Data-independent acquisition (DIA) is a high-throughput, unbiased mass spectrometry data acquisition method which has good quantitative reproducibility and is friendly to low-abundance proteins. It becomes the preferred choice for clinical proteomic studies especially for large cohort studies in recent years. The mass-spectrometry (MS)/MS spectra generated by DIA is usually heavily mixed with fragment ion information of multiple peptides, which makes the protein identification and quantification more difficult. Currently, DIA data analysis methods fall into two main categories, namely peptide-centric and spectrum-centric. The peptide-centric strategy is more sensitive for identification and more accurate for quantification. Thus, it has become the mainstream strategy for DIA data analysis, which includes four key steps: building a spectral library, extracting ion chromatogram, feature scoring and statistical quality control. This work reviews the peptide-centric DIA data analysis procedure, introduces the corresponding algorithms and software tools, and summarizes the improvements for the existing algorithms. Finally, the future development directions are discussed.
Humans ; Proteomics/methods* ; Reproducibility of Results ; Peptides/chemistry* ; Software ; Algorithms ; Tandem Mass Spectrometry/methods* ; Proteome/analysis*

The systematic and in-depth study of phosphoproteome rely on highly reproducible and specific phosphopeptide enrichment methods. At present, a variety of enrichment methods have been developed based on different principles, and these methods often display different selectivity and specificity. It is therefore very important to select the most suitable enrichment method according to different research purposes. This review summarized the phosphopeptide enrichment based on affinity chromatography, immunoprecipitation, chemical derivatization, chromatography and other newly developed methods. The advantages and disadvantages of these methods, as well as the related optimization and improvement strategies, were discussed in detail. In addition, we also briefly summarized the progress of the combination of phosphopeptide enrichment and fractionation methods developed in recent years.
Phosphopeptides/metabolism* ; Proteomics/methods* ; Titanium/chemistry* ; Chromatography, Affinity ; Proteome ; Phosphorylation

In the present study,fresh Guangdilong( GD),originating from Pheretima aspergillum,was taken as the object. The total proteins from GD were firstly separated by SDS-PAGE according to their molecular weights and in-gel digestion was then performed.After that,the peptides were analyzed by nano LC/orbitrap fusion lumos high resolution mass spectrometry( nano LC/orbitrap fusion lumos HR-MS). Protein identification was implemented by comparison with Annelida. fasta database using Proteome Discoverer software.As a result,386 proteins were tentatively identified,including chain F,globin B chain,glyceraldehyde-3-phosphate dehydrogenase,fibrinolytic protein,and so on. Most of the proteins took part in cell structure and energy metabolism,and fibrinolytic protein and lombricine kinase might be related to fibrinolytic activity. Protein classification based on gene ontology was carried out using PANTHER and KEGG for metabolic pathway enrichment. The results indicated that these proteins were related to diverse signal transduction pathways,including metabolic pathways,central carbon metabolism,biosynthesis of amino acids,ribosome,glycolysis,citrate cycle( TCA cycle),and so on. This study would lay the foundation for the further research on the proteins in GD and also their functions.
Animals ; Chromatography, Liquid ; Electrophoresis, Polyacrylamide Gel ; Gene Ontology ; Mass Spectrometry ; Oligochaeta ; chemistry ; Proteome ; Proteomics

Under different red (R):blue (B) photon flux ratios, the growth performance of rapeseed (Brassica napus L.) is significantly different. Rapeseed under high R ratios shows shade response, while under high B ratios it shows sun-type morphology. Rapeseed under monochromatic red or blue light is seriously stressed. Transcriptomic and proteomic methods were used to analyze the metabolic pathway change of rapeseed (cv. "Zhongshuang 11") leaves under different R:B photon flux ratios (including 100R:0B%, 75R:25B%, 25R:75B%, and 0R:100B%), based on digital gene expression (DGE) and two-dimensional gel electrophoresis (2-DE). For DGE analysis, 2054 differentially expressed transcripts (|log₂(fold change)|≥1, q<0.005) were detected among the treatments. High R ratios (100R:0B% and 75R:25B%) enhanced the expression of cellular structural components, mainly the cell wall and cell membrane. These components participated in plant epidermis development and anatomical structure morphogenesis. This might be related to the shade response induced by red light. High B ratios (25R:75B% and 0R:100B%) promoted the expression of chloroplast-related components, which might be involved in the formation of sun-type chloroplast induced by blue light. For 2-DE analysis, 37 protein spots showed more than a 2-fold difference in expression among the treatments. Monochromatic light (ML; 100R:0B% and 0R:100B%) stimulated accumulation of proteins associated with antioxidation, photosystem II (PSII), DNA and ribosome repairs, while compound light (CL; 75R:25B% and 25R:75B%) accelerated accumulation of proteins associated with carbohydrate, nucleic acid, amino acid, vitamin, and xanthophyll metabolisms. These findings can be useful in understanding the response mechanisms of rapeseed leaves to different R:B photon flux ratios.
Brassica napus/radiation effects* ; Brassica rapa/radiation effects* ; Carbon/chemistry* ; Chloroplasts/radiation effects* ; Computational Biology ; Electrophoresis, Gel, Two-Dimensional ; Gene Expression Regulation, Plant/radiation effects* ; Image Processing, Computer-Assisted ; Light ; Mass Spectrometry ; Metabolic Networks and Pathways ; Nitrogen/chemistry* ; Photons ; Photosystem II Protein Complex/genetics* ; Plant Leaves/radiation effects* ; Plant Proteins/genetics* ; Proteome ; Ribosomes ; Transcription, Genetic ; Transcriptome

OBJECTIVE: Hyperstimulation methods are broadly used for in vitro fertilization (IVF) in patients with infertility; however, the side effects associated with these therapies, such as ovarian hyperstimulation syndrome (OHSS), have not been well studied. N-glycoproteomes are subproteomes used for the remote sensing of ovarian stimulation in follicular growth. Glycoproteomic variation in human follicular fluid (hFF) has not been evaluated. In this study, we aimed to identify and quantify the glycoproteomes and N-glycoproteins (N-GPs) in natural and stimulated hFF using label-free nano-liquid chromatography/electrospray ionization-quad time-of-flight mass spectrometry. METHODS: For profiling of the total proteome and glycoproteome, pooled protein samples from natural and stimulated hFF samples were selectively isolated using hydrazide chemistry to obtain the total proteomes and glycoproteomes. N-GPs were validated by the consensus sequence N-X-S/T (92.2% specificity for the N-glycomotif at p<0.05). All data were compared between natural versus hyperstimulated hFF samples. RESULTS: We detected 41 and 44 N-GPs in the natural and stimulated hFF samples, respectively. Importantly, we identified 11 N-GPs with greater than two-fold upregulation in stimulated hFF samples compared to natural hFF samples. We also validated the novel N-GPs thyroxine-binding globulin, vitamin D-binding protein, and complement proteins C3 and C9. CONCLUSION: We identified and classified N-GPs in hFF to improve our understanding of follicular physiology in patients requiring assisted reproduction. Our results provided important insights into the prevention of hyperstimulation side effects, such as OHSS.
Chemistry ; Complement System Proteins ; Consensus Sequence ; Female ; Fertilization in Vitro* ; Follicular Fluid* ; Humans* ; In Vitro Techniques* ; Infertility ; Mass Spectrometry ; Ovarian Hyperstimulation Syndrome ; Ovulation Induction ; Physiology ; Proteome ; Proteomics ; Reproduction ; Sensitivity and Specificity ; Thyroxine-Binding Globulin ; Up-Regulation ; Vitamin D-Binding Protein

Reversible phosphorylation plays a crucial role in regulating protein activities and functions. Sexual reproduction directly affects yield of most agricultural crops. As the male reproductive organ, anther generates microspores (pollen), delivering gametes (sperms) to complete double fertilization in higher plants. Here, we took the advantage of Nano UHPLC-MS/MS to analyze maize (Zea mays, B73) early anthers at proteomic and phosphoproteomic levels, to explore the protein and phosphorylation modification regulatory networks controlling maize anther development. Our proteomic analysis identified 3 016 unique peptides, belonging to 1 032 maize proteins. MapMan analysis revealed variously potential proteins associated with maize anther development, such as receptor-like kinases (GRMZM2G082823_P01 and GRMZM5G805485_P01). Using phospho-peptides enriched by TiO2 affinity chromatography, our phosphoproteomic analysis detected 257 phospho-peptides from 210 phosphoproteins, discovering 223 phosphosites. Compared to the 86 maize phosphoproteins collected in the Plant Protein Phosphorylation Data Base (P3DB), we found that 203 phosphoproteins and 218 phosphosites were not revealed before. Further bioinformatics analysis revealed that phosphorylation of 14-3-3 proteins, kinases, phosphatases, transcription factors, cell cycle and chromatin structure related proteins might play important roles in regulating normal anther development in maize. Our findings not only enlarged the maize phosphoproteome data, but also provided information for analyzing the molecular mechanism controlling maize anther development at genetic and biochemical levels.
Crops, Agricultural ; chemistry ; Phosphoproteins ; chemistry ; Phosphorylation ; Plant Proteins ; chemistry ; Pollen ; chemistry ; Proteome ; Tandem Mass Spectrometry ; Zea mays ; chemistry

