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1.
Anal Methods ; 16(27): 4590-4598, 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-38920099

RESUMO

Protein S-acylation is an important lipid modification and plays a series of biological functions. As a classic proteomic method for S-acylated proteome analysis, the acyl-biotin exchange and its derivative methods are known to be very labour-intensive and time-consuming all the time, and will result in significant sample loss. Multiple methanol-chloroform precipitations are involved in order to remove the substances that would interfere with enrichment and identification including detergents, the residual reduction and alkylation reagents. Here, we developed a rapid and convenient method for S-acylation proteomics by combining a dissolvable tube gel and the classic ABE method, a Dissolvable Gel based One-Tube sample Treatment method (DGOTT) method. The protein fixation rate, impact of the gel size on analysis performance and feasibility for analyzing complex samples were evaluated. This method enabled the alkylation and chemical substitution reactions to be conducted in a single EP tube, and convenient removal of interferents through gel washing, which could obviously simplify operations and shorten the sample treatment duration. Finally, we identified a total of 1625 potential S-acylated proteins from 800 µg of mouse brain cerebral cortex proteins. We believe that our method could offer potential for high-throughput analysis of protein S-acylation.


Assuntos
Proteômica , Acilação , Animais , Proteômica/métodos , Camundongos , Resinas Acrílicas/química , Eletroforese em Gel de Poliacrilamida , Córtex Cerebral/química
2.
Talanta ; 276: 126300, 2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-38795647

RESUMO

N-myristoylation is one of the most widespread and important lipidation in eukaryotes and some prokaryotes, which is formed by covalently attaching various fatty acids (predominantly myristic acid C14:0) to the N-terminal glycine of proteins. Disorder of N-myristoylation is critically implicated in numerous physiological and pathological processes. Here, we presented a method for purification and comprehensive characterization of endogenous, intact N-glycine lipid-acylated peptides, which combined the negative selection method for N-terminome and the nanographite fluoride-based solid-phase extraction method (NeS-nGF SPE). After optimizing experimental conditions, we conducted the first global profiling of the endogenous and heterogeneous modification states for N-terminal glycine, pinpointing the precise sites and their associated lipid moieties. Totally, we obtained 76 N-glycine lipid-acylated peptides, including 51 peptides with myristate (C14:0), 10 with myristoleate (C14:1), 6 with tetradecadienoicate (C14:2), 5 with laurate (C12:0) and 4 with lauroleate (C12:1). Therefore, our proteomic methodology could significantly facilitate precise and in-depth analysis of the endogenous N-myristoylome and its heterogeneity.


Assuntos
Ácido Mirístico , Extração em Fase Sólida , Extração em Fase Sólida/métodos , Ácido Mirístico/química , Ácido Mirístico/análise , Proteômica/métodos , Fluoretos/química , Fluoretos/análise , Glicina/química , Glicina/análise , Peptídeos/química , Peptídeos/análise
3.
Cell Rep ; 43(2): 113720, 2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38308845

RESUMO

LMNA gene mutation can cause muscular dystrophy, and post-translational modification plays a critical role in regulating its function. Here, we identify that lamin A is palmitoylated at cysteine 522, 588, and 591 residues, which are reversely catalyzed by palmitoyltransferase zinc finger DHHC-type palmitoyltransferase 5 (ZDHHC5) and depalmitoylase α/ß hydrolase domain 7 (ABHD7). Furthermore, the metabolite lactate promotes palmitoylation of lamin A by inhibiting the interaction between it and ABHD7. Interestingly, low-level palmitoylation of lamin A promotes, whereas high-level palmitoylation of lamin A inhibits, murine myoblast differentiation. Together, these observations suggest that ABHD7-mediated depalmitoylation of lamin A controls myoblast differentiation.


Assuntos
Lamina Tipo A , Distrofias Musculares , Animais , Camundongos , Diferenciação Celular , Lamina Tipo A/metabolismo , Distrofias Musculares/genética , Mioblastos/metabolismo , Processamento de Proteína Pós-Traducional
4.
Analyst ; 149(4): 1111-1120, 2024 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-38170640

RESUMO

Protein S-acylation is an important lipid modification characteristic for heterogeneity in the acyl chain and dynamicity in the acylation/deacylation cycle. Most S-acylproteomic research has been limited by indirect identification of modified proteins/peptides without attached fatty acids, resulting in the failure to precisely characterize S-acylated sites with attached fatty acids. The study of S-acylation turnover is still limited at the protein level. Herein, aiming to site-specifically profile both the heterogeneity and the turnover of S-acylation, we first developed a site-specific strategy for intact S-acylated peptide analysis by introducing an acid cleavable bioorthogonal tag into a metabolic labelling method (ssMLCC). The cleavable bioorthogonal tag allowed for the selective enrichment and efficient MS analysis of intact S-acylated peptides so that S-acylated sites and their attached fatty acids could be directly analysed, enabling the precise mapping of S-acylated sites, as well as circumventing false positives from previous studies. Moreover, 606 S-palmitoylated (C16:0) sites of 441 proteins in HeLa cells were identified. All types of S-acylated peptides were further characterized by an open search, providing site-specific profiling of acyl chain heterogeneity, including S-myristoylation, S-palmitoylation, S-palmitoleylation, and S-oleylation. Furthermore, site-specific monitoring of S-palmitoylation turnover was achieved by coupling with pulse-chase methods, facilitating the detailed observation of the dynamic event at each site in multi-palmitoylated proteins, and 85 rapidly cycling palmitoylated sites in 79 proteins were identified. This study provided a strategy for the precise and comprehensive analysis of protein S-acylation based on intact S-acylated peptide analysis, contributing to the further understanding of its complexity and biological functions.


Assuntos
Ácidos Graxos , Proteínas , Humanos , Células HeLa , Proteínas/metabolismo , Ácidos Graxos/metabolismo , Peptídeos/metabolismo , Acilação
5.
J Affect Disord ; 347: 437-444, 2024 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-38000472

RESUMO

OBJECTIVE: This pilot study was designed to investigate the antidepressant effects of dexmedetomidine (DEX), a selective α2-adrenergic receptor agonist, in patients with treatment-resistant depression (TRD). The antidepressant effects of dexmedetomidine was compared with ECT, which is widely used in clinical practice for treatment of patients with TRD. METHODS: Seventy six patients with TRD were randomly assigned to receive 10 sessions of DEX infusions or electroconvulsive therapy (ECT) treatment. The primary outcome was the changes of depression severity determined by the improvement of 24-item Hamilton Depression Rating Scale (HDRS-24). The second outcomes were the rates of therapeutic response (reduction in HDRS-24 ≥ 50 %) and remission (HDRS-24 ≤ 10 and reduction in HDRS-24 ≥ 60 %) at posttreatment and after 3 months of follow-up visits. RESULTS: We found that 10 sessions of DEX infusions or ECT treatments significantly improved HDRS-24 scores at posttreatment and after 3 months of follow-up visits compared with the baseline. In addition, there was no significant difference between DEX infusions and ECT treatments regarding HDRS-24 at these evaluating points. Furthermore, the depression severity dropped to mild after 2 sessions of DEX infusion. In contrast, at least 6 sessions of ECT treatment were needed to achieve a same level. Finally, the rates of therapeutic response and remission were comparable between the two groups. No serious adverse events were observed. CONCLUSIONS: Based on current published evidence, we conclude that DEX exhibits rapid and durable antidepressant properties similar to ECT but with fewer side effects.


Assuntos
Dexmedetomidina , Eletroconvulsoterapia , Humanos , Dexmedetomidina/uso terapêutico , Depressão/terapia , Projetos Piloto , Resultado do Tratamento , Antidepressivos/uso terapêutico
7.
Anal Chem ; 95(35): 13055-13063, 2023 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-37611173

RESUMO

S-acylation is a widespread lipidation form in eukaryotes in which various fatty acids can be covalently attached to specific cysteine residues. However, due to the low reactivity of the lipid moieties and lack of specific antibodies, purification of intact S-acylated peptides remains challenging. Here, we developed a pretreatment method for direct separation and global analysis of endogenously intact S-acylated peptides by nanographite fluoride-based solid-phase extraction (nGF-SPE), together with the investigation and optimization of the enrichment procedure as well as the LC-MS/MS analysis process. Consequently, we performed the first global profiling of endogenously intact S-acylated peptides, with 701 S-palmitoylated peptides from HeLa cell lysates in a restricted search. Furthermore, coupling the nGF-SPE method with open search mode, altogether 1119 intact S-acylated peptides were identified with the attached palmitate, palmitoleate, myristate, and octanoate chain, respectively, providing a global insight into the endogenously heterogeneous modification state. Notably, we found and validated that S-palmitoleoylation (C16:1) provided less affinity toward lipid rafts compared with S-palmitoylation (C16:0). This study developed the first straightforward way to characterize endogenously intact S-acylated peptides on a proteome-wide scale, providing the modified residues together with their attached lipid moieties simultaneously, which paves the way for further understanding of protein S-acylation.


Assuntos
Fluoretos , Espectrometria de Massas em Tandem , Animais , Humanos , Cromatografia Líquida , Células HeLa , Acilação , Diferenciação Celular , Mamíferos
8.
Eur J Radiol ; 166: 111003, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37506477

RESUMO

PURPOSE: To assess the continuous-time random-walk (CTRW) model's diagnostic value in breast lesions and to explore the associations between the CTRW parameters and breast cancer pathologic factors. METHOD: This retrospective study included 85 patients (70 malignant and 18 benign lesions) who underwent 3.0T MRI examinations. Diffusion-weighted images (DWI) were acquired with 16b-values to fit the CTRW model. Three parameters (Dm, α, and ß) derived from CTRW and apparent diffusion coefficient (ADC) from DWI were compared among the benign/malignant lesions, molecular prognostic factors, and molecular subtypes by Mann-Whitney U test. Spearman correlation was used to evaluate the associations between the parameters and prognostic factors. The diagnostic performance was assessed by the area under the receiver operating characteristic curve (AUC) based on the diffusion parameters. RESULTS: All parameters, ADC, Dm, α, and ß were significantly lower in the malignant than benign lesions (P < 0.05). The combination of all the CTRW parameters (Dm, α, and ß) provided the highest AUC (0.833) and the best sensitivity (94.3%) in differentiating malignant status. And the positive status of estrogen receptor (ER) and progesterone receptor (PR) showed significantly lower ß compared with the negative counterparts (P < 0.05). The high Ki-67 expression produced significantly lower Dm and ADC values (P < 0.05). Additionally, combining multiple CTRW parameters improved the performance of diagnosing molecular subtypes of breast cancer. Moreover, Spearman correlations analysis showed that ß produced significant correlations with ER, PR and Ki-67 expression (P < 0.05). CONCLUSIONS: The CTRW parameters could be used as non-invasive quantitative imaging markers to evaluate breast lesions.


Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Prognóstico , Estudos Retrospectivos , Antígeno Ki-67 , Sensibilidade e Especificidade , Interpretação de Imagem Assistida por Computador/métodos , Reprodutibilidade dos Testes , Imageamento por Ressonância Magnética , Imagem de Difusão por Ressonância Magnética/métodos , Receptores de Estrogênio , Mama/patologia
9.
Acad Radiol ; 30(1): 40-46, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-35577699

RESUMO

RATIONALE AND OBJECTIVES: To explore the feasibility of differentiating three predominant metastatic tumor types using lung computed tomography (CT) radiomics features based on supervised machine learning. MATERIALS AND METHODS: This retrospective analysis included 252 lung metastases (LM) (from 78 patients), which were divided into the training (n = 176) and test (n = 76) cohort randomly. The metastases originated from colorectal cancer (n = 97), breast cancer (n = 87), and renal carcinoma (n = 68). An additional 77 LM (from 35 patients) were used for external validation. All radiomics features were extracted from lung CT using an open-source software called 3D slicer. The least absolute shrinkage and selection operator (LASSO) method selected the optimal radiomics features to build the model. Random forest and support vector machine (SVM) were selected to build three-class and two-class models. The performance of the classification model was evaluated with the area under the receiver operating characteristic curve (AUC) by two strategies: one-versus-rest and one-versus-one. RESULTS: Eight hundred and fifty-one quantitative radiomics features were extracted from lung CT. By LASSO, 23 optimal features were extracted in three-class, and 25, 29, and 35 features in two-class for differentiating every two of three LM (colorectal cancer vs. renal carcinoma, colorectal cancer vs. breast cancer, and breast cancer vs. renal carcinoma, respectively). The AUCs of the three-class model were 0.83 for colorectal cancer, 0.79 for breast cancer, and 0.91 for renal carcinoma in the test cohort. In the external validation cohort, the AUCs were 0.77, 0.83, and 0.81, respectively. Swarmplot shows the distribution of radiomics features among three different LM types. In the two-class model, high accuracy and AUC were obtained by SVM. The AUC of discriminating colorectal cancer LM from renal carcinoma LM was 0.84, and breast cancer LM from colorectal cancer LM and renal carcinoma LM were 0.80 and 0.94, respectively. The AUCs were 0.77, 0.78, and 0.84 in the external validation cohort. CONCLUSION: Quantitative radiomics features based on Lung CT exhibited good discriminative performance in LM of primary colorectal cancer, breast cancer, and renal carcinoma.


Assuntos
Neoplasias da Mama , Carcinoma de Células Renais , Neoplasias Colorretais , Neoplasias Renais , Neoplasias Pulmonares , Humanos , Feminino , Estudos Retrospectivos , Tomografia Computadorizada por Raios X/métodos , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias da Mama/diagnóstico por imagem , Neoplasias Renais/diagnóstico por imagem , Neoplasias Colorretais/diagnóstico por imagem
10.
Methods Mol Biol ; 2603: 59-69, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36370270

RESUMO

Cysteine-SILAC enables the detection and quantification of protein S-palmitoylation, an important protein posttranslational modification. Here we describe the cell culture, protein extraction, selective enrichment, mass spectrometry, and data analysis for palmitoylated proteins from cell samples by this method.


Assuntos
Cisteína , Lipoilação , Cisteína/metabolismo , Espectrometria de Massas , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo
11.
Comput Intell Neurosci ; 2022: 9751988, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36531926

RESUMO

Purpose: To develop and validate a clinical-radiomics nomogram based on clinical risk factors and CT radiomics feature to predict hypertensive intracerebral hemorrhage (HICH) prognosis. Methods: A total of 195 patients with HICH treated in our hospital from January 2018 to January 2022 were retrospectively enrolled and randomly divided into two cohorts for training (n = 138) and validation (n = 57) according to the ratio of 7 : 3. All CT radiomics features were extracted from intrahematomal, perihematomal, and combined intra- and perihematomal regions by using free open-source software called 3D slicer. The least absolute shrinkage and selection operator method was used to select the optimal radiomics features, and the radiomics score (Rad-score) was calculated. The relationship between Rad-score, clinical risk factors, and the HICH prognosis was analyzed by univariate and multivariate logistic regression analyses, and the clinical-radiomics nomogram was built. The area under the receiver operating characteristic curve (AUC) and decision curve analysis (DCA) were used to evaluate the performance of the clinical-radiomics nomogram in predicting the prognosis of HICH. Results: A total of 1702 radiomics features were extracted from the CT images of each patient for analysis. By univariate and stepwise multivariate logistic regression analyses, age, sex, RBC, serum glucose, D-dimer level, hematoma volume, and midline shift were clinical risk factors for the prognosis of HICH. Rad-score and clinical risk factors developed the clinical-radiomics nomogram. The nomogram showed the highest predictive efficiency in the training cohort (AUC = 0.95, 95% confidence interval (CI), 0.92 to 0.98) and the validation cohort (AUC = 0.90, 95% CI, 0.82 to 0.98). The calibration curve indicated that the clinical-radiomics nomogram had good calibration. DCA showed that the nomogram had high applicability in clinical practice. Conclusions: The clinical-radiomics nomogram incorporated with the radiomics features and clinical risk factors has good potential in predicting the prognosis of HICH.


Assuntos
Hemorragia Intracraniana Hipertensiva , Humanos , Nomogramas , Estudos Retrospectivos , Fatores de Risco , Tomografia Computadorizada por Raios X
12.
Front Oncol ; 12: 922185, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36158700

RESUMO

Purpose: To develop and validate a clinical-radiomics nomogram based on radiomics features and clinical risk factors for identification of human epidermal growth factor receptor 2 (HER2) status in patients with breast cancer (BC). Methods: Two hundred and thirty-five female patients with BC were enrolled from July 2018 to February 2022 and divided into a training group (from center I, 115 patients), internal validation group (from center I, 49 patients), and external validation group (from centers II and III, 71 patients). The preoperative MRI of all patients was obtained, and radiomics features were extracted by a free open-source software called 3D Slicer. The Least Absolute Shrinkage and Selection Operator regression model was used to identify the most useful features. The radiomics score (Rad-score) was calculated by using the radiomics signature-based formula. A clinical-radiomics nomogram combining clinical factors and Rad-score was developed through multivariate logistic regression analysis. The performance of the nomogram was evaluated using receiver operating characteristic (ROC) curve and decision curve analysis (DCA). Results: A total of 2,553 radiomics features were extracted, and 21 radiomics features were selected as the most useful radiomics features. Multivariate logistic regression analysis indicated that Rad-score, progesterone receptor (PR), and Ki-67 were independent parameters to distinguish HER2 status. The clinical-radiomics nomogram, which comprised Rad-score, PR, and Ki-67, showed a favorable classification capability, with AUC of 0.87 [95% confidence internal (CI), 0.80 to 0.93] in the training group, 0.81 (95% CI, 0.69 to 0.94) in the internal validation group, and 0.84 (95% CI, 0.75 to 0.93) in the external validation group. DCA illustrated that the nomogram was useful in clinical practice. Conclusions: The nomogram combined with Rad-score, PR, and Ki-67 can identify the HER2 status of BC.

13.
Front Neurosci ; 16: 974096, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36033623

RESUMO

Purpose: To build CT perfusion (CTP)-based delta-radiomics models to identify collateral vessel formation after revascularization in patients with moyamoya disease (MMD). Methods: Fifty-three MMD patients who underwent CTP and digital subtraction angiography (DSA) examination were retrospectively enrolled. Patients were divided into good and poor groups based on postoperative DSA. CTP parameters, such as mean transit time (MTT), time to drain (TTD), time to maximal plasma concentration (Tmax), and flow extraction product (FE), were obtained. CTP efficacy in evaluating surgical treatment were compared between the good and poor groups. The changes in the relative CTP parameters (ΔrMTT, ΔrTTD, ΔrTmax, and ΔrFE) were calculated to evaluate the differences between pre- and postoperative CTP values. CTP parameters were selected to build delta-radiomics models for identifying collateral vessel formation. The identification performance of machine learning classifiers was assessed using area under the receiver operating characteristic curve (AUC). Results: Of the 53 patients, 36 (67.9%) and 17 (32.1%) were divided into the good and poor groups, respectively. The postoperative changes of ΔrMTT, ΔrTTD, ΔrTmax, and ΔrFE in the good group were significantly better than the poor group (p < 0.05). Among all CTP parameters in the perfusion improvement evaluation, the ΔrTTD had the largest AUC (0.873). Eleven features were selected from the TTD parameter to build the delta-radiomics model. The classifiers of the support vector machine and k-nearest neighbors showed good diagnostic performance with AUC values of 0.933 and 0.867, respectively. Conclusion: The TTD-based delta-radiomics model has the potential to identify collateral vessel formation after the operation.

