Glyceraldehyde-derived advanced glycation end products (AGEs) play an important role in the pathogenesis of many diseases including cancer. Accumulation of intracellular AGEs could stimulate cancer induction and facilitate cancer progression. We evaluated the toxic effect of glyceraldehyde-derived intracellular AGEs on normal and malignant pancreatic ductal cells by assessing the cell viability, toxicity, and oxidative stress, followed by proteomic analysis. Our functional studies showed that pancreatic cancer cells (PANC-1 and MIA PaCa-2) were more resistant to glyceraldehyde treatment compared to normal pancreatic ductal epithelial cells (HPDE), while cytotoxicity effects were observed in all cell types. Furthermore, using ¹³C isotopic labeled glyceraldehyde, the proteomic data revealed a dose-dependent increment of the number of glycation adducts in both these cell types. HPDE cells showed a higher number of intracellular AGEs compared to cancer cells. At a molecular level, the glycations in the lysine residues of proteins showed a concurrent increase with the concentration of the glyceraldehyde treatment, while the arginine glycations appeared to be less affected by the glyceraldehyde doses. Further pathway analysis of these glycated proteins suggested that the glycated proteins participate in important biological processes that are major hallmarks of cancer initiation and progression, including metabolic processes, immune response, oxidative stress, apoptosis, and S100 protein binding.

Dissecting the proteome of cell types and states at single-cell resolution, while being highly challenging, has significant implications in basic science and biomedicine. Mass spectrometry (MS)-based single-cell proteomics represents an emerging technology for system-wide, unbiased profiling of proteins in single cells. However, significant challenges remain in analyzing an extremely small amount of proteins collected from a single cell, as a proteome-wide amplification of proteins is not currently feasible. Here, we report an integrated spectral library-based single-cell proteomics (SLB-SCP) platform that is ultrasensitive and well suited for a large-scale analysis. To overcome the low MS/MS signal intensity intrinsically associated with a single-cell analysis, this approach takes an alternative approach by extracting a breadth of information that specifically defines the physicochemical characteristics of a peptide from MS1 spectra, including monoisotopic mass, isotopic distribution, and retention time (hydrophobicity), and uses a spectral library for proteomic identification. This conceptually unique MS platform, coupled with the DIRECT sample preparation method, enabled identification of more than 2000 proteins in a single cell to distinguish different proteome landscapes associated with cellular types and heterogeneity. We characterized individual normal and cancerous pancreatic ductal cells (HPDE and PANC-1, respectively) and demonstrated the substantial difference in the proteomes between HPDE and PANC-1 at the single-cell level. A significant upregulation of multiple protein networks in cancer hallmarks was identified in the PANC-1 cells, functionally discriminating the PANC-1 cells from the HPDE cells. This integrated platform can be built on high-resolution MS and widely accepted proteomic software, making it possible for community-wide applications.

As a well-known glycolysis inhibitor for anticancer treatment, 2-Deoxy-D-glucose (2DG) inhibits the growth and survival of cancer cells by interfering with the ATP produced by the metabolism of D-glucose. In addition, 2DG inhibits protein glycosylation in vivo by competing with D-mannose, leading to endoplasmic reticulum (ER) stress and unfolded protein responses in cancer cells. However, the molecular details underlying the impact of 2DG on protein glycosylation remain largely elusive. With an integrated approach to glycoproteomics and proteomics, we characterized the 2DG-induced alterations in N-glycosylation, as well as the cascading impacts on the whole proteome using the HT29 colorectal cancer cell line as a model system. More than 1700 site-specific glycoforms, represented by unique intact glycopeptides (IGPs), were identified. The treatment of 2DG had a broad effect on the N-glycoproteome, especially the high-mannose types. The glycosite occupancy of the high-mannose N-glycans decreased the most compared with the sialic acid and fucose-containing N-glycans. Many of the proteins with down-regulated high-mannose were implicated in functional networks related to response to topologically incorrect protein, integrin-mediated signaling, lysosomal transport, protein hydroxylation, vacuole, and protein N-glycosylation. The treatment of 2DG also functionally disrupted the global cellular proteome, evidenced by significant up-regulation of the proteins implicated in protein folding, endoplasmic reticulum, mitochondrial function, cellular respiration, oxidative phosphorylation, and translational termination. Taken together, these findings reveal the complex changes in protein glycosylation and expression underlying the various effects of 2DG on cancer cells, and may provide insightful clues to inform therapeutic development targeting protein glycosylation.