Peptide Code-on-a-Microplate for Protease Activity Analysis via MALDI-TOF Mass Spectrometric Quantitation

A peptide-encoded microplate was proposed for MALDI-TOF mass spectrometric (MS) analysis of protease activity. The peptide codes were designed to contain a coding region and the substrate of protease for enzymatic cleavage, respectively, and an internal standard method was proposed for the MS quantitation of the cleavage products of these peptide codes. Upon the cleavage reaction in the presence of target proteases, the coding regions were released from the microplate, which were directly quantitated by using corresponding peptides with one-amino acid difference as the internal standards. The coding region could be used as the unique “Protease ID” for the identification of corresponding protease, and the amount of the cleavage product was used for protease activity analysis. Using trypsin and chymotrypsin as the model proteases to verify the multiplex protease assay, the designed “Trypsin ID” and “Chymotrypsin ID” occurred at <i>m</i>/<i>z</i> 761.6 and 711.6. The logarithm value of the intensity ratio of “Protease ID” to internal standard was proportional to trypsin and chymotrypsin concentration in a range from 5.0 to 500 and 10 to 500 nM, respectively. The detection limits for trypsin and chymotrypsin were 2.3 and 5.2 nM, respectively. The peptide-encoded microplate showed good selectivity. This proposed method provided a powerful tool for convenient identification and activity analysis of multiplex proteases

Attachment of CSP to hepatocytes.

<p><b>A</b>, Purified GFP-<i>Pf</i>CSP or mutant proteins were incubated with HepG2 cells. Binding of the proteins to the cells was quantified by using flow cytometry to detect GFP fluorescence. Analyzed data are shown on the right. The data are representative of at least four repetitions. <b>B</b>, Purified GFP-<i>Pf</i>CSP or mutant proteins were incubated with HepG2 cells. GFP on the cell surface was detected by live cell imaging. Nuclei were stained with Hoechst 33258 for identification of individual cells. Approximately 300 cells were counted for each sample. The data represent at least three repetitions.</p

Dual Quinone Tagging for MALDI-TOF Mass Spectrometric Quantitation of Cysteine-Containing Peptide

A dual quinone tagging strategy is designed for quantitation of cysteine-containing peptide (CCP) with MALDI-TOF mass spectrometry. The quinone compounds can rapidly and specifically bind to the thiol group of cysteine residues by a Michael addition reaction, which is used to identify both CCP and the number of cysteine residues in CCP through the direct observation of untagged and tagged products. After reduced with DL-dithiothreitol, the intramolecular disulfide bond can also be identified. Using benzoquinone (BQ) and methyl-<i>p</i>-benzoquinone (MBQ) as dual tags and a peptide with an amino acid sequence of SSDQFRPDDCT (C-pep1) as a model target, respectively, the quantitation strategy is performed through the intensity ratio of MBQ-tagged C-pep1 to BQ-tagged C-pep1 as the internal standard. The logarithm value of the intensity ratio is proportional to C-pep1 concentration in a range from 5.0 to 5000 nM. The limit of detection is as low as 2.0 nM. The proposed methodology provides a novel tool for rapid characterization, identification, and quantitation of biomolecules containing thiol reactive sites and has a promising application in the large-scale detection and analysis of cysteine-containing biomolecules

GFP-PfCSP interacts with heparin.

<p><b>A</b>, Domain structures of full-length and recombinant <i>Pf</i>CSP. The termini are indicated. SP, signal peptide; NTD, N-terminal domain; CTD, C-terminal domain; RI, region I; RII+, region II plus; RIII, region III; GPI, GPI anchor sequence. <b>B</b>, Sequence alignment of residues preceding the region I in the NTD of CSP. Residues in <i>Pf</i>CSP are numbered, and residues mutated are highlighted in bold. Mutations that affect heparin binding are colored in red and mutation that does not is colored in cyan. Basic residues in other CSPs that are near region I are colored in orange. The region I is highlighted by a yellow box. Peptides that have been tested for heparin binding are underlined. <b>C</b>,<b>D</b>, Heparin binding of GFP-<i>Pf</i>CSP (<b>C</b>) and GFP alone (<b>D</b>). ~150 μg of purified protein was applied to the heparin column, and samples were analyzed by SDS-PAGE, followed by Coomassie staining. Domain structure of the protein used is shown on the left. I, input; FT, flow-through. <b>E-N</b>, as in <b>C</b>, but with GFP-tagged CSP mutants. Arrowhead indicates a contaminant. All data were confirmed by at least three independent experiments using three independently purified batches of proteins. Data shown are from a representative experiment.</p

Homotypic interactions between CSP termini.

