Rapid and High-Sensitive Phosphoproteomics Elucidated the Spatial Dynamics of the Mouse Brain.

Recent developments in phosphoproteomics have enabled signaling studies where over 10,000 phosphosites can be routinely identified and quantified. Yet, current analyses are limited in sample size, reproducibility, and robustness, hampering experiments that involve low-input samples such as rare cells and fine-needle aspiration biopsies. To address these challenges, we introduced a simple and rapid phosphorylation enrichment method (miniPhos) that uses a minimal amount of the sample to get enough information to decipher biological significance. The miniPhos approach completed the sample pretreatment within 4 h and high effectively collected the phosphopeptides in a single-enrichment format with an optimized enrichment process and miniaturized system. This resulted in an average of 22,000 phosphorylation peptides quantified from 100 µg of proteins and even confidently localized over 4500 phosphosites from as little as 10 µg of peptides. Further application was carried out on different layers of mouse brain micro-sections; our miniPhos method provided quantitative information on protein abundance and phosphosite regulation for the most relevant neurodegenerative diseases, cancers, and signaling pathways in the mouse brain. Surprisingly, the phosphoproteome exhibited more spatial variations than the proteome in the mouse brain. Overall, spatial dynamics of phosphosites are integrated with proteins to gain insights into crosstalk of cellular regulation at different layers, thereby facilitating a more comprehensive understanding of mouse brain development and activity.

Cancer Serum Atlas-Supported Precise Pan-Targeted Proteomics Enable Multicancer Detection.

The wide dynamic range of serum proteome restrained discovery of clinically interested proteins in large cohort studies. Herein, we presented a high-sensitivity, high-throughput, and precise pan-targeted serum proteomic strategy for highly efficient cancer serum proteomic research and biomarker discovery. We constructed a resource of over 2000 cancer-secreted proteins, and the standard MS assays and spectra of at least one synthetic unique peptide per protein were acquired and documented (Cancer Serum Atlas, www.cancerserumatlas.com). Then, the standard peptide-anchored parallel reaction monitoring (SPA-PRM) method was developed with support of the Cancer Serum Atlas, achieving precise quantification of cancer-secreted proteins with high throughput and sensitivity. We directly quantified 325 cancer-related serum proteins in 288 serums of four cancer types (liver, stomach, lung, breast) and controls with the pan-targeted strategy and discovered considerable potential biomarker benefits for early detection of cancer. Finally, a proteomic-based multicancer detection model was built, demonstrating high sensitivity (87.2%) and specificity (100%), with 73.8% localization accuracy for an independent test set. In conclusion, the Cancer Serum Atlas provides a wide range of potential biomarkers that serve as targets and standard assays for systematic and highly efficient serological studies of cancer. The Cancer Serum Atlas-supported pan-targeted proteomic strategy enables highly efficient biomarker discovery and multicancer detection and thus can be a powerful tool for liquid biopsy.

Matricellular Protein Cilp1 Promotes Myocardial Fibrosis in Response to Myocardial Infarction.

[Figure: see text].

An Integrated Strategy Reveals Complex Glycosylation of Erythropoietin Using Mass Spectrometry.

The characterization of therapeutic glycoproteins is challenging due to the structural heterogeneity of the therapeutic protein glycosylation. This study presents an in-depth analytical strategy for glycosylation of first-generation erythropoietin (epoetin beta), including a developed mass spectrometric workflow for N-glycan analysis, bottom-up mass spectrometric methods for site-specific N-glycosylation, and a LC-MS approach for O-glycan identification. Permethylated N-glycans, peptides, and enriched glycopeptides of erythropoietin were analyzed by nanoLC-MS/MS, and de-N-glycosylated erythropoietin was measured by LC-MS, enabling the qualitative and quantitative analysis of glycosylation and different glycan modifications (e.g., phosphorylation and O-acetylation). The newly developed Python scripts enabled the identification of 140 N-glycan compositions (237 N-glycan structures) from erythropoietin, especially including 8 phosphorylated N-glycan species. The site-specificity of N-glycans was revealed at the glycopeptide level by pGlyco software using different proteases. In total, 114 N-glycan compositions were identified from glycopeptide analysis. Moreover, LC-MS analysis of de-N-glycosylated erythropoietin species identified two O-glycan compositions based on the mass shifts between non-O-glycosylated and O-glycosylated species. Finally, this integrated strategy was proved to realize the in-depth glycosylation analysis of a therapeutic glycoprotein to understand its pharmacological properties and improving the manufacturing processes.

Puromycin-Modified Silica Microsphere-Based Nascent Proteomics Method for Rapid and Deep Nascent Proteome Profile.

Nascent proteome is crucial in directly revealing how the expression of a gene is regulated on a translation level. In the nascent protein identification, puromycin capture is one of the pivotal methods, but it is still facing the challenge in the deep profiling of nascent proteomes due to the low abundance of most nascent proteins. Here, we describe the synthesis of puromycin-modified silica microspheres (PMSs) as the sorbent of dispersive solid-phase microextraction and the establishment of the PMS-based nascent proteomics (PMSNP) method for efficient capture and analysis of nascent proteins. The modification efficiency of puromycin groups on silica microspheres reached 91.8% through the click reaction. After the optimization and simplification of PMSNP, more than 3500 and 3900 nascent proteins were rapidly identified in HeLa cells and mouse brains within 13.5 h, respectively. The PMSNP method was successfully applied to explore changes in the translation process in a biological stress model, namely, the lipopolysaccharide-stimulated HeLa cells. Biological functional analyses revealed the unique characters of the nascent proteomes and exhibited the superiority of the PMSNP in the identification of low abundance and secreted nascent proteins, thus demonstrating the sensitivity and immediacy of the PMSNP method.

Quantitative proteomics reveals stage-specific protein regulation of triple negative breast cancer.

BACKGROUNDS: Triple negative breast cancer (TNBC) is a heterogeneous disease with more aggressive clinical courses than other subtypes of breast cancer. In this study, we performed high-resolution mass spectrometry-based quantitative proteomics with TNBC clinical tissue specimens to explore the early and sensitive diagnostic signatures and potential therapeutic targets for TNBC patients. METHODS: We performed an iTRAQ labeling coupled LC-MS/MS approach to explore the global proteome in tumor tissues and corresponding para-tumor tissues from 24 patients with grade I-II and grade III primary TNBC. Relative peptide quantification and protein identification were performed by Proteome Discoverer™ software with Mascot search engine. Differentially expressed proteins were analyzed by bioinformatic analyses, including GO function classification annotation and KEGG enrichment analysis. Pathway analyses for protein-protein interactions and upstream regulations of differentially expressed candidates were performed by Ingenuity Pathway Analysis (IPA) software. RESULTS: Totally, 5401 unique proteins were identified and quantified in different stage of TNBCs. 845 proteins were changed in patients with grade I or II TNBC, among which 304 were up-regulated and 541 were down-regulated. Meanwhile, for patients with grade III TNBC, 358 proteins were increased and 651 proteins were decreased. Comparing to para-cancerous tissues, various signaling pathways and metabolic processes, including PPAR pathways, PI3K-Akt pathway, one-carbon metabolism, amino acid synthesis, and lipid metabolism were activated in TNBC cancer tissues. Death receptor signaling was significantly activated in grade I-II TNBCs, however, remarkably inhibited in grade III TNBCs. Western blot experiments were conducted to validate expression levels of CYCS, HMGA1 and XIAP with samples from individual patients. CONCLUSIONS: Overall, our proteomic data presented precise quantification of potential signatures, signaling pathways, regulatory networks, and characteristic differences in each clinicopathological subgroup. The proteome provides complementary information for TNBC accurate subtype classification and therapeutic targets research.

