Peptide- and Protein-Level Combined Strategy for Analyzing Newly Synthesized Proteins by Integrating Tandem Orthogonal Proteolysis with Cleavable Bioorthogonal Tagging.

A newly synthesized proteome reflects perturbations sensitively and maintains homeostasis in cells. To investigate the low abundant newly synthesized proteins (NSPs) from a complex background proteome, an enrichment process with high selectivity and reliability is essential. Here, we have developed a strategy to realize comprehensive analysis of NSPs by integrating tandem orthogonal proteolysis (TOP) with cleavable bioorthogonal tagging (CBOT) called TOP-CBOT. A solid-phase-conjugated probe with a clickable moiety and a protease-cleavable site was designed, which allowed NSPs to be covalently captured along with tandem release by trypsin and orthogonal tobacco etch virus (TEV) protease. Our method has integrated the advantages of protein-level and peptide-level enrichment. Trypsin digests larger number of peptides from the recovered proteins for NSPs identification and quantification, while the specific tag-contained peptides from TEV data set enabled further NSPs confirmation. Integrating information from two complementary data sets, the reliability in NSPs identification and quantitation were remarkably enhanced. A total of 3699 proteins were recovered in the trypsin data set. Additionally, 1931 proteins were confirmed as NSPs with 5019 identified peptides in the TEV data set, over 90% of which were overlapped with the tryptic data set. Our strategy was further applied to profile NSP degradation kinetics during rapamycin-induced macroautophagy. The newly synthesized proteome displayed varied alteration of degradation rates among stimulation and more than half of NSPs showed decreased half-lives during autophagy.

Nascent Glycoproteome Reveals That N-Linked Glycosylation Inhibitor-1 Suppresses Expression of Glycosylated Lysosome-Associated Membrane Protein-2.

Glycosylation inhibition has great potential in cancer treatment. However, the corresponding cellular response, protein expression and glycosylation changes remain unclear. As a cell-permeable small-molecule inhibitor with reduced cellular toxicity, N-linked glycosylation inhibitor-1 (NGI-1) has become a great approach to regulate glycosylation in mammalian cells. Here for the first time, we applied a nascent proteomic method to investigate the effect of NGI-1 in hepatocellular carcinoma (HCC) cell line. Besides, hydrophilic interaction liquid chromatography (HILIC) was adopted for the enrichment of glycosylated peptides. Glycoproteomic analysis revealed the abundance of glycopeptides from LAMP2, NICA, and CEIP2 was significantly changed during NGI-1 treatment. Moreover, the alterations of LAMP2 site-specific intact N-glycopeptides were comprehensively assessed. NGI-1 treatment also led to the inhibition of Cathepsin D maturation and the induction of autophagy. In summary, we provided evidence that NGI-1 repressed the expression of glycosylated LAMP2 accompanied with the occurrence of lysosomal defects and autophagy.

Nascent Proteome and Glycoproteome Reveal the Inhibition Role of ALG1 in Hepatocellular Carcinoma Cell Migration.

Asparagine-linked glycosylation protein 1 homolog (ALG1) participates in the initial stage of protein N-glycosylation and N-glycosylation has been implicated in the process of hepatocellular carcinoma (HCC) progression. However, whether ALG1 plays a role in human HCC remains unknown. In this study, the expression profile of ALG1 in tumorous and corresponding adjacent non-tumor tissues was analyzed. The relationship of ALG1 expression with clinical features and prognosis of HCC patients was also evaluated using immuno-histochemical method. Here we found ALG1 decreased in HCC tissues compared with adjacent normal liver tissues, which predicted an unfavorable prognosis. Combined with RNA interference, nascent proteome and glycoproteome were determined systematically in Huh7 cell line. Bioinformatics analysis indicated that the differentially expressed proteins participating in the response of ALG1 knockdown were most significantly associated with cell-cell adhesion. Functional studies confirmed that knockdown of ALG1 reduced cell adhesion capacity, and promoted cell migration. Furthermore, down-regulation of H8N2 (on N-glycosite N651) and H5N4S2F1 (on N-glycosite N692) from N-cadherin was identified as a feature of ALG1 knockdown. Our findings revealed that ALG1 controlled the expression of glycosylated N-cadherin and played a role in HCC migration, with implications for prognosis. Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-022-00050-5.

Enhancing Comprehensive Analysis of Newly Synthesized Proteins Based on Cleavable Bioorthogonal Tagging.

Protein synthesis and degradation responding to environmental cues is critical for understanding the mechanisms involved. Chemical proteomics introducing bioorthogonal tagging into proteins and isolation by biotin affinity purification is applicable for enrichment of newly synthesized proteins (NSPs). Current enrichment methods based on biotin-streptavidin interaction lack efficiency to release enriched NSPs under mild conditions. Here we designed a novel method for enriching newly synthesized peptides by click chemistry followed by release of enriched peptides via tryptic digestion based on cleavable bioorthogonal tagging (CBOT). CBOT-modified peptides can further enhance identification in mass spectrometry analysis and provide a confirmation by small mass shift. Our method achieved an improvement in specificity (97.1%) and sensitivity for NSPs in cell lysate, corresponding to profiling at a depth of 4335 NSPs from 2 mg of starting materials in a single LC-MS/MS run. In addition, the CBOT strategy can quantify NSPs when coupling a pair of isotope-labeled azidohomoalanine (AHA/hAHA) with decent reproducibility. Furthermore, we applied it to analyze newly synthesized proteomes in the autophagy process after 6 h rapamycin stimulation in cells, 2910 NSPs were quantified, and 337 NSPs among them were significantly up- and down-regulated. We envision CBOT as an effective and alternative approach for bioorthogonal chemical proteomics to study stimuli-sensitive subsets.

Analysis of Serum Paraoxonase 1 Using Mass Spectrometry and Lectin Immunoassay in Patients With Alpha-Fetoprotein Negative Hepatocellular Carcinoma.

The diagnosis of AFP (alpha-fetoprotein)-negative HCC (hepatocellular carcinoma) mostly relies on imaging and pathological examinations, and it lacks valuable and practical markers. Protein N-glycosylation is a crucial post-translation modifying process related to many biological functions in an organism. Alteration of N-glycosylation correlates with inflammatory diseases and infectious diseases including hepatocellular carcinoma. Here, serum N-linked intact glycopeptides with molecular weight (MW) of 40-55 kDa were analyzed in a discovery set (n = 40) including AFP-negative HCC and liver cirrhosis (LC) patients using label-free quantification methodology. Quantitative lens culinaris agglutin (LCA) ELISA was further used to confirm the difference of glycosylation on serum PON1 in liver diseases (n = 56). Then, the alteration of site-specific intact N-glycopeptides of PON1 was comprehensively assessed by using Immunoprecipitation (IP) and mass spectrometry based 16O/18O C-terminal labeling quantification method to distinguish AFP-negative HCC from LC patients in a validation set (n = 64). Totally 195 glycopeptides were identified using a dedicated search engine pGlyco. Among them, glycopeptides from APOH, HPT/HPTR, and PON1 were significantly changed in AFP-negative HCC as compared to LC. In addition, the reactivity of PON1 with LCA in HCC patients with negative AFP was significantly elevated than that in cirrhosis patients. The two glycopeptides HAN253WTLTPLK (H5N4S2) and (H5N4S1) corresponding to PON1 were significantly increased in AFP-negative HCC patients, as compared with LC patients. Variations in PON1 glycosylation may be associated with AFP-negative HCC and might be helpful to serve as potential glycomic-based biomarkers to distinguish AFP-negative HCC from cirrhosis.