14.
Sci China Life Sci ; 65(10): 2017-2030, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35366151

RESUMO

Epithelial ovarian cancer (EOC) exhibits strong dependency on the tricarboxylic acid (TCA) cycle and oxidative phosphorylation to fuel anabolic process. Here, we show that malate dehydrogenase 2 (MDH2), a key enzyme of the TCA cycle, is palmitoylated at cysteine 138 (C138) residue, resulting in increased activity of MDH2. We next identify that ZDHHC18 acts as a palmitoyltransferase of MDH2. Glutamine deprivation enhances MDH2 palmitoylation by increasing the binding between ZDHHC18 and MDH2. MDH2 silencing represses mitochondrial respiration as well as ovarian cancer cell proliferation both in vitro and in vivo. Intriguingly, re-expression of wild-type MDH2, but not its palmitoylation-deficient C138S mutant, sustains mitochondrial respiration and restores the growth as well as clonogenic capability of ovarian cancer cells. Notably, MDH2 palmitoylation level is elevated in clinical cancer samples from patients with high-grade serous ovarian cancer. These observations suggest that MDH2 palmitoylation catalyzed by ZDHHC18 sustains mitochondrial respiration and promotes the malignancy of ovarian cancer, yielding possibilities of targeting ZDHHC18-mediated MDH2 palmitoylation in the treatment of EOC.


Assuntos
Malato Desidrogenase , Neoplasias Ovarianas , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Cisteína , Feminino , Glutamina , Humanos , Lipoilação , Malato Desidrogenase/química , Malato Desidrogenase/metabolismo , Respiração , Ácidos Tricarboxílicos
15.
Anal Chem ; 94(13): 5213-5220, 2022 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-35333042

RESUMO

Comprehensive analysis of protein glycation is important for better understanding of its formation mechanism and biological significance. The current preconcentration methods of glycated proteome mainly depend on the reversible combination of boronic acid and cis-dihydroxy group by pH adjustment, but it has inherent limitations (e.g., poor specificity and time-consuming). Herein, for the first time, a novel enrichment method for glycated peptides is proposed based on the reversible chemical reaction between aldehyde and 1,2-aminothiol groups, in which oxidized glycated peptides are captured onto the magnetic nanoparticles via thiazolidine chemistry and then released by palladium-mediated cleavage. The method is rapid, with excellent selectivity (even at a 1:1000 molar ratio of glycated peptides/nonglycated peptides) and high sensitivity (1 fmol/µL). As a good evidence, 1549 glycated peptides were identified from glycated human serum with 94.6% specificity, providing a powerful technique for high-throughput analysis of glycated peptides.


Assuntos
Paládio , Proteoma , Glicosilação , Humanos , Peptídeos , Tiazolidinas
16.
RSC Chem Biol ; 2(1): 192-205, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-34458782

RESUMO

Programmed cell death protein 1 (PD-1) is a crucial anticancer target, but the relatively low response rate and acquired resistance to existing antibody drugs highlight an urgent need to develop alternative targeting strategies. Here, we report the palmitoylation of PD-1, discover the main DHHC enzyme for this modification, reveal the mechanism of its effect on PD-1 protein stability, and rationally develop a peptide for targeting PD-1 expression. Palmitoylation promoted the trafficking of PD-1 to the recycling endosome, thus preventing its lysosome-dependent degradation. Palmitoylation of PD-1, but not of PD-L1, promoted mTOR signaling and tumor cell proliferation, and targeting palmitoylation displayed significant anti-tumor effects in a three-dimensional culture system. A peptide was designed to competitively inhibit PD-1 palmitoylation and expression, opening a new route for developing PD-1 inhibitors and combinatorial cancer immunotherapy.

17.
Anal Chem ; 93(10): 4398-4407, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33661625

RESUMO

Glycation plays a pathogenic role in many age-related degenerative pathological conditions, such as diabetes, end-stage renal diseases, and cardiovascular diseases. Mass spectrometry-based qualitative and quantitative analysis methods have been greatly developed and contribute to our understanding of protein glycation. However, it is still challenging to sensitively and accurately quantify endogenous glycated proteome in biological samples. Herein, we proposed an integrated and robust quantitative strategy for comprehensive profiling of early-stage glycated proteome. In this strategy, a filter-assisted sample preparation method was applied to reduce sample loss and improve reproducibility of sample preparation, contributing to high-throughput analysis and accurate quantification of endogenous glycated proteins with low abundance. Standard glycated peptides were spiked and performed the subsequent process together with complex samples both in label-free quantification and multiple reaction monitoring (MRM) analysis, contributing to the improvement of quantitative accuracy. In parallel, a novel approach was developed for the synthesis of heavy isotope-labeled glycated peptides used in MRM analysis. By this way, a total of 1128 endogenous glycated peptides corresponding to 203 serum proteins were identified from 60 runs of 10 pairs of hemodialysis patients with and without cardiovascular complications, and 234 glycated peptides corresponding to 63 proteins existed in >70% runs, among which 17 peptides were discovered to be differentially glycated (P < 0.05, fold-change > 1.5 or <0.67). Furthermore, we validated the glycation difference of four target peptides in 46 serum samples using MRM analysis, which were consistent with our results of label-free quantification.


Assuntos
Proteínas Sanguíneas , Diálise Renal , Biomarcadores , Humanos , Espectrometria de Massas , Reprodutibilidade dos Testes
18.
Anal Chem ; 92(23): 15317-15322, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33174720

RESUMO

S-Nitrosylation is an important post-translational modification that occurs on cysteine amino acid and regulates signal transduction in diverse cell processes. Dysregulation of protein nitrosylation has shown close association with cardiovascular and neurological diseases, thus demanding further precise and in-depth understanding. Mass spectrometry-based proteomics has been the method of choice for analyzing S-nitrosylated (SNO-) proteins. However, due to their extremely low expression level and rapid turnover rate, quantitative analysis of the S-nitrosylation at the proteomic level remains challenging. Herein, we developed a novel approach termed FluoroTRAQ, which combined the fluorous solid-phase extraction of SNO-peptides and iTRAQ labeling for the quantitative analysis of the SNO-proteome with high sensitivity and specificity. This new analytical strategy was subsequently applied to examine the dynamic SNO-proteome changes of human umbilical vein endothelial cells upon in vitro S-nitrosoglutathione induction. Our data identified a number of novel SNO-proteins and revealed their temporal modulation as validated by biotin switch assay. Our study offered a practical approach for quantitative analysis of protein S-nitrosylation.


Assuntos
Espectrometria de Massas , Óxido Nítrico/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/isolamento & purificação , Proteínas/metabolismo , Extração em Fase Sólida , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Cinética , Proteômica
19.
Anal Chem ; 92(12): 8315-8322, 2020 06 16.
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 the lysine residue, all resulting peptides except naturally acetylated N-terminal peptides contain free amino groups and can be removed by coupling with AminoLink Resin. Therefore, the naturally acetylated N-terminal peptides were left in solution and enriched for further liquid chromatography-tandem mass spectrometry (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 toward 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 the 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.


Assuntos
Aminas/metabolismo , Lisina/metabolismo , Resinas Sintéticas/metabolismo , Acetilação , Aminas/química , Células HeLa , Humanos , Lisina/química , Resinas Sintéticas/análise
20.
Adv Exp Med Biol ; 1248: 399-424, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32185719

RESUMO

The ligands and receptors in immune checkpoint signaling are typically transmembrane proteins, which may be regulated by palmitoylation as a reversible lipid modification. Our recent work demonstrated that palmitoylation reduces the lysosomal degradation of PD-L1 trafficking and may present a new therapeutic target. To facilitate future investigations on palmitoylation and immune checkpoints, here we summarize the molecular roles of palmitoylation on protein stability, trafficking, membrane association, and protein-protein interaction. The biological effects of palmitoylation are exemplified by well-studied substrates such as Ras, EGFR, and Wnt proteins. Finally, the strategies for targeting protein palmitoylation are discussed to facilitate future translational studies.


Assuntos
Lipoilação , Palmitatos/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico , Transdução de Sinais , Humanos , Lisossomos/metabolismo , Proteínas/química , Proteínas/metabolismo
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