A Multi-Omics Study of Human Testis and Epididymis.

The human testis and epididymis play critical roles in male fertility, including the spermatogenesis process, sperm storage, and maturation. However, the unique functions of the two organs had not been systematically studied. Herein, we provide a systematic and comprehensive multi-omics study between testis and epididymis. RNA-Seq profiling detected and quantified 19,653 in the testis and 18,407 in the epididymis. Proteomic profiling resulted in the identification of a total of 11,024 and 10,386 proteins in the testis and epididymis, respectively, including 110 proteins that previously have been classified as MPs (missing proteins). Furthermore, Five MPs expressed in testis were validated by the MRM method. Subsequently, multi-omcis between testis and epididymis were performed, including biological functions and pathways of DEGs (Differentially Expressed Genes) in each group, revealing that those differences were related to spermatogenesis, male gamete generation, as well as reproduction. In conclusion, this study can help us find the expression regularity of missing protein and help related scientists understand the physiological functions of testis and epididymis more deeply.

Multilayered N-Glycoproteome Profiling Reveals Highly Heterogeneous and Dysregulated Protein N-Glycosylation Related to Alzheimer's Disease.

Protein N-glycosylation is ubiquitous in the brain and is closely related to cognition and memory. Alzheimer's disease (AD) is a multifactorial disorder that lacks a clear pathogenesis and treatment. Aberrant N-glycosylation has been suggested to be involved in AD pathology. However, the systematic variations in protein N-glycosylation and their roles in AD have not been thoroughly investigated due to technical challenges. Here, we applied multilayered N-glycoproteomics to quantify the global protein expression levels, N-glycosylation sites, N-glycans, and site-specific N-glycopeptides in AD (APP/PS1 transgenic) and wild-type mouse brains. The N-glycoproteomic landscape exhibited highly complex site-specific heterogeneity in AD mouse brains. The generally dysregulated N-glycosylation in AD, which involved proteins such as glutamate receptors as well as fucosylated and oligomannose glycans, were explored by quantitative analyses. Furthermore, functional studies revealed the crucial effects of N-glycosylation on proteins and neurons. Our work provides a systematic multilayered N-glycoproteomic strategy for AD and can be applied to diverse biological systems.

Fast and precise quantification of serum biomarkers and simultaneous recognition of multiple diseases enabled by a stable isotope-labelled peptides assisted high-throughput MRM strategy.

Serum/plasma holds promise as an important source of disease-related proteins and even biomarkers in clinical practice. However, the discovery of biomarker candidates in serum/plasma remains challenging. In this study, we constructed an MS strategy that enables the fast and precise quantification of serum biomarkers through coupling a high-throughput scheduled MRM strategy with a stable isotope-labelled (SIL) peptide panel from more than 500 plasma proteins as internal standards. With this strategy, we discovered relevant serum proteins of atherosclerosis (AS), lung cancer (LC) and breast cancer (BC), which can simultaneously recognize these diseases. The results indicate that the powerful strategy we constructed has the potential for serum biomarker screening and disease detection.

Regulation of maternal phospholipid composition and IP(3)-dependent embryonic membrane dynamics by a specific fatty acid metabolic event in C. elegans.

Natural fatty acids (FAs) exhibit vast structural diversity, but the functional importance of FA variations and the mechanism by which they contribute to a healthy lipid composition in animals remain largely unexplored. A large family of acyl-CoA synthetases (ACSs) regulates FA metabolism by esterifying FA to coenyzme A. However, little is known about how particular FA-ACS combinations affect lipid composition and specific cellular functions. We analyzed how the activity of ACS-1 on branched chain FA C17ISO impacts maternal lipid content, signal transduction, and development in Caenorhabditis elegans embryos. We show that expression of ACS-1 in the somatic gonad guides the incorporation of C17ISO into certain phospholipids and thus regulates the phospholipid composition in the zygote. Disrupting this ACS-1 function causes striking defects in complex membrane dynamics, including exocytosis and cytokinesis, leading to early embryonic lethality. These defects are suppressed by hyperactive IP(3) signaling, suggesting that C17ISO and ACS-1 functions are necessary for optimal IP(3) signaling essential for early embryogenesis. This study shows a novel role of branched chain FAs whose functions in humans and animals are unknown and uncovers a novel intercellular regulatory pathway linking a specific FA-ACS interaction to specific developmental events.

