We report here using a novel technology-electrospray-assisted laser desorption ionization (ELDI)/mass spectrometry-for the rapid and sensitive detection of the major proteins that exist in dried biological fluids (e.g., blood, tears, saliva, serum), bacterial cultures, and tissues (e.g., porcine liver and heart) under ambient conditions. This technique required essentially no sample pretreatment. The proteins in the samples were desorbed using a pulsed nitrogen laser without the assistance of an organic matrix. The desorbed protein molecules were then post-ionized through their fusion into the charged solvent droplets produced from the electrospray of an acidic methanol solution; electrospray ionization (ESI) proceeded from the newly formed droplets to generate the ESI-like protein ions. This new ionization approach combines some of the features of electrospray ionization with those of matrix-assisted laser desorption ionization (MALDI), that is, sampling of a solid surface with spatial resolution, generating ESI-like mass spectra of the desorbed proteins, and operating under ambient conditions.
A starlike water-soluble fullerene derivative, hexa(sulfonbutyl)fullerene (C60[(CH2)4SO3-]6; HSBF), consisting of a C60 cage covalently bonded with six negatively charged sulfonate arms, was synthesized and used to selectively precipitate positively charged surfactants, amino acids, peptides, and proteins. The affinity of HSBF to the analytes depends on the charge, structure, and hydrophobic characteristics of the analytes. The ion pair precipitate was easily removed from the solution by centrifugation. After washing, the precipitate was redissolved in the solvent or buffer solution and the analyte was characterized by laser desorption ionization-time-of-flight mass spectrometry (LD-TOF). HSBF shows strong optical absorbance in the UV range, so no additional organic matrix was required to conduct LD-TOF analysis of small analytes. For the solution that contained five quaternary amines differing only in alkyl chain length, HSBF exhibits the highest affinity to the amine with the longest alkyl chain. Only the arginine signal was detected from the solution that contained 14 amino acids. The peptides with arginine as the end groups interacted most strongly with HSBF and could be selectively precipitated from a solution of a mixture of five peptides. The signals associated with a trace amount of charged peptides derived from the digestion of proteins by trypsin were greatly enhanced after concentration with HSBF. Among eight proteins in the sample solution, insulin had the strongest affinity to the HSBF and exhibited the strongest signal on the matrix-assisted laser desorption/ionization mass spectrum.
This study demonstrated the feasibility of performing protein analysis with ultralow sample volume by combining a tungsten oxide nanowire (TON) fiber with a miniaturized electrospray ionization interface. An increase in wettability of the tugsten surface after growing randomly oriented TON on its surface allows strong adhesion of approximately 50 nL of the methanol solution at its tip. Under the influence of a high electric field, electrospray from a Taylor cone on the adhered methanol solution was observed and the multiply charged ions of protein molecules predissolved in the solution were detected.
This paper reports development of a non-mechanical electrospray ionization (ESI) method to generate electrospray from a droplet deposited on an optical fiber coated with a thin gold or Nafion film. Modification of the surface of the optical fiber in this manner increases its wettability, such that a droplet of the aqueous sample solution can adhere sufficiently strongly to the tip of the fiber. The aqueous sample solution was deposited near the tip of the fiber with a micropipette. When a high voltage (2,000 V) was applied to the fiber by electrical connection through the gold film, the sample solution moved and hung at the tip of the fiber. Simultaneously, ESI was generated from the sample droplet. Multiply charged peptide and protein ions were detected by connecting the ESI source to a quadrupole mass analyzer.
We describe an innovative approach - using a high concentration of trypsin-modified magnetic nanoparticles (TMNPs) - for the rapid and efficient digestion of proteins at elevated temperature. The required digestion time could be reduced to less than 10 s. After digestion, the TMNPs were collected magnetically from the sample solution for reuse and the digested peptides were characterized using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Protein digestion was optimized when using the TMNPs (5 microg/microL) at 57 degrees C; a significantly high peptide coverage was achieved for protein identification (e.g., 98% for lysozyme). Although a high concentration of TMNPs was used for digestion, the short digestion time led to much lower amounts of trypsin peptides being produced through self-digestion. As a result, interference in the mass spectrometric detection of the peptide ions was reduced significantly.
Hepatocellular carcinoma (HCC) has been recognized worldwide as one of the major causes of cancer death. The medicinal fungus Antrodia cinnamomea (A. cinnamomea) has been served as a functional food for liver protection. The aim of the present study was to investigate the potential activity of A. cinnamomea extracts as a safe booster for the anticancer activity of sorafenib, a multi-kinase inhibitor approved for the treatment of HCC. The biologically active triterpenoids in the ethanolic extracts of A. cinnamomea (EAC) were initially identified by HPLC/LC/MS then the different extracts and sorafenib were assessed in vitro and in vivo. EAC could effectively sensitize HCC cells to low doses of sorafenib, which was perceived via the ability of the combination to repress cell viability and to induce cell cycle arrest and apoptosis in HCC cells. The ability of EAC to enhance sorafenib activity was mediated through targeting mitogen-activated protein (MAP) kinases, modulating cyclin proteins expression and inhibiting cancer cell invasion. Moreover, the proposed combination significantly suppressed ectopic tumor growth in mice with high safety margins compared to single-agent treatment. Thus, this study highlights the advantage of combining EAC with sorafenib as a potential adjuvant therapeutic strategy against HCC.
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