Comprehensive Profiling of N‑Linked Glycosylation Sites in HeLa Cells Using Hydrazide Enrichment

The adenocarcinoma cell line HeLa serves as a model system for cancer research in general and cervical cancer in particular. In this study, hydrazide enrichment in combination with state-of-the art nanoLC−MS/MS analysis was used to profile N-linked glycosites in HeLa cells. N-Linked glycoproteins were selectively enriched in HeLa cells by the hydrazide capture method, which isolates all glycoproteins independent of their glycans. Nonglycosylated proteins were removed by extensive washing. N-Linked glycoproteins were identified with the specific NXT/S motif and deamidated asparagine (N). Deglycosylation was carried out in both H_2 (^16)O and H_2 ^(18)O to confirm the deamidation. NanoLC−MS/MS analysis indicated that the method selectively enriched at least 100 fold N-linked glycosites in HeLa cells. When both the membrane and cytosolic fractions were used, a total of 268 unique N-glycosylation sites were identified corresponding to 106 glycoproteins. Bioinformatic analysis revealed that most of the glycoproteins identified are known to have an impact on cancer and have been proposed as biomarkers

Functional characteristics of neonatal rat β cells with distinct markers

Neonatal β cells are considered developmentally immature and hence less glucose responsive. To study the acquisition of mature glucose responsiveness, we compared glucose-regulated redox state, insulin synthesis, and secretion of β cells purified from neonatal or 10-week-old rats with their transcriptomes and proteomes measured by oligonucleotide and LC-MS/MS profiling. Lower glucose responsiveness of neonatal β cells was explained by two distinct properties: higher activity at low glucose and lower activity at high glucose. Basal hyperactivity was associated with higher NAD(P)H, a higher fraction of neonatal β cells actively incorporating (3)H-tyrosine, and persistently increased insulin secretion below 5 mM glucose. Neonatal β cells lacked the steep glucose-responsive NAD(P)H rise between 5 and 10 mM glucose characteristic for adult β cells and accumulated less NAD(P)H at high glucose. They had twofold lower expression of malate/aspartate-NADH shuttle and most glycolytic enzymes. Genome-wide profiling situated neonatal β cells at a developmental crossroad: they showed advanced endocrine differentiation when specifically analyzed for their mRNA/protein level of classical neuroendocrine markers. On the other hand, discrete neonatal β cell subpopulations still expressed mRNAs/proteins typical for developing/proliferating tissues. One example, delta-like 1 homolog (DLK1) was used to investigate whether neonatal β cells with basal hyperactivity corresponded to a more immature subset with high DLK1, but no association was found. In conclusion, the current study supports the importance of glycolytic NADH-shuttling in stimulus function coupling, presents basal hyperactivity as novel property of neonatal β cells, and provides potential markers to recognize intercellular developmental differences in the endocrine pancrea