A new panel of pancreatic cancer biomarkers discovered using a mass spectrometry-based pipeline.

This corrects the article DOI: 10.1038/bjc.2017.85.

In Situ Synthesis of Magnetic Mesoporous Phenolic Resin for the Selective Enrichment of Glycopeptides.

Protein glycosylation is a significant participant in a mass of biological processes, which is a pivotal protein post-translational modification. Due to the low contents of glycopeptides compared with nonglycopeptides and the microheterogeneity of glycosylation sites, highly selective enrichment methods for the purification of glycopeptides are required for the comprehensive characterization of glycoproteomics. In this work, a type of magnetic mesoporous phenolic resin (MMP) was prepared using branched polyethylenimine (PEI) as a cross-linker from a homogeneous magnetic Fe3O4@SiO2 solution in a resorcinol/formaldehyde monomer aqueous system via an in situ emulsion polymerization procedure. The results showed that MMP exhibited good biocompatibility, a mesoporous structure, nitrogen-containing functionality, excellent hydrophilicity, and solvent resistance by using multiple characterization methods. By taking advantage of the interaction between hydrophilic groups on the MMP and glycan components on the glycopeptides, the acquired MMP was utilized to the selective capture of N-glycopeptides (human IgG or HRP tryptic digests/BSA proteins = 1:50), good recovery yield (70.18-97.23%), superior binding capacity (400 mg g-1), and excellent reproducibility. Based on the outstanding performance in standard glycoproteins tryptic digests enrichment, MMP was further used to capture N-glycopeptides from tryptic digests of human serum. A total of 15 unique N-glycopeptides were identified from an ultralow sample volume (0.025 µL) of human serum. Overall, we identified 356 unique N-glycopeptides corresponding to 119 glycoproteins from human serum (0.35 µL) in the overlap of three replicate analyses. All the results have demonstrated that MMP has great potential in large-scale N-glycoproteomics research.

Functional lipidomics: Palmitic acid impairs hepatocellular carcinoma development by modulating membrane fluidity and glucose metabolism.

Lipids are essential cellular components and energy sources of living organisms, and altered lipid composition is increasingly recognized as a signature of cancer. We performed lipidomic analysis in a series of hepatocellular carcinoma (HCC) cells and identified over 1,700 intact lipids originating from three major lipid categories. Comparative lipidomic screening revealed that 93 significantly changed lipids and decreased palmitic acyl (C16:0)-containing glycerophospholipids were positively associated with metastatic abilities of HCC cells. Furthermore, both in vitro and in vivo experiments demonstrated that C16:0 incubation specifically reduced malignant cell proliferation, impaired cell invasiveness, and suppressed tumor growth in mouse xenograft models. Biochemical experiments demonstrated that C16:0 treatment decreased cell membrane fluidity and limited glucose metabolism. A phosphoproteomics approach further revealed such C16:0 incubation attenuated phosphorylation levels of mammalian target of rapamycin (mTOR) and signal transducer and activator of transcription 3 (STAT3) pathway proteins. Multiple reaction monitoring analysis of 443 lipid molecules showed 8 reduced C16:0-containing lipids out of total 10 altered lipids when cancer tissues were compared with adjacent nontumor tissues in a cohort of clinical HCC specimens (P < 0.05). CONCLUSION: These data collectively demonstrate the biomedical potential of using altered lipid metabolism as a diagnostic marker for cancerous cells and open an opportunity for treating aggressive HCCs by targeting altered C16:0 metabolism. (Hepatology 2017;66:432-448).

