scholarly journals Characterization of the Ubiquitin-Modified Proteome Regulated by Transient Forebrain Ischemia

2013 ◽  
Vol 34 (3) ◽  
pp. 425-432 ◽  
Author(s):  
Masahiro Iwabuchi ◽  
Huaxin Sheng ◽  
JWill Thompson ◽  
Liangli Wang ◽  
Laura G Dubois ◽  
...  
Keyword(s):  
Protein Modification ◽  
Internal Standard ◽  
Forebrain Ischemia ◽  
Label Free ◽  
Cellular Processes ◽  
Damage Repair ◽  
Liquid Chromatography Tandem Mass ◽  
Posttranslational Protein Modification ◽  
Neuronal Functions

Ubiquitylation is a posttranslational protein modification that modulates various cellular processes of key significance, including protein degradation and DNA damage repair. In animals subjected to transient cerebral ischemia, ubiquitin-conjugated proteins accumulate in Triton-insoluble aggregates. Although this process is widely considered to modulate the fate of postischemic neurons, few attempts have been made to characterize the ubiquitin-modified proteome in these aggregates. We performed proteomics analyses to identify ubiquitylated proteins in postischemic aggregates. Mice were subjected to 10 minutes of forebrain ischemia and 4 hours of reperfusion. The hippocampi were dissected, aggregates were isolated, and trypsin-digested after spiking with GG-BSA as internal standard. K- ε-GG-containing peptides were immunoprecipitated and analyzed by label-free quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. We identified 1,664 peptides to 520 proteins containing at least one K- ε-GG. Sixty-six proteins were highly ubiquitylated, with 10 or more K- ε-GG peptides. Based on selection criteria of greater than fivefold increase and P<0.001, 763 peptides to 272 proteins were highly enriched in postischemic aggregates. These included proteins involved in important neuronal functions and signaling pathways that are impaired after ischemia. Results of this study could serve as an important platform to uncover the mechanisms linking insoluble ubiquitin aggregates to the functions of postischemic neurons.

scholarly journals Label-free methods for optical in vitro characterization of protein–protein interactions

2021 ◽  
Author(s):  
Fabian Soltermann ◽  
Weston B. Struwe ◽  
Philipp Kukura
Keyword(s):  
Protein Interactions ◽  
Label Free ◽  
Protein Protein Interactions ◽  
Cellular Processes ◽  
P Protein ◽  
In Vitro Characterization ◽  
And Function

Protein–protein interactions are involved in the regