Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 103
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Anal Chem ; 2020 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-32433867

RESUMO

Protein N-terminal acetylation (Nα-acetylation) is one of the most common modifications in both eukaryotes and prokaryotes. Although studies have shown that Nα-acetylation plays important roles in protein assembly, stability and location, the physiological role has not been fully elucidated. Therefore, a robust and large-scale analytical method is important for a better understanding of Nα-acetylation. Here, an enrichment strategy was presented based on LysN digestion and amine-reactive resin capture to study naturally acetylated protein N termini. Since LysN protease cleaves at the amino-terminus of lysine residue, all resulting peptides except naturally acetylated N terminal peptides contain free amino groups and can be removed by coupling with AminoLinkTM Resin. Therefore, the naturally acetylated N-terminal peptides were left in solution and enriched for further LC-MS/MS analysis. The method was very simple and fast, which contained no additional chemical derivatization except protein reduction and alkylation necessarily needed in bottom-up proteomics. It could be used to study acetylated N termini from complex biological samples without bias towards different peptides with various physicochemical properties. The enrichment specificity was above 99% when it was applied in HeLa cell lysates. Neo-N termini generated by endogenous degradation could be directly distinguished without use of stable-isotope labeling because no chemical derivatization was introduced in this method. Furthermore, this method was highly complementary to the traditional analytical methods for protein N termini based on trypsin only with ArgC-like activity. Therefore, the described method was beneficial to naturally acetylated protein N termini profiling.

2.
J Proteome Res ; 2020 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-32248692

RESUMO

Exosomes, a subtype of extracellular vesicles secreted by mammalian cells with a typical size range of 30-150 nm, have been implicated in many biological processes as intercellular communication carriers. The isolation of exosomes is an essential and challenging step before subsequent analysis and functional studies, due to the complexity of body fluids, as well as the small size and low density of exosomes. Ultracentrifugation (UC) and size exclusion chromatography (SEC) are two methods that have been extensively used for exosomes isolation in biological studies in recent years. In this work, we compared the characteristics of urinary exosomes extracted with SEC and UC methods in detail. Results showed that the SEC isolation method was superior to UC in the recovery of exosomal particles and proteins. The results of proteomics analysis showed that more purified exosomes were extracted with the SEC method. We also observed that parts of exosomes were ruptured and precipitated insufficiently during UC isolations. It not only led to a low recovery of exosome proteins but also resulted in a considerable loss of exosomal particles. Moreover, the exosomal rupture and particle loss in UC could not be avoided by resuspension of the exosomal particles. Our results also showed that exosomes from SEC purifications possessed a high internalization capability from 4 to 6 h when incubated with EA.hy926 and HCV29 cell lines.

3.
Anal Chim Acta ; 1100: 174-181, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31987138

RESUMO

For mass spectrometry (MS)-based N-glycoproteomics, selective enrichment of N-glycopeptides prior to MS analysis is a crucial step to reduce sample complexity. Enrichment based on covalent coupling is as an increasingly attractive strategy due to the unbiased and highly specific features. However, most of current covalent coupling reactions for N-glycopeptides enrichment are still limited by long coupling time and harsh coupling conditions. Herein, we developed a thiazolidine formation-based approach for ultrafast and highly efficient solid-phase extraction of N-Glycoproteome. With the use of facile synthesis of Cys-terminated magnetic nanoparticles, the oxidized glycan moieties on glycopeptides could be selectively captured by the ß-amino thiols groups on the surface of magnetic nanoparticles through thiazolidine formation. The coupling could be achieved within 30 min under mild condition, eliminating the addition of toxic catalyst or sample-destroying reducing agent. Also, the great enrichment performance for N-glycopeptides were obtained in terms of sensitivity (low fmol levels), selectivity (extracting N-glycopeptides from the mixture of glycopeptides and non-glycopeptides at a 1:100 molar ratio) and reproducibility (CVs<26%). Finally, this proposed method was successfully demonstrated by analyzing the N-glycoproteome from 2 µL human serum, which offers an alternative purification method for analysis of N-glycoproteome from complex biological samples.