Study on behaviors and performances of universal N-glycopeptide enrichment methods.

Glycosylation is a crucial process in protein biosynthesis. However, the analysis of glycopeptides through MS remains challenging due to the microheterogeneity and macroheterogeneity of the glycoprotein. Selective enrichment of glycopeptides from complex samples prior to MS analysis is essential for successful glycoproteome research. In this work, we systematically investigated the behaviors and performances of boronic acid chemistry, ZIC-HILIC, and PGC of glycopeptide enrichment to promote understanding of these methods. We also optimized boronic acid chemistry and ZIC-HILIC enrichment methods and applied them to enrich glycopeptides from mouse liver. The intact N-glycopeptides were interpreted using the in-house analysis software pGlyco 2.0. We found that boronic acid chemistry in this study preferred to capture glycopeptides with high mannose glycans, ZIC-HILIC enriched most N-glycopeptides and did not show significant preference during enrichment and PGC was not suitable for separating glycopeptides with a long amino acid sequence. We performed a detailed study on the behaviors and performances of boronic acid chemistry, ZIC-HILIC, and PGC enrichment methods and provide a better understanding of enrichment methods for further glycoproteomics research.

A new panel of pancreatic cancer biomarkers discovered using a mass spectrometry-based pipeline.

BACKGROUND: Pancreatic carcinoma (PC) is an aggressive malignancy that lacks strategies for early detection. This study aimed to develop a coherent, high-throughput and non-discriminatory pipeline for the novel clinical biomarker discovery of PC. METHODS: We combined mass spectrometry (MS)-intensive methods such as isobaric tags for relative and absolute quantitation with two-dimensional liquid chromatography-tandem mass spectrometry (iTRAQ-2DLC-MS/MS), 1D-targeted LC-MS/MS, prime MRM (P-MRM) and stable isotope dilution-based MRM (SID-MRM) to analyse serum samples from healthy people (normal control, NC), patients with benign diseases (BD) and PC patients to identify novel biomarkers of PC. RESULTS: On the basis of the newly developed pipeline, we identified >1000 proteins, verified 142 differentially expressed proteins and finally targeted four proteins for absolute quantitation in 100 serum samples. The novel biomarker panel of apolipoprotein E (APOE), inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3), apolipoprotein A-I (APOA1), apolipoprotein L1 (APOL1), combining with CA19-9, statistically-significantly improved the sensitivity (95%) and specificity (94.1%), outperforming CA19-9 alone, for the diagnosis of PC. CONCLUSIONS: We developed a highly efficient pipeline for biomarker discovery, verification and validation, with each step systematically informing the next. A panel of proteins that might be clinically relevant biomarkers for PC was found.

Site-specific structural characterization of O-glycosylation and identification of phosphorylation sites of recombinant osteopontin.

Osteopontin (OPN) plays a key role in multiple physiological and pathological processes such as cytokine production, mineralization, inflammation, immune responses, and tumorigenesis. Post-translational modifications (PTMs) of OPN significantly affect its structure and biological properties; however, site-specific characterization of O-glycosylation in human OPN has not been reported. In this work, we profiled the overall glycan pattern of human recombinant OPN using a lectin array and completed detailed structural analysis of O-glycopeptides by mass spectrometry (MS). We detected 28 O-glycopeptides from 7 O-glycosylation regions of human OPN, occupied by highly heterogeneous O-glycans. These O-glycans carried, in part, functionally relevant epitopes such as T antigens (Galß1-3GalNAcα1-), sialyl-Tn antigens, sialyl-T antigens, and sialyl-Le(x/a) antigens [Neuα2-3Galß1-4(Fucα1-3)GlcNAc/Neuα2-3Galß1-3(Fucα1-4)GlcNAc]. MS(3) spectra of the generated O-glycopeptides showed cleavages of the peptide backbone and provided essential information on the peptide sequence. Furthermore, 26 phosphorylation sites were identified by reverse-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS), including a novel one (Y209). We provide a detailed, site-specific structural characterization of O-glycosylation and identify the phosphorylation sites of OPN. These data lay the foundation for further research into the role of oligosaccharides and phosphorylation of recombinant human OPN. This article is part of a Special Issue entitled: Medical Proteomics.