Protein ubiquitination is one of the most important and widely exist protein post-translational modifications in eukaryotic cells, which takes the ubiquitin and ubiquitin chains as signal molecules to covalently modify other protein substrates. It plays an important roles in the control of almost all of the life processes, including gene transcription and translation, signal transduction and cell-cycle progression, besides classical 26S protesome degradation pathway. Varied modification sites in the same substrates as well as different types of ubiquitin linkages in the same modification sites contain different structural information, which conduct different signal or even determine the fate of the protein substrates in the cell. Any abnormalities in ubiquitin chain formation or its modification process may cause severe problem in maintaining the balance of intracellular environment and finally result in serious health problem of human being. In this review, we discussed the discovery, genetic characteristics and the crystal structure of the ubiquitin. We also emphasized the recent progresses of the assembly processes, structure and their biological function of ubiquitin chains. The relationship between the disregulation and related human diseases has also been discussed. These progress will shed light on the complexity of proteome, which may also provide tools in the new drug research and development processes.
Humans ; Proteome ; Ubiquitin ; chemistry ; Ubiquitination

To understand molecular modules related to polyunsaturated fatty acids (PUFA) synthesis and eventually produce PUFA at high efficiency, we developed a protein complex analysis technology in Synechocystis sp. PCC 6803, and applied it to identify possible partner proteins interacting with the key enzymes that catalyze PUFA biosynthesis. We first constructed a recombinant expression of protein of slr1609 encoding the fatty acid activation enzyme, by fusing 3xFLAG tag with the target protein. Then we verified its expression by Western blotting targeting 3xFLAG tag. To maximize purification of Slr1609 protein complex, we optimized the protein expression conditions of Slr1609 in Synechocystis in a 5 L fermenter by monitoring its gene expression using RT-qPCR. The purification of the Slr1609 protein complexes was demonstrated by a Native-PAGE analysis. Finally, LC-MS/MS proteomic analysis allowed identification of the possible partner proteins interacting with Slr1609.
Bacterial Proteins ; chemistry ; Chromatography, Liquid ; Fatty Acid Synthases ; chemistry ; Fatty Acids, Unsaturated ; biosynthesis ; Proteome ; chemistry ; Proteomics ; Synechocystis ; enzymology ; Tandem Mass Spectrometry

Animals ; Databases, Protein ; Humans ; Models, Molecular ; Protein Interaction Domains and Motifs ; Protein Interaction Mapping ; methods ; Protein Interaction Maps ; Proteins ; chemistry ; metabolism ; Proteome ; chemistry ; metabolism

Platycodin D (PD), a triterpenoid saponin isolated from Platycodonis Radix, is a famous Chinese herbal medicine that has been shown to have anti-proliferative effects in several cancer cell lines. The aim of this study was to determine the changes in cellular proteins after the treatment of hepatocellular carcinoma HepG2 cells with PD using proteomics approaches. The cell viability was determined using the MTT assay. The proteome was analyzed by two-dimensional difference gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Western blot analysis was used to confirm the expression of changed proteins. Our results showed that PD inhibited the proliferation of HepG2 cells in concentration- and time-dependent manners. Sixteen proteins were identified to be up-regulated in PD-treated HepG2 cells, including ATP5H, OXCT1, KRT9, CCDC40, ERP29, RCN1, ZNF175, HNRNPH1, HSP27, PA2G4, PHB, BANF1, TPM3, ECH1, LGALS1, and MYL6. Three proteins (i.e., RPS12, EMG1, and KRT1) decreased in HepG2 cells after treatment with PD. The changes in HSP27 and PHB were further confirmed by Western blotting. In conclusion, our results shed new lights on the mechanisms of action for the anti-cancer activity of PD.
Antineoplastic Agents, Phytogenic ; pharmacology ; therapeutic use ; Apoptosis ; Blotting, Western ; Campanulaceae ; chemistry ; Carcinoma, Hepatocellular ; drug therapy ; metabolism ; Cell Proliferation ; Cell Survival ; Hep G2 Cells ; Humans ; Liver Neoplasms ; drug therapy ; metabolism ; Phytotherapy ; Plant Extracts ; pharmacology ; therapeutic use ; Proteome ; metabolism ; Proteomics ; Saponins ; pharmacology ; therapeutic use ; Triterpenes ; pharmacology ; therapeutic use ; Up-Regulation