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Cancer cell metastasis is a major cause of cancer death. Unfortunately, the underlying molecular mechanisms remain unknown, which results in the lack of efficient diagnosis, therapy and prevention approaches. Nevertheless, the dysregulation of the cancer cell secretome is known to play key roles in tumor transformation and progression. The majority of proteins in the secretome are secretory proteins and membrane-released proteins, and, mostly, the glycosylated proteins. Until recently, few studies have explored protein N-glycosylation changes in the secretome, although protein glycosylation has received increasing attention in the study of tumor development processes. Here, the N-glycoproteins in the secretome of two human hepatocellular carcinoma (HCC) cell lines with low (MHCC97L) or high (HCCLM3) metastatic potential were investigated with a in-depth characterization of the N-glycosites by combining two general glycopeptide enrichment approaches, hydrazide chemistry and zwitterionic hydrophilic interaction chromatography (zic-HILIC), with mass spectrometry analysis. A total of 1,213 unique N-glycosites from 611 N-glycoproteins were confidently identified. These N-glycoproteins were primarily localized to the extracellular space and plasma membrane, supporting the important role of N-glycosylation in the secretory pathway. Coupling label-free quantification with a hierarchical clustering strategy, we determined the differential regulation of several N-glycoproteins that are related to metastasis, among which AFP, DKK1, FN1, CD151 and TGFβ2 were up-regulated in HCCLM3 cells. The inclusion of the well-known metastasis-related proteins AFP and DKK1 in this list provides solid supports for our study. Further western blotting experiments detecting FN1 and FAT1 confirmed our discovery. The glycoproteome strategy in this study provides an effective means to explore potential cancer biomarkers.

CeO2 nanoparticles (NPs) have been shown to cause lung fibrosis, however, the exact underlying molecular mechanisms are poorly understood. In this study, we have conducted a mass spectrometry-based global metabolomic analysis of human bronchial epithelial BEAS-2B cells treated by CeO2 NPs with different aspect ratios and assessed their toxicity on the bronchial epithelial cells by various cell-based functional assays. Although CeO2 NPs at doses ranging from 12.5 μg/mL to 25 μg/mL displayed low cytotoxicity on the bronchial epithelial cells, the metabolomic analysis revealed a number of metabolites in the cellular metabolic pathways of sphingosine-1-phosphate, fatty acid oxidation, inflammation, etc. were significantly altered by CeO2 NPs, especially those with high aspect ratios. More importantly, the robustness of metabolomics findings was further successfully validated in mouse models upon acute and chronic exposures to CeO2 NPs. Mechanistically, CeO2 NPs upregulated transforming growth factor beta-1 (TGF-β1) levels in BEAS-2B cells in an aspect ratio-dependent manner through enhancing the expression of early growth response protein 1 (EGR-1). In addition, both in vitro and in vivo studies demonstrated that CeO2 NPs significantly induced the expression of sphingosine kinase 1 (SHPK1), phosphorylated Smad2/3 and lung fibrosis markers. Moreover, targeting SPHK1, TGFβ receptor or Smad3 phosphorylation significantly attenuated the fibrosis-promoting effects of CeO2 NPs, and SPHK1-S1P pathway exerted a greater effect on the TGF-β1-mediated lung fibrosis compared to the conventional Smad2/3 pathway. Collectively, our studies have identified the metabolomic changes in BEAS-2B cells exposed to CeO2 NPs with different aspect ratios and revealed the subtle changes in metabolic activities that traditional approaches might have missed. More importantly, we have discovered a previously unknown molecular mechanism underlying CeO2 NP-induced lung fibrosis with different aspect ratios, shedding new insights on the environmental hazard potential of CeO2 NPs.

