Laser Cleavable Probes-Based Cell Surface Engineering for <i>in Situ</i> Sialoglycoconjugates Profiling by Laser Desorption/Ionization Mass Spectrometry

Cell-surface sialoglycoconjugates (sialoglycoproteins and sialoglycolipids) play important roles in cell–cell interactions and related tumor metastasis process. Although there have been some analytical methods to evaluate the sialoglycoconjugates, an effective method providing both qualitative and quantitative information is still deficient. Here we establish an extraction-free, sensitive, and high-throughput platform to realize <i>in situ</i> detection of the cell-surface sialoglycoconjugates on various cell lines, e.g., cancer and normal cells by laser desorption/ionization mass spectrometry (LDI MS). In this proposal, azide groups were introduced into the ends of cell-surface sialoglycoconjugates by the biorthogonal method, and then the sialoglycoconjugates were armed with a laser-cleavable probe (Tphsene) through click chemistry. We can easily get the probes signal under laser irradiation, which reflected the presence of cell-surface sialoglycoconjugates. Different cell lines were discriminated simultaneously, and the LDI relative quantification agreed with fluorescent results. Besides, a linear quantitation relationship in the range of 100 fmol to 100 pmol was obtained with a designed and synthesized internal standard (phTsane) added. A detection limit of 5 fmol was obtained with good reproducibility. Based on the quantitative and high-throughput ability, we conducted pharmacodynamics study of drug (tunicamycin) on cancer cells. In addition, we found the tag was safe from sweet-spot effect of matrix adding. The simultaneous detection of sialoglycoconjugates and metabolites was therefore achieved. We believe that this laser cleavable probes-based cell-surface engineering for sialoglycoconjugates platform means great significance to diagnosis, prognosis, and therapeutic purposes. Besides, this strategy can be applied to other glycoconjugates which is hard to detect and the related disease processes when more corresponding chemically modified sugar substrates and exact biorthogonal reactions are developed

Utilizing a Mini-Humidifier To Deposit Matrix for MALDI Imaging

MALDI mass spectrometry imaging (MALDI-MSI) is a powerful tool to study endogenous metabolites. The process of matrix deposition is crucial for a high-quality imaging result. Commercial instruments for matrix deposition are expensive. Low-cost methods like airbrushing will generate matrix crystals that are too large for high-spatial-resolution imaging. Sublimation may cause some compounds to go undetected because of the lack of solvent. Herein, we utilized a mini-humidifier, costing less than 5 dollars, to deposit matrix for MALDI-MSI. Compared with Imageprep, a commercialized instrument, our device based on the humidifier provided higher sensitivity and much smaller matrix crystals with diameters of less than 10 μm. High-quality ion images with 10 μm spatial resolution were obtained using our method. The enhancement of sensitivity by the humidifier could provide a sufficient amount of ions to perform tandem mass imaging. We also performed MALDI-MS/MS imaging to separate two lipids in mouse brain

<i>N</i>‑Phenyl-2-naphthylamine as a Novel MALDI Matrix for Analysis and in Situ Imaging of Small Molecules

Due to its strong ultraviolet absorption, low background interference in the small molecular range, and salt tolerance capacity, <i>N</i>-phenyl-2-naphthylamine (PNA) was developed as a novel matrix in the present study for analysis and imaging of small molecules by matrix-assisted laser desorption/ionization mass spectrometry time-of-fight (MALDI-TOF MS). The newly developed matrix displayed good performance in analysis of a wide range of small-molecule metabolites including free fatty acids, amino acids, peptides, antioxidants, and phospholipids. In addition, PNA-assisted LDI MS imaging of small molecules in brain tissue of rats subjected to middle cerebral artery occlusion (MCAO) revealed unique distributions and changes of 89 small-molecule metabolites including amino acids, antioxidants, free fatty acids, phospholipids, and sphingolipids in brain tissue 24 h postsurgery. Fifty-nine of the altered metabolites were identified, and all the changed metabolites were subject to relative quantitation and statistical analysis. The newly developed matrix has great potential application in the field of biomedical research