Carboxylated/oxidized diamond nanoparticles (nominal size 100 nm) exhibit exceptionally high affinity for proteins through both hydrophilic and hydrophobic forces. The affinity is so high that proteins in dilute solution can be easily captured by diamonds, simply separated by centrifugation, and directly analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). No preseparation of the adsorbed molecules from diamonds is required for the mass spectrometric analysis. Compared to conventional MALDI-TOF-MS, an enhancement in detection sensitivity by more than 2 orders of magnitude is achieved for dilute solution containing cytochrome c, myoglobin, and albumin because of preconcentration of the probed molecules. The lowest concentration detectable is 100 pM for a 1-mL solution. Aside from the enhanced sensitivity, the overall performance of this technique does not show any sign of deterioration for highly contaminated protein solutions, and furthermore, no significant peak broadening and band shift were observed in the mass spectra. The promise of this new method for clinical proteomics research is demonstrated with an application to human blood serum.
For the characterization of protein sequences and post‐translational modifications by MS, the ‘top‐down’ proteomics approach utilizes molecular and fragment ion mass data obtained by ionizing and dissociating a protein in the mass spectrometer. This requires more complex instrumentation and methodology than the far more widely used ‘bottom‐up’ approach, which instead uses such data of peptides from the protein's digestion, but the top‐down data are far more specific. The ESI MS spectrum of a 14 protein mixture provides full separation of its molecular ions for MS/MS dissociation of the individual components. False‐positive rates for the identification of proteins are far lower with the top‐down approach, and quantitation of multiply modified isomers is more efficient. Bottom‐up proteolysis destroys the information on the size of the protein and the connectivities of the peptide fragments, but it has no size limit for protein digestion. In contrast, the top‐down approach has a ∼ 500 residue, ∼ 50 kDa limitation for the extensive molecular ion dissociation required. Basic studies indicate that this molecular ion intractability arises from greatly strengthened electrostatic interactions, such as hydrogen bonding, in the gas‐phase molecular ions. This limit is now greatly extended by variable thermal and collisional activation just after electrospray (‘prefolding dissociation’). This process can cleave 287 inter‐residue bonds in the termini of a 1314 residue (144 kDa) protein, specify previously unidentified disulfide bonds between eight of 27 cysteines in a 1714 residue (200 kDa) protein, and correct sequence predictions in two proteins, one of 2153 residues (229 kDa).
Carbon nanodots were applied for the first time as a new matrix for the analysis of low-molecular-weight compounds by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in both positive- and negative-ion modes. A wide range of small molecules including amino acids, peptides, fatty acids, as well as β-agonists and neutral oligosaccharides were analyzed by MALDI MS with carbon nanodots as the matrix, and the lowest 0.2 fmol limits-of-detection were obtained for octadecanoic acid. Clear sodium and potassium adducts and deprotonated signals were produced in positive- and negative-ion modes. Furthermore, the glucose and uric acid in real samples were quantitatively determined by the internal standard method with the linear range of 0.5-9 mM and 0.1-1.8 mM (R(2) > 0.999), respectively. This work gives new insight into the application of carbon nanodots and provides a general approach for rapid analysis of low-molecular-weight compounds.
Unsatisfactory post-stroke recovery has long been a negative factor in the prognosis of ischemic stroke due to the lack of pharmacological treatments. Mesenchymal stem cells (MSCs)-based therapy has recently emerged as a promising strategy redefining stroke treatment; however, its effectiveness has been largely restricted by insufficient therapeutic gene expression and inadequate cell numbers in the ischemic cerebrum. Herein, a non-viral and magnetic field-independent gene transfection approach is reported, using magnetosome-like ferrimagnetic iron oxide nanochains (MFIONs), to genetically engineer MSCs for highly efficient post-stroke recovery. The 1D MFIONs show efficient cellular uptake by MSCs, which results in highly efficient genetic engineering of MSCs to overexpress brainderived neurotrophic factor for treating ischemic cerebrum. Moreover, the internalized MFIONs promote the homing of MSCs to the ischemic cerebrum by upregulating CXCR4. Consequently, a pronounced recovery from ischemic stroke is achieved using MFION-engineered MSCs in a mouse model.
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