Posttranslational modifications of antibody products affect their stability, charge distribution, and drug activity and are thus a critical quality attribute. The comprehensive mapping of antibody modifications and different charge isomers (CIs) is of utmost importance, but is challenging. We intended to quantitatively characterize the posttranslational modification status of CIs of antibody drugs and explore the impact of posttranslational modifications on charge heterogeneity. The CIs of antibodies were fractionated by strong cation exchange chromatography and verified by capillary isoelectric focusing-whole column imaging detection, followed by stepwise structural characterization at three levels. First, the differences between CIs were explored at the intact protein level using a top-down mass spectrometry approach; this showed differences in glycoforms and deamidation status. Second, at the peptide level, common modifications of oxidation, deamidation, and glycosylation were identified. Peptide mapping showed nonuniform deamidation and glycoform distribution among CIs. In total, 10 N-glycoforms were detected by peptide mapping. Finally, an in-depth analysis of glycan variants of CIs was performed through the detection of enriched glycopeptides. Qualitative and quantitative analyses demonstrated the dynamics of 24 N-glycoforms. The results revealed that sialic acid modification is a critical factor accounting for charge heterogeneity, which is otherwise missed in peptide mapping and intact molecular weight analyses. This study demonstrated the importance of the comprehensive analyses of antibody CIs and provides a reference method for the quality control of biopharmaceutical analysis. [Display omitted] • A stepwise investigation on the diversity and dynamics of modifications of antibody charge isomers (CIs) was performed. • The results from SCX-HPLC and cIEF-WCID supported each other. • In-depth analysis on glycan variants of CIs was achieved by analyzing the enriched glycopeptides. • Sialic acid and deamidation modifications were critical factors for the charge heterogeneity. [ABSTRACT FROM AUTHOR]

We report a new reagent that is capable of both chemical derivatization and selective enrichment of azide-labeled PTM peptides for sensitive identification by mass spectrometry (MS). Facile sample recovery, enhanced ionization and fragmentation in MS of the enriched PTM peptides are achieved, which leads to the identification of 3293 O-GlcNAc peptides and the location of 1706 sites in HeLa cells and efficiently expands the current mapping scale. [ABSTRACT FROM AUTHOR]

Liquid biopsies using body fluids have gained much attention in recent years due to their multiple advantages in clinical diagnosis, such as less/non-invasive collection, suitability for longitudinal disease monitoring, and better representation of tumor heterogeneity. As an attractive choice for liquid biopsy, urine proteins and their post-translational modifications (PTMs) have the potential to offer significant insights into physiological variations and pathological changes in the human body. However, due to the intrinsically large variability of urine proteins and their PTMs among different individuals, there is a high demand for strategies for high-throughput analysis of a large number of samples to obtain a comprehensive view and a reliable reference interval of the urine proteome. In this work, we proposed a new urine phosphoproteome sample processing strategy that combines fast protein extraction, efficient multiple immobilized-proteases digestion, and tandemly connected centrifugal tips device-based facile phosphopeptide enrichment & fractionation. This strategy is capable of paralleled sample processing with an approximate five-fold reduction in processing time and is therefore particularly suitable for handling a large number of urine samples. Totally, we identified 4196 phosphosites in human urine proteins by mass spectrometry in replicated tests, a number which is dozens of times larger than those previously reported. Therefore, this strategy may have great potential in urine-based phosphoprotein biomarker screening and drug response studies. [ABSTRACT FROM AUTHOR]

Detailed characterization of glycoprotein structures requires determining both the sites of glycosylation as well as the glycan structures associated with each site. In this work, we developed an analytical strategy for characterization of intact N-glycopeptides in complex proteome samples. In the first step, tryptic glycopeptides were enriched using ZIC-HILIC. Secondly, a portion of the glycopeptides was treated with endoglycosidase H (Endo H) to remove high-mannose (Man) and hybrid N-linked glycans. Thirdly, a fraction of the Endo H-treated glycopeptides was further subjected to PNGase F treatment in O water to remove the remaining complex glycans. The intact glycopeptides and deglycosylated peptides were analyzed by nano-RPLC-MS/MS, and the glycan structures and the peptide sequences were identified by using the Byonic or pFind tools. Sequential digestion by endoglycosidase provided candidate glycosites information and indication of the glycoforms on each glycopeptide, thus helping to confine the database search space and improve the confidence regarding intact glycopeptide identification. We demonstrated the effectiveness of this approach using RNase B and IgG and applied this sequential digestion strategy for the identification of glycopeptides from the HepG2 cell line. We identified 4514 intact glycopeptides coming from 947 glycosites and 1011 unique peptide sequences from HepG2 cells. The intensity of different glycoforms at a specific glycosite was obtained to reach the occupancy ratios of site-specific glycoforms. These results indicate that our method can be used for characterizing site-specific protein glycosylation in complex samples. [ABSTRACT FROM AUTHOR]

Deep and efficient proteolysis is the critical premise in mass spectrometry-based bottom-up proteomics. It is difficult for traditional in-solution digestion to meet the requirement unless prolonged digestion time and enhanced enzyme dosage are employed, which makes the whole workflow time-consuming and costly. The abovementioned problems could be effectively ameliorated by anchoring many proteases on solid supports. In this work, covalent organic framework-coated magnetic graphene (MG@TpPa-1) was designed and prepared as a novel enzyme carrier for the covalent immobilization of trypsin with a high degree of loading (up to 268 μg mg). Profiting from the advantages of magnetic graphene and covalent organic frameworks, the novel trypsin bioreactor was successfully applied for the enzymatic digestion of a model protein with dramatically improved digestion efficiency, stability, and reusability. Complete digestion could be achieved in a time period as short as 2 min. For the digestion of proteins extracted from Amygdalus pedunculata, a total of 2833 protein groups were identified, which was slightly more than those obtained by 12 h of in-solution digestion (2739 protein groups). All of the results demonstrate that MG@TpPa-1-trypsin is an excellent candidate for sample preparation in a high-throughput proteomics analysis. [ABSTRACT FROM AUTHOR]

Abstract Isotope-tracing facilitates the understanding of metabolic regulation in biological systems. Depending on the selection of tracers, some essential metabolites cannot be traced. A comprehensive understanding of the regulated pathways can only be achieved with focus beyond labeled metabolites. The isotope tracing assisted metabolic profiling described here is a platform for high throughput mapping of isotope labeled metabolites with simultaneous metabolomics profiling. This approach incorporates an in-house MS/MS library for metabolite identification and ID-based quantitation. An "Isotopic" software was developed to generate potential labeled isotopomers. Using this platform, a total of 394 metabolites were reliably identified based on MS/MS confirmation in 3 million 293 T cells, among which 54 and 43 metabolites were discovered to carry extensive labels (>2%) from 13C 6 -glucose and 13C 5 -glutamine respectively. Citrate flowing into malate shuttle was also observed. More interestingly, the rate-limiting step in NAD and UDP-GlcNAc biosynthesis was clearly observed according to time course labeling. In HSP60 knockdown cell lines, enhanced purine and pyrimidine biosynthesis were confirmed by the abundance and labeling percentages of intermediate metabolites. Graphical abstract Image 1 Highlights • Isotope Tracing Assisted Metabolic Profiling Platform allowed simultaneously profile isotope labeled and unlabeled metabolites. • In-house MS/MS library and "Isotopic" software were incorporated to achieve high throughput analysis. • Mapping information of traced metabolites from 13C 6 -glucose and 13C 5 -glutamine in 293T cells was provided as resources. • Metabolic regulations of HSP60 knockdown were observed based on Isotope Tracing Assisted Metabolic Profiling. [ABSTRACT FROM AUTHOR]

