treatment (20, 60 and 120 min after the drug exposure, three replicates) to compare the protein expression profiles using two complementary quantitative proteomic techniques, 2D-DIGE and iTRAQ. To the best of our knowledge, this is the first comprehensive longitudinal investigation describing the effect of curcumin treatment on B. subtilis proteome. The proteomics analysis revealed several interesting targets such UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1, putative septation protein SpoVG and ATP-dependent Clp protease proteolytic subunit. Further, in silico pathway analysis using DAVID and KOBAS has revealed modulation of pathways related to the fatty acid metabolism and cell wall synthesis, which are crucial for cell viability. Our findings revealed that curcumin treatment lead to inhibition of the cell wall and fatty acid synthesis in addition to differential expression of many crucial proteins involved in modulation of bacterial metabolism. Findings obtained from proteomics analysis were further validated using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) assay for respiratory activity, resazurin assay for metabolic activity and membrane integrity assay by potassium and inorganic phosphate leakage measurement. The gene expression analysis of selected cell wall biosynthesis enzymes has strengthened the proteomics findings and indicated the major effect of curcumin on cell division

Additional file 3: of Variation in the ribosome interacting loop of the Sec61α from Giardia lamblia

Tertiary structure of GlSec61α obtained by homology modeling based on 2WWB (a & b) and 3J7Q (c & d), followed by molecular dynamic simulation for 30 ns, with (b & d) or without (a & c) docked RNA. Other details are same as those described in Fig. 1b. The corresponding RMSD graphs are shown below each structure. The templates used for modeling (2WWB and 3J7Q) had several other protein/peptide chains and RNA fragments, all of which exerted a constraint on the protein conformation. Thus, although the homology modeled structures resembled that conformation of the template, the above-mentioned constraints were absent in our simulations as only a fragment of the RNA was used for docking. As a result, during simulation, the structures relaxed in the first few nanoseconds and this resulted in a rise of the RMSD before subsequent stabilization. Hence observed increase of RMSD does not reflect any destabilization. (PPTX 1691 kb

La Petite presse : journal quotidien... / [rédacteur en chef : Balathier Bragelonne]

17 novembre 18791879/11/17 (A13,N4941)

Comprehensive analysis of temporal alterations in cellular proteome of Bacillus subtilis under curcumin treatment.

Curcumin is a natural dietary compound with antimicrobial activity against various gram positive and negative bacteria. This study aims to investigate the proteome level alterations in Bacillus subtilis due to curcumin treatment and identification of its molecular/cellular targets to understand the mechanism of action. We have performed a comprehensive proteomic analysis of B. subtilis AH75 strain at different time intervals of curcumin treatment (20, 60 and 120 min after the drug exposure, three replicates) to compare the protein expression profiles using two complementary quantitative proteomic techniques, 2D-DIGE and iTRAQ. To the best of our knowledge, this is the first comprehensive longitudinal investigation describing the effect of curcumin treatment on B. subtilis proteome. The proteomics analysis revealed several interesting targets such UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1, putative septation protein SpoVG and ATP-dependent Clp protease proteolytic subunit. Further, in silico pathway analysis using DAVID and KOBAS has revealed modulation of pathways related to the fatty acid metabolism and cell wall synthesis, which are crucial for cell viability. Our findings revealed that curcumin treatment lead to inhibition of the cell wall and fatty acid synthesis in addition to differential expression of many crucial proteins involved in modulation of bacterial metabolism. Findings obtained from proteomics analysis were further validated using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) assay for respiratory activity, resazurin assay for metabolic activity and membrane integrity assay by potassium and inorganic phosphate leakage measurement. The gene expression analysis of selected cell wall biosynthesis enzymes has strengthened the proteomics findings and indicated the major effect of curcumin on cell division

Schematic representation of experimental strategy for temporal proteome analysis of <i>B</i>. <i>subtilis</i> under curcumin treatment by iTRAQ-based quantitative proteomics.

<p><b>(A)</b> Samples processed in triplicate were pooled from control, 20, 60 and 120 min curcumin treated cultures and labelled with iTRAQ reagent 114, 115, 116 and 117, respectively. The labelled peptides were fractionated in OFFGEL fractionators using high resolution (24 cm; 3–10 pH) IPG strips and each fraction was desalted using C18 tips. Desalted fractions were subjected to LTQ-Orbitrap Velos mass spectrometer for protein identification and quantitation. <b>(B)</b> Representative MS/MS spectrum of a few selected differentially expressed proteins identified after curcumin treatment. UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1 (MurAA), ATP-dependent zinc metalloprotease FtsH, Septum site-determining protein (DivIVA), and 3-oxoacyl-[acyl-carrier-protein] synthase 3 protein 1 (FabHB). Inset showing the iTRAQ reporter ion intensities for representative peptides in control and curcumin treated samples. <b>(C)</b> S-curve analysis exhibiting distribution of the differentially expressed proteins in <i>B</i>. <i>subtilis</i> after 20, 60 and 120 min of curcumin treatment identified using Q-TOF (average of three triplicate runs). <b>(D)</b> S-curve analysis exhibiting distribution of the differentially expressed proteins in 20, 60 and 120 min curcumin treated <i>B</i>. <i>subtilis</i> identified using LTQ-orbitrap.</p

List of differentially expressed proteins in <i>B</i>. <i>subtilis</i> due to curcumin treatment obtained from DIGE analysis and its comparison with iTRAQ analysis^$.

<p> This is a partial list having selected candidates with >1.5 fold change and complete list is provided in S2 Table

#: Proteins unique in DIGE, Bold: Same trend in both DIGE and iTRAQ (Orbitrap data);

* or NS: No significant change in iTRAQ in Orbitrap data (less than 1.2 fold up and down);

NI- Not identified in iTRAQ analysis.

List of differentially expressed proteins in B. subtilis due to curcumin treatment obtained from DIGE analysis and its comparison with iTRAQ analysis</a>.</p</sup>