Mapping and analyzing the human liver proteome: progress and potential.

INTRODUCTION: The liver is an important organ in humans. Hepatocellular carcinoma (HCC) is one of the deadliest cancers in the world. Progress in the Human Liver Proteome Project (HLPP) has improved understanding of the liver and the liver cancer proteome. AREAS COVERED: Here, we summarize the recent progress in liver proteome modification profiles, proteomic studies in liver cancer, proteomic study in the search for novel liver cancer biomarkers and drug targets, and progress of the Chromosome Centric Human Proteome Project (CHPP) in the past five years in the Institutes of Biomedical Sciences (IBS) of Fudan University. Expert commentary: Recent advances and findings discussed here provide great promise of improving the outcome of patients with liver cancer.

Self-aspiration sampling extractive electrospray ionization mass spectrometry (EESI-MS) for high-throughput analysis of liquid samples.

RATIONALE: Extractive electrospray ionization mass spectrometry (EESI-MS) was invented as a typical ambient mass spectrometry method (AMS) and has been used for analyzing complex liquid samples. Here, we designed a Venturi effect-based self-aspiration sampling device and applied it to the EESI-MS for high-throughput analysis of liquid sample. METHODS: A special concentric nebulizer was designed and employed to produce a suction force for the direct aspiration of liquid samples, followed by ionization and detection. This sample aspiration process was explained and optimized using computational fluid dynamics (CFD) analysis. Experiment data were recorded to exhibit the sensitivity, memory effect, inter-day reproducibility, throughput, and applicability of the self-aspiration sampling EESI-MS. RESULTS: The limit of detection (LOD) of this method was determined as 4.5 × 10(-10)  g/mL (S/N = 3) for caffeine, and the sample throughput and relative standard deviation (RSD) for full scan mode can reach 0.67 samples/s and 4.76%, respectively. Even for MS/MS mode, the frequency can still be kept at 0.4 samples/s (RSD = 4.71%). Inter-day RSD examined in 1 week was below 10% for the signal of characteristic fragment ions of reserpine. Moreover, based on this method, the amount of caffeine in instant coffee was determined as 4.7%. This device was also proven to be suitable for the protein/peptide analysis. CONCLUSIONS: These experiment results, especially the amazing results on sample throughput and inter-day RSD, suggest that we provide a valuable device which can be used for the direct high-throughput qualitative/quantitative mass spectrometry analysis of real liquid samples in ambient. Copyright © 2016 John Wiley & Sons, Ltd.

Transcriptome and proteome of human hepatocellular carcinoma reveal shared metastatic pathways with significant genes.

Previously isolated pathways screened from individual genes were investigated at either the transcriptional or translational level; however, the consistency between the pathways screened at the gene expression levels was obscure in metastatic human hepatocellular carcinoma (HCC). To elucidate this question, we performed a transcriptomic (16,353 genes) and proteomic (7861 proteins) analysis simultaneously on six metastatic HCC cell lines against two nonmetastatic HCC cell lines, with all HBV traceable and close genetic-backgrounds for a comparative study. The quantitative and integrated results showed that significant genes were screened differentially with 351 transcripts from the transcriptome and 304 proteins from the proteome, with limited overlapping genes (7%). However, we discovered that these discrete 351 transcripts and 304 proteins screened share extrusive significant-pathways/networks with a 77% overlap, including active TGF-ß, RAS, NFκB, and Wnt, and inactive HNF4A, which are responsible for HCC metastasis. We conclude that the discrete, but significant genes predicted by either ome play intrinsically important roles in the linkage of responsible pathways shared by both omes in HCC metastasis.