Human serum has been intensively studied to identify biomarkers via global proteomic analysis. The altered O -glycoproteome is associated with human pathological state including cancer, inflammatory and degenerative diseases and is an attractive source of disease biomarkers. Because of the microheterogeneity and macroheterogeneity of O -glycosylation, site-specific O -glycosylation analysis in human serum is still challenging. Here, we developed a systematic strategy that combined multiple enzyme digestion, multidimensional separation for sample preparation and high-resolution tandem MS with Byonic software for intact O -glycopeptide characterization. We demonstrated that multiple enzyme digestion or multidimensional separation can make sample preparation more efficient and that EThcD is not only suitable for the identification of singly O -glycosylated peptides (50.3%) but also doubly (21.2%) and triply (28.5%) O -glycosylated peptides. Totally, with the strict scoring criteria, 499 non-redundant intact O -glycopeptides, 173 O -glycosylation sites and 6 types of O -glycans originating from 49 O -glycoprotein groups were identified in human serum, including 121 novel O -glycosylation sites. Currently, this is the largest data set of site-specific native O -glycoproteome from human serum samples. We expect that the strategies developed by this study will facilitate in-depth analyses of native O -glycoproteomes in human serum and provide opportunities to understand the functional roles of protein O -glycosylation in human health and diseases. Biological significance The altered O -glycoproteome is associated with human pathological state and is an attractive source of disease biomarkers. However, site-specific O -glycosylation analysis is challenging because of the microheterogeneity (different glycoforms attached to one glycosylation site) and macroheterogeneity (site occupancy) of O -glycosylation. In this work, we developed a systematic strategy for intact O -glycopeptide characterization. This study took advantage of the inherent properties of the new fragmentation method called EThcD, which provides more complete fragmentation information about O -glycosylated peptides and a more confident site localization of O -glycans than collision-induced dissociation (HCD). We demonstrated that multiple enzyme digestion or multidimensional separation can make sample preparation more efficient and that EThcD was not only suitable for the identification of singly O -glycosylated peptides (50.3%) but also doubly (21.2%) and triply (28.5%) O -glycosylated peptides. Finally, we got a largest data set of site-specific native O -glycoproteome from human serum samples. Furthermore, quantitative analysis of intact O -glycopeptides from the serum samples of IgA nephropathy (IgAN) patients and healthy donors was performed, and the results showed the potential of the strategy to discover O -glycosylation biomarkers. We expect that the strategies developed by this study will facilitate in-depth analyses of native O -glycoproteomes in human serum and lead to exciting opportunities to understand the functional roles of protein O -glycosylation in human health and diseases. [ABSTRACT FROM AUTHOR]

Protein N -glycosylation is one of the most important post-translational modifications, participating in many key biological and pathological processes. Large-scale and precise identification of N -glycosylated proteins and peptides is especially beneficial for understanding their biological functions and for discovery of new clinical biomarkers and therapeutic drug targets. However, protein N -glycosylation is microheterogeneous and low abundant in living organisms, therefore specific enrichment of N -glycosylated proteins/peptides before mass spectrometry analysis is a prerequisite. In this work, we developed a new type of polymer hybrid graphene oxide (GO) by in situ growth of hydrazide-functionalized hydrophilic polymer chains on the GO surface (GO-PAAH) for selective N -glycopeptide enrichment and identification by mass spectrometry. The densely attached and low steric hindrance hydrazide groups as well as the highly hydrophilic nature of GO-PAAH facilitate N -glycopeptide enrichment by the combination of hydrazide capturing and HILIC interaction. Taking advantage of the unique features of GO-PAAH, all of the three N -glycopeptides of bovine fetuin were successfully enriched and identified with significantly enhanced signal intensities from a digest mixture of bovine fetuin and bovine serum albumin at a mass ratio of 1:100, demonstrating the excellent enrichment selectivity of GO-PAAH. Furthermore, a total of 507 N -glycosylation sites and 480 N -glycopeptides in 232 N -glycoproteins were enriched and identified from 10 μL of human serum by three replicates using this novel enrichment material, which is nearly two times higher than the commercial hydrazide resin based method (280 N -glycosylation sites, 261 N -glycopeptides and 144 N -glycoproteins in three experiments). Among the identified, 95 N -glycosylation sites were not reported in the Uniprot database, and 106 N -glycoproteins were disease related in the Nextprot database, indicating the potential of this new enrichment material in global mapping of protein N -glycosylation. [ABSTRACT FROM AUTHOR]