<p><b>A</b>, <b>B</b>, Purified GFP-<i>Pf</i>CSPΔC or GFP alone was incubated with HA-αTSR. Immunoprecipitation (IP) was performed using anti-GFP (<b>A</b>) or anti-HA (<b>B</b>) antibodies. The samples were analyzed by SDS-PAGE and immunoblotting (IB) with anti-HA or anti-GFP antibodies. <b>C</b>, as in <b>A</b>, but with GFP-<i>Pf</i>CSP instead of GFP. <b>D</b>, as in <b>B</b>, but with GFP-<i>Pf</i>CSP instead of GFP.</p

Total flavonoids from Ampelopsis megalophylla suppress proliferation of vascular smooth muscle cells in vivo and in vitro

<div><p>ABSTRACT Various benefits of flavonoids for ameliorating cardiovascular diseases have been demonstrated. However, the lowering effects on blood pressure caused by antiproliferative potentials of flavonoids in vascular smooth muscle cells are rare. In this study, the antihypertensive effects of total flavonoids from Ampelopsis megalophylla were investigated. The dynamic pressure values and the rate of media thickness versus lumen diameter were measured by the tail-cuff system and H&E staining in vivo, respectively. The mRNA expressions of ACE, Ang II, eNOS, c-Myc, cyclin D1 and p27Kip1 in thoracic aorta or A7r5 cells were measured by qPCR, respectively. The protein expressions of c-Myc, Cyclin D1, p27Kip1 and β-catenin in tissues or A7r5 cells were measured by Western blot assay. Total flavonoids of A. megalophylla (TFAM) reduced the expressions of ACE and Ang II, and elevated the content of eNOS in thoracic aorta cells of SHRs. Furthermore, TFAM decreased the mRNA and protein expressions of c-Myc and cyclin D1 by repressing the Wnt/β-catenin-mediated TCF/LEF transcriptional activation both in vivo and in vitro, which is synergetic with the up-regulation of p27Kip1 expression. Our study provided evidence for developing flavonoids from A. megalophylla as herbal supplements to prevent against cardiovascular diseases by suppressing vascular remodeling.</p></div

Antiproliferative effect of urolithin A, the ellagic acid-derived colonic metabolite, on hepatocellular carcinoma HepG2.2.15 cells by targeting Lin28a/let-7a axis

<div><p>An abnormality in the Lin28/let-7a axis is relevant to the progression of hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC), which could be a novel therapeutic target for this malignant tumor. The present study aimed to investigate the antiproliferative and anti-invasive effects of urolithin A in a stable full-length HBV gene integrated cell line HepG2.2.15 using CCK-8 and transwell assays. The RNA and protein expressions of targets were assessed by quantitative PCR and western blot, respectively. Results revealed that urolithin A induced cytotoxicity in HepG2.2.15 cells, which was accompanied by the cleavage of caspase-3 protein and down-regulation of Bcl-2/Bax ratio. Moreover, urolithin A suppressed the protein expressions of Sp-1, Lin28a, and Zcchc11, and elevated the expression of microRNA let-7a. Importantly, urolithin A also regulated the Lin28a/let-7a axis in transient HBx-transfected HCC HepG2 cells. Furthermore, urolithin A decelerated the HepG2.2.15 cell invasion, which was involved in suppressing the let-7a downstream factors HMGA2 and K-ras. These findings indicated that urolithin A exerted the antiproliferative effect by regulating the Lin28a/let-7a axis and may be a potential supplement for HBV-infected HCC therapy.</p></div

SLAWD expression during the different developmental stages of <i>S. litura</i>.

<p>The different letters above the columns indicate significant differences in <i>AWD</i> gene expression during <i>S. litura</i> development (<i>P</i><0.05).</p

Thermoresponsive Arrays Patterned via Photoclick Chemistry: Smart MALDI Plate for Protein Digest Enrichment, Desalting, and Direct MS Analysis

Sample desalting and concentration are crucial steps before matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis. Current sample pretreatment approaches require tedious fabrication and operation procedures, which are unamenable to high-throughput analysis and also result in sample loss. Here, we report the development of a smart MALDI substrate for on-plate desalting, enrichment, and direct MS analysis of protein digests based on thermoresponsive, hydrophilic/hydrophobic transition of surface-grafted poly­(<i>N</i>-isopropylacrylamide) (PNIPAM) microarrays. Superhydrophilic 1-thioglycerol microwells are first constructed on alkyne–silane-functionalized rough indium tin oxide substrates based on two sequential thiol-yne photoclick reactions, whereas the surrounding regions are modified with hydrophobic 1<i>H</i>,1<i>H</i>,2<i>H</i>,2<i>H</i>-perfluorodecanethiol. Surface-initiated atom-transfer radical polymerization is then triggered in microwells to form PNIPAM arrays, which facilitate sample loading and enrichment of protein digests by concentrating large-volume samples into small dots and achieving on-plate desalting through PNIPAM configuration change at elevated temperature. The smart MALDI plate shows high performance for mass spectrometric analysis of cytochrome <i>c</i> and neurotensin in the presence of 1 M urea and 100 mM NaHCO₃, as well as improved detection sensitivity and high sequence coverage for α-casein and cytochrome <i>c</i> digests in femtomole range. The work presents a versatile sample pretreatment platform with great potential for proteomic research

Phenotypic analysis of <i>S. litura</i> after RNAi and DA treatment.

<p>A: Percentage mortality 24 h after RNAi or DA treatment; B: Percentage of normal pupation; C: Percentage of normal eclosion; D: Cumulative mortality of <i>S. litura</i>. The different letters above the columns indicate significant differences in <i>SLAWD</i> expression after RNAi or DA treatment (<i>P</i><0.05).</p