4.
Anal Chem ; 92(1): 867-874, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31751117

RESUMO

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.

5.
Talanta ; 206: 120178, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514832

RESUMO

A facile and mild approach was carried out to synthesize citrate acid-magnetic ferroferric oxide for glycopeptide analysis. The material was synthesized successfully and applied in glycopeptide identification from human saliva, indicating that this method could be a promising tool for glycopeptidome analysis, which also enlightened the simple fabrication of hydrophilic materials in analytical science.


Assuntos
Ácido Cítrico/química , Glicopeptídeos/análise , Nanopartículas de Magnetita/química , Saliva/química , Cromatografia Líquida/métodos , Glicoproteínas/química , Humanos , Limite de Detecção , Fragmentos de Peptídeos/análise , Proteólise , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Tripsina/química
6.
Talanta ; 207: 120313, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31594600

RESUMO

Peptidomics research is of great significance for discovering potential biomarkers and monitoring human diseases. As a kind of common clinical biofluid, saliva known for its noninvasive collection and easy accessibility has been widely used in peptidomics research. In this article, we combined immobilized metal ions affinity chromotography (IMAC) with mesoporous material and proposed the copper ion doped magnetic mesoporous silica material (denoted as Fe3O4@mSiO2-Cu2+) which had a large surface area of 221 m2 g-1 and pore volume of 0.20 cm3 g-1. By immobilizing copper ions onto the mesopore walls, the standard peptide Angiotensin II could be identified in an extremely low concentration of 0.1 fmol µl-1 and in a mass ratio of 1:500 (Angiotensin II:BSA, m/m), which indicated significant sensitivity and a great size-exclusive ability. In addition, the introduction of polydopamine (PDA) made Fe3O4@mSiO2-Cu2+ more hydrophilic and biocompatible which could improve the profiling of endogenous peptides in bio-sample. Finally, 131 endogenous peptides were identified in human saliva after enrichment with Fe3O4@mSiO2-Cu2+. Therefore, Fe3O4@mSiO2-Cu2+ nanoparticles provided a promising candidate protocol for biomarker discovery.


Assuntos
Cromatografia Líquida , Cobre/química , Nanopartículas de Magnetita/química , Peptídeos/análise , Saliva/química , Dióxido de Silício/química , Espectrometria de Massas em Tandem , Angiotensina II/química , Animais , Bovinos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Peptídeos/química , Porosidade , Soroalbumina Bovina/química
7.
FASEB J ; 33(11): 13040-13050, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31487196

RESUMO

Embryonic stem cells (ESCs) are pluripotent stem cells with the ability to self-renew and to differentiate into any cell types of the 3 germ layers. Recent studies have demonstrated that there is a strong connection between mitochondrial function and pluripotency. Here, we report that methyltransferase like (Mettl) 17, identified from the clustered regularly interspaced short palindromic repeats knockout screen, is required for proper differentiation of mouse embryonic stem cells (mESCs). Mettl17 is located in mitochondria through its N-terminal targeting sequence and specifically interacts with 12S mitochondrial ribosomal RNA (mt-rRNA) as well as small subunits of mitochondrial ribosome (MSSUs). Loss of Mettl17 affects the stability of both 12S mt-rRNA and its associated proteins of MSSUs. We further showed that Mettl17 is an S-adenosyl methionine (SAM)-binding protein and regulates mitochondrial ribosome function in a SAM-binding-dependent manner. Loss of Mettl17 leads to around 70% reduction of m4C840 and 50% reduction of m5C842 of 12S mt-rRNA, revealing the first regulator of the m4C840 and indicating a crosstalk between the 2 nearby modifications. The defects of mitochondrial ribosome caused by deletion of Mettl17 lead to the impaired translation of mitochondrial protein-coding genes, resulting in significant changes in mitochondrial oxidative phosphorylation and cellular metabolome, which are important for mESC pluripotency.-Shi, Z., Xu, S., Xing, S., Yao, K., Zhang, L., Xue, L., Zhou, P., Wang, M., Yan, G., Yang, P., Liu, J., Hu, Z., Lan, F. Mettl17, a regulator of mitochondrial ribosomal RNA modifications, is required for the translation of mitochondrial coding genes.

