Journal: International Journal of Proteomics

Article Title: An Internal Standard-Assisted Synthesis and Degradation Proteomic Approach Reveals the Potential Linkage between VPS4B Depletion and Activation of Fatty Acid β -Oxidation in Breast Cancer Cells

doi: 10.1155/2013/291415

Figure Lengend Snippet: Using internal standard-assisted synthesis and degradation mass spectrometry (iSDMS) to study the roles of EGF on global protein synthesis and degradation. SKBR3_shVPS4B and the parental SKBR3 cells were first labeled with 13 C 6 -arginine (Arg6) and D 4 -lysine (Lys4) medium (labeled in red). After overnight serum starvation, Arg6/Lys4-labeled cells were stimulated with 100 ng/mL EGF in medium supplemented with regular arginine (Arg0) and lysine (Lys0) for 0, 2, 6, 12, and 24 hr (labeled in blue). Protein isolated from SKBR3_shVPS4B cells labeled with 13 C 6 15 N 4 -arginine (Arg10) and 13 C 6 15 N 2 -lysine (Lys8)—internal standard (labeled in green)—was spiked into each sample at a ratio of 1 : 3 (wt/wt). The mixtures were digested by the Filter Aided Sample Preparation (FASP) procedure, followed by strong anion exchange (SAX) peptide fractionation. Peptides were analyzed by online LC-MS/MS using an LTQ-Orbitrap mass spectrometer. The relative abundance of the newly synthesized ( A l ) or preexisting peptides ( A m ) was defined as the ratio of mass spectrometric peak intensities of the unlabeled peptides ( I l ) or Arg6/Lys4-labeled peptides ( I m ) to the intensities of the Arg10/Lys8-labeled peptides ( I h ), respectively.

Article Snippet: To prepare the “heavy-” labeled internal standard, SKBR3_shVPS4B cells were metabolically labeled with 13 C 6 15 N 4 -arginine (Arg10) and 13 C 6 15 N 2 -lysine (Lys8) by culturing in DMEM “heavy” SILAC medium supplemented with 28 mg/L 13 C 6 15 N 4 -arginine (Arg10, purity 97–99%, Cambridge Isotope Laboratories), 72 mg/L 13 C 6 15 N 4 -lysine (Lys8, purity 97–99%, Cambridge Isotope Laboratories), 10% dialyzed FBS, and 1% antibiotic antimycotic solution (Invitrogen).

Techniques: Mass Spectrometry, Labeling, Isolation, Sample Prep, Peptide Fractionation, Liquid Chromatography with Mass Spectroscopy, Synthesized

Journal: Brain research

Article Title: PrP-grafted antibodies bind certain amyloid β-protein aggregates, but do not prevent toxicity

doi: 10.1016/j.brainres.2018.12.038

Figure Lengend Snippet: Primary antibodies and their antigens, dilutions, and source

Article Snippet: 266 , Aβ 13–28 , 3 μg/ml (MSD) , Janssen , Johnson-Wood et al., 1997.

Techniques: Enzyme-linked Immunosorbent Assay