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Glycosylation is a major protein post-translational modification whose dysregulation has been associated with many diseases. Herein, an on-tissue chemical derivatization strategy based on positively charged hydrazine reagent (Girard's reagent P) coupled with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was developed for analysis of N-glycans from FFPE treated tissue sections. The performance of the proposed approach was evaluated by analysis of monosaccharides, oligosaccharides, N-glycans released from glycoproteins, as well as MS imaging of N-glycans from human cancer tissue sections. The results demonstrated that the signal-to-noise ratios for target saccharides were notably improved after chemical derivatization, in which signals were enhanced by 230-fold for glucose and over 28-fold for maltooctaose. Improved glycome coverage was obtained for N-glycans derived from glycoproteins and tissue samples after chemical derivatization. Furthermore, on-tissue derivatization was applied for MALDI-MSI of N-glycans from human laryngeal cancer and ovarian cancer tissues. Differentially expressed N-glycans among the tumor region, adjacent normal tissue region, and tumor proximal collagen stroma region were imaged, revealing that high-mannose type N-glycans were predominantly expressed in the tumor region. Overall, our results indicate that the on-tissue labeling strategy coupled with MALDI-MSI shows great potential to spatially characterize N-glycan expression within heterogeneous tissue samples with enhanced sensitivity. This study provides a promising approach to better understand the pathogenesis of cancer related aberrant glycosylation, which is beneficial to the design of improved clinical diagnosis and therapeutic strategies.

Despite extensive efforts on probing the mechanism of Alzheimer’s disease (AD) and enormous investments into AD drug development, the lack of effective disease-modifying therapeutics and the complexity of the AD pathogenesis process suggest a great need for further insights into alternative AD drug targets. Herein, we focus on the chiral effects of truncated amyloid beta (Aβ) and offer further structural and molecular evidence for epitope region-specific, chirality-regulated Aβ fragment self-assembly and its potential impact on receptor-recognition. A multidimensional ion mobility-mass spectrometry (IM-MS) analytical platform and in-solution kinetics analysis reveal the comprehensive structural and molecular basis for differential Aβ fragment chiral chemistry, including the differential and cooperative roles of chiral Aβ N-terminal and C-terminal fragments in receptor recognition. Our method is applicable to many other systems and the results may shed light on the potential development of novel AD therapeutic strategies based on targeting the D-isomerized Aβ, rather than natural L-Aβ.

CBT-Cys click condensation reaction has a high second-order reaction rate constant and has found wide applicability in recent years.

Tyrosine kinase and phosphatase are two important, antagonistic enzymes in organisms. Development of noninvasive approach for sensing their activity with high spatial and temporal resolution remains challenging. Herein, we rationally designed a hydrogelator Nap-Phe-Phe(CF3)-Glu-Tyr-Ile-OH (1a) whose supramolecular hydrogel (i.e., Gel 1a) can be subjected to tyrosine kinase-directed disassembly, and its phosphate precursor Nap-Phe-Phe(CF3)-Glu-Tyr(H2PO3)-Ile-OH (1b), which can be subjected to alkaline phosphatase (ALP)-instructed self-assembly to form supramolecular hydrogel Gel 1b, respectively. Mechanic properties and internal fibrous networks of the hydrogels were characterized with rheology and cryo transmission electron microscopy (cryo-TEM). Disassembly/self-assembly of their corresponding supramolecular hydrogels conferring respective "On/Off" (19)F NMR/MRI signals were employed to sense the activity of these two important enzymes in vitro and in cell lysates for the first time. We anticipate that our new (19)F NMR/magnetic resonance imaging (MRI) method would facilitate pharmaceutical researchers to screen new inhibitors for these two enzymes without steric hindrance.

The identification of endogenous proteins as well as their binding to metal ions in living cells is determined by combining pulsed electrophoretic separations with nanoelectrospray ionization followed by mass spectrometric detection. This approach avoids problems resulting from the complicated cellular environment. In this manner, we demonstrate the rapid identification (300 ms or less) of intact proteins from living E. coli cells including the complexation of calmodulin with calcium ion. The latter showed different binding states from those observed in in vitro studies. These observations also reveal in vitro measurements do not necessarily represent the actual situation in living cells. We conclude that the attempted in situ measurement of intracellular proteins with minimal sampling processes should be preferred.

In situ living cell protein analysis would enable the structural identification and functional interrogation of intracellular proteins in native cellular environments. Previously, we have presented an in situ mass spectrometry (MS) strategy to identify protein and protein/metal ion complex with relatively small molecular weight ( Anal. Chem. 2016, 88, 10860-10866). However, it is still challenging to directly identify larger proteins and protein/ligand complexes in cell, due to numerous nonspecific bindings of ligands, solvents, and other cellular constituents. Here we present a versatile single-step mass spectrometric strategy, "in-cell" mass spectrometry ("in-cell" MS), for in situ protein identification and dynamic protein-ligand interaction monitoring directly from living cells. "In-cell" MS combined all-ion-fragmentation mode with our previous method; thus, on a high-resolution MS instrument, we can greatly improve the signal/noise ratio of the larger proteins and protein/ligand complexes. Meanwhile, we also achieved a much wider mass range for protein complex and detection of 17 proteins with molecular weight ranging from 4 to 44 kDa. In addition, "in-cell" MS could also monitor dynamic protein interactions in living cells. Calcium-regulated calmodulin-melittin interaction was tested to demonstrate the proof of concept. "In-cell" MS provides an alternative for in situ analysis of living cells, which might contribute to rapid protein analysis and quality control in biochemistry laboratories, protein engineering, and even protein industry.