8.
Mol Cell Proteomics ; 18(11): 2262-2272, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31501225

RESUMO

N-glycosylation alteration has been reported in liver diseases. Characterizing N-glycopeptides that correspond to N-glycan structure with specific site information enables better understanding of the molecular pathogenesis of liver damage and cancer. Here, unbiased quantification of N-glycopeptides of a cluster of serum glycoproteins with 40-55 kDa molecular weight (40-kDa band) was investigated in hepatitis B virus (HBV)-related liver diseases. We used an N-glycopeptide method based on 18O/16O C-terminal labeling to obtain 82 comparisons of serum from patients with HBV-related hepatocellular carcinoma (HCC) and liver cirrhosis (LC). Then, multiple reaction monitoring (MRM) was performed to quantify N-glycopeptide relative to the protein content, especially in the healthy donor-HBV-LC-HCC cascade. TPLTAN 205ITK (H5N5S1F1) and (H5N4S2F1) corresponding to the glycopeptides of IgA2 were significantly elevated in serum from patients with HBV infection and even higher in HBV-related LC patients, as compared with healthy donor. In contrast, the two glycopeptides of IgA2 fell back down in HBV-related HCC patients. In addition, the variation in the abundance of two glycopeptides was not caused by its protein concentration. The altered N-glycopeptides might be part of a unique glycan signature indicating an IgA-mediated mechanism and providing potential diagnostic clues in HBV-related liver diseases.

9.
Anal Chem ; 91(15): 9986-9992, 2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31313914

RESUMO

Protein sialylation is ubiquitous and essential in a wide range of biological processes. Herein, a mass defect-based chemical-directed proteomics method (MdCDPM) was presented for targeted analysis of intact sialylglycopeptides (SGPs). The process starts by specific oxidation of dihydroxy in sialic acid to aldehyde, which was then chemically labeled by two arginine isotopologues (Arg-15N4 and Arg-D4, differs by 36 mDa). The equally mixed precursor partners, spacing tens of mDa apart, enable the direct recognition of SGPs in MS1 level and benefit the subsequent targeted MS2 characterization. The mass envelope of two labeled forms falling into a narrow m/z window strengthens recognition uniqueness greatly, and the proposed 1:1 intensity ratio of doublets will not be readily distorted. More important, such subtle mass differences permit multiple sialic acids labeling without additional complexity of precursor patterns. Also, the partner m/z shifts detail the number of sialic acids contained in the precursor species. By applying MdCDPM, femtomole quantities of SGPs could be detected from total cell lysates, even at a signal-to-noise ratio of as low as 3:1. In addition, assays were performed to estimate the false positive rate and demonstrated high confidence of MdCDPM. Furthermore, it was designed and successfully exploited to analyze SGPs in human serum, which highlighted the feasibility of this strategy for biological applications.

10.
Talanta ; 204: 367-371, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31357307

RESUMO

A rapid and efficient method to isolate global N-termini is presented. Utilizing laser-assisted proteolysis and Fe3O4 microsphere, protein N-termini could be isolated in 4 h. The amino-blocked protein was digested by trypsin assisted by laser radiation, shortening the digest time from overnight to 40 s. Non-N-terminal peptides were characterized by a tryptic free amino in their N-term, which could be derived with sulfhydryl by traut' s reagent efficiently and then coupled with Fe3O4 microspheres nearly completely in less than 4 h. The rapid method was beneficial for the identification of unstable N-termini in short-lived proteins. Human serum albumin was studied as a model. The N-terminus was successfully isolated from the digest within 4 h. Also, 2011 N-terminal peptides out of 936 proteins in mouse liver proteome sample were identified using liquid chromatography-tandem mass spectrometer (LC-MS/MS). This method was demonstrated as a facile and efficient N-termini enrichment method for targeted protein N-termini analysis, especially those with short half-life.


Assuntos
Óxido Ferroso-Férrico/química , Peptídeos/análise , Domínios Proteicos , Proteoma/química , Análise de Sequência de Proteína/métodos , Sequência de Aminoácidos , Animais , Cromatografia Líquida/métodos , Óxido Ferroso-Férrico/síntese química , Humanos , Raios Infravermelhos , Lasers , Fígado/química , Camundongos , Microesferas , Peptídeos/química , Proteólise/efeitos da radiação , Proteoma/efeitos da radiação , Proteômica/métodos , Albumina Sérica Humana/química , Albumina Sérica Humana/efeitos da radiação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas em Tandem/métodos
11.
Anal Chem ; 91(10): 6498-6506, 2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31025853

RESUMO

Analysis of protein C termini is very important for functional annotations of proteomes, while proteome-wide C termini analysis still poses substantial challenges. Here we described a simple and robust strategy for specific isolation of protein C termini based on LysC digestion and site-selective dimethylation to deplete N-terminal and internal peptides by scavenger materials. The performance of LysC digestion and conditions of site-selective dimethylation and resin coupling were discussed in detail. Then the strategy was successfully applied to the characterization of protein C termini of HeLa cells. A total of 781 protein C termini were identified with a 300 µg digest in our study, among which 38.9% were actually not identifiable using current trypsin digestion-based methods due to their inappropriate peptide length for MS analysis, indicating that our method was highly complementary to the existing methods. The enrichment procedure was rapid and easy to operate and could afford a very good identification efficiency by obtaining the largest C termini data set of the human proteome with the least sample loading. This method was without bias toward physicochemical properties of peptides. Moreover, a peptide-centric database was first introduced to analyze protein C termini, which effectively improved the accuracy and speed of the database search. Therefore, our method can be used to effectively and selectively isolate protein C termini and contributes to the global annotation of C terminomes.

12.
Talanta ; 199: 254-261, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30952254

RESUMO

N-glycosylation is deeply involved in many biological processes, and approximately 50% of mammalian proteins are predicted to be glycosylated. Many large-scale studies have been carried out to reveal the glycosylation status involved in different physiological pathologies across species. However, the lack of a highly specific and high-throughput N-glycosylated enrichment method not only results in extended time requirements but also limits the depth of mapping when handling a large number of samples. In this study, we firstly optimized traditional zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) enrichment and found that using of 70% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA) as the enrichment buffer, 2800 g as the washing speed and 600 µL as the washing volume achieved the best specificity, which is higher than 75%. On this basis, we developed a multi-parallel enrichment strategy assisted by a filter-coated 96-well plate, which achieved high specificity and high throughput simultaneously. This strategy allowed us to enrich large numbers of fractionated samples from hepatocellular carcinoma (HCC) cell lines in less than 2 h. Its good specificity helped us achieve in-depth mapping of the N-glycoproteome in metastatic HCC cell lines. A total of 5466 N-glycosites from 2383 glycoproteins were identified, among which 1900 N-glycosites were unannotated in UniProt. The in-depth glycoproteome mapping provides insight into the N-glycosylation status in HCC cell lines with differences in metastatic potential and contributes to biomarker discovery.


Assuntos
Carcinoma Hepatocelular/química , Glicopeptídeos/química , Ensaios de Triagem em Larga Escala , Neoplasias Hepáticas/química , Proteoma/análise , Carcinoma Hepatocelular/metabolismo , Cromatografia Líquida , Glicopeptídeos/metabolismo , Glicosilação , Humanos , Neoplasias Hepáticas/metabolismo , Espectrometria de Massas , Mapeamento de Interação de Proteínas , Proteoma/metabolismo , Células Tumorais Cultivadas
13.
Anal Chem ; 91(8): 5235-5243, 2019 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-30892874

RESUMO

4-Hydroxy-2-nonenal (HNE)-modified proteins are closely associated with cellular functions and diseases, so qualitative and quantitative analysis of HNE-modified proteins is very necessary in order to further understand their structures and molecular functions. In this study, we described a six-plex isobaric labeling affinity purification (SiLAP) method based on the interaction of aminoxyTMT six-plex and anti-TMT antibody resin to identify and quantify the HNE modifications simultaneously. The labeling efficiency, ionization efficiency of the aminoxyTMT-tagged peptides, and reliability of the quantification method were investigated in detail. The mass tags were labeled on the modification sites, which could also significantly increase the ionization efficiency, contributing to site-specific identification and quantification of HNE peptides. The SiLAP strategy possessed high sensitivity, accuracy, and good reproducibility to qualitatively and quantitatively analyze HNE-modified proteins/peptides, which could be used to analyze both endogenously and exogenously modified proteins. Using the SiLAP strategy, 2257 HNE-modified peptides mapping 1121 proteins were collectively quantified, which was the largest data set of HNE-modified proteins with detailed modification sites, and 101 proteins were found to be differentially modified by HNE in six liver cell lines. At the same time, 33 endogenously HNE-modified peptides mapping 33 proteins were identified with modification sites.

14.
Anal Chim Acta ; 1058: 107-116, 2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-30851844

RESUMO

Increasing researches proved that abnormal glycosylation is strongly correlated with many diseases. Specially, site-specific glycosylation and its associated heterogeneity are closely related to the function and activity of the glycoprotein. However, intact N-glycopeptide analysis still faces great challenges because the presence of highly abundant non-glycosylated peptides would suppress the ionization of lowly abundant glycopeptides. In the present study, we developed a practical intact tryptic N-glycopeptide enrichment method using acrylamide-agarose composite gel that combined the size exclusion chromatography and hydrophilic (named SELIC) effects, aimed to remove the detergent rapidly and effectively, as well as enrich intact N-glycopeptides while extracting peptides. This is a useful tool to facilitate the intact N-glycopeptides analysis of complex protein mixtures, particularly for samples that extracted from formalin-fixed and paraffin-embedded (FFPE) tissues by SDS. Using this method, we successfully identified 700 site-specific intact tryptic N-glycopeptides corresponding to 261 glycosylation sites on 191 glycoproteins from FFPE thymoma tissues.


Assuntos
Acrilamida/química , Cromatografia em Gel/métodos , Glicopeptídeos/análise , Sefarose/química , Timoma/química , Neoplasias do Timo/química , Animais , Glicopeptídeos/química , Glicoproteínas/química , Glicosilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Proteólise , Tripsina/química
15.
Anal Bioanal Chem ; 411(18): 4141-4149, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30762101

RESUMO

Exosomes are cell-derived functional microparticles which exist in most body fluids. They carry abundant signaling molecules to transfer information between cells and microenvironment. Research on exosomes' heterogeneity and constitute variations has been a heated topic in recent years. In this work, size-dependent sub-proteome analysis of urinary exosomes was investigated by size exclusion chromatography (SEC) firstly. The particle size of urinary exosomes is distributed in four main ranges naturally. We found out that these fractions contained sub-proteomes with great difference in constitution. In each fraction, 206, 134, 157, and 276 unique proteins were identified by LC-MS/MS. Differential expression of exosomal markers such as TSG101, CD9, CD63, and caveolin-1 was observed in these fractions by western blots. Biological function annotation indicated that the proteins identified in each fraction were involved in different molecular and cellular processes. It is proven that SEC can serve as an efficient analytical tool for exosomes isolation and fractionation. This work provides a new strategy to classify exosomes into sub-populations for comprehensive study of heterogeneous functionalities. Graphical abstract ᅟ.


Assuntos
Exossomos , Proteoma , Urina/química , Western Blotting , Cromatografia em Gel/métodos , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Microscopia Eletrônica de Transmissão , Espectrometria de Massas em Tandem/métodos
16.
Nanoscale ; 11(8): 3701-3709, 2019 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-30742181

RESUMO

The highly effective analysis of glycopeptides from complex biological samples is an attractive and critical topic all the time. In this study, a novel thickness-controlled hydrophilic Mg-metal organic frameworks (Mg-MOFs) coating-functionalized magnetic graphene composite (MagG@Mg-MOFs-1C) was prepared for the capture of the glycopeptides. The as-synthesized composite exhibits an ultralow limit of detection (0.1 fmol µL-1), a perfect size-exclusion effect (HRP digests/BSA protein/HRP protein, 1 : 500 : 500, w/w/w), and a high binding capacity (150 mg g-1), satisfying reusability and high recovery in the recognition of glycopeptides due to its outstanding characteristics including strong magnetic property, large surface area (617 m2 g-1), plenty of affinity sites, and excellent hydrophilicity. Furthermore, the MagG@Mg-MOFs-1C composite was successfully applied to selectively enriched glycopeptides in human urine. More excitingly, 406 N-glycosylation peptides corresponding to 185 glycoproteins were identified in the urine of the bladder cancer patients, in which these identified glycoproteins include the potential biomarkers (α-2-macroglobulin, complement C4-B, and α-1-antitrypsin) for the bladder cancer. This study suggests that the hydrophilic porous MOFs-functionalized composite has a great potential in the large-scale characterization of the low-abundance biomolecules in urine, opening a new avenue for the rapid and convenient diagnosis of the disease.


Assuntos
Glicopeptídeos/urina , Grafite/química , Magnésio/química , Magnetismo , Estruturas Metalorgânicas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Cromatografia Líquida de Alta Pressão , Glicosilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Limite de Detecção , Ligação Proteica , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia
17.
Anal Bioanal Chem ; 411(2): 403-411, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30478513

RESUMO

Despite the importance of tobacco (Nicotiana tabacum) in agriculture and model organism investigations, the proteomic changes that occur in the tobacco leaf as it matures remain to be explored. In this study, an isobaric tags for relative and absolute quantification (iTRAQ) strategy was applied to investigate the proteomic profiles of K326 and Honghua Dajinyuan (HD) tobacco leaves at four growth stages. The proteomic profile varied with growth stage in both K326 and HD. Gene ontology (GO) classification was used to identify the biological processes that showed the greatest changes in protein expression between growth stages of HD and K326. Moreover, the number of differentially expressed proteins was greater in HD than in K326, especially during the rosette growth stage and the fast-growing stage. The galactose metabolism and glycosphingolipid biosynthesis-globo series pathways appeared only during the rosette growth stage of HD. It therefore appears that these pathways may be correlated with tobacco mosaic disease. The identification of these pathways should prove useful in investigations of the pathogenesis of tobacco mosaic virus. Graphical abstract ᅟ.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Proteômica/métodos , Tabaco/metabolismo , Transcriptoma , Tabaco/genética , Tabaco/crescimento & desenvolvimento
18.
Anal Chem ; 90(24): 14303-14308, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30500165

RESUMO

The identification and quantification of low-abundant proteins are always impeded by high-abundant proteins in proteomic analysis because of the extreme complexity of peptide mixtures and wide dynamic range of protein abundances. Here, we developed a novel approach to enrich and quantify N-terminal glycine peptides through sortase A mediated ligation. This strategy was based on the formation of a covalent bond between the sortase A recognition motif LPXTG and a N-terminal glycine residue. Also, the quantification was achieved by introducing isotopically labeled threonine in the motif LPXTG. In this strategy, both the enrichment of N-terminal glycine peptides and the stable isotope labeling were achieved in a single step. We applied this approach for the proteome analysis of MCF-7 cell line. It was demonstrated a significant reduction in sample complexity via highly selective and efficient enrichment of N-terminal glycine peptides, thereby detecting lots of less abundant proteins and enhancing proteome coverage. In comparison to the untreated sample, an increase of 34% of proteins was additionally identified. Furthermore, 97% of proteins were successfully quantified with high accuracy. In summary, this quantitative N-terminal glycine peptides enrichment strategy is expected for high-throughput qualitative and quantitative proteomic analysis as a complementary approach to conventional shotgun proteomics.


Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Glicina/química , Peptídeos/química , Peptídeos/metabolismo , Proteômica/métodos , Sequência de Aminoácidos , Humanos , Células MCF-7
19.
Anal Chem ; 90(23): 14003-14010, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30375851

RESUMO

In our previous work, we have demonstrated an integrated proteome analysis device (iPAD-100) to analyze proteomes from 100 cells. (1) In this work, for the first time, a novel integrated device for single-cell analysis (iPAD-1) was developed to profile proteins in a single cell within 1 h. In the iPAD-1, a selected single cell was directly sucked into a 22 µm i.d. capillary. Then the cell lysis and protein digestion were simultaneously accomplished in the capillary in a 2 nL volume, which could prevent protein loss and excessive dilution. Digestion was accelerated by using elevated temperature with ultrasonication. The whole time of cell treatment was 30 min. After that, single-cell digest peptides were transferred into an LC column directly through a true zero dead volume union, to minimize protein transfer loss. A homemade 22 µm i.d. nano-LC packing column with 3 µm i.d. ESI tip was used in the device to achieve ultrasensitive detection. A 30 min elution program was applied to analysis of the single-cell proteome. Therefore, the total time needed for a single-cell analysis was only 1 h. In an analysis of 10 single HeLa cells, a maximum of 328 proteins were identified in one cell by using an Orbitrap Fusion Tribrid MS instrument, and the detection limit was estimated at around 1.7-170 zmol. Such a sensitivity of the iPAD-1 was ∼120-fold higher than that of our previously developed iPAD-100 system. (1) Prominent cellular heterogeneity in protein expressive profiling was observed. Furthermore, we roughly estimated the phases of the cell cycle of tested HeLa cells by the amount of core histone proteins.


Assuntos
Proteoma/análise , Análise de Célula Única/instrumentação , Células HeLa , Humanos
20.
Curr Protoc Cell Biol ; 78(1): 5.8.1-5.8.8, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-30040186

RESUMO

Mass spectrometry-based proteomic technology experienced remarkable advancement in the past decades. However, their application was still hampered by the complexity of sample preparation. Conventional strategies for sample preparation incorporate multiple time-consuming steps, including cell lysis, protein extraction, protease cleavage, and desalting. Thus, we explored a simplified method (the cell-absorb method) during which living cells were absorbed into vacuum-dried polyacrylamide gel and directly digested in gel into peptides for subsequent LC-MS/MS analysis. As a consequence, both of the steps for cell lysis and protein extraction involved in traditional protocol were skipped. In addition to the decrease in time, more proteins were identified. Indeed, 3022 proteins were identified by the cell-absorb method. Meanwhile, only 2642 and 2420 proteins were identified by the classical SDS-PAGE based method and the reported gel absorption-based method, respectively. The cell-absorb method exhibited apparent advantage in terms of the depth of proteome coverage. Furthermore, the number of proteins identified show excellent reproducibility with a CV (coefficient of variation) of 0.03 among three replicates using the cell-absorb method. These advantages suggest that cell-absorb method is a promising choice for mapping the whole proteome of cells. © 2018 by John Wiley & Sons, Inc.


Assuntos
Métodos Analíticos de Preparação de Amostras , Espectrometria de Massas/métodos , Alquilação , Animais , Linhagem Celular , Géis/química , Humanos , Peptídeos/metabolismo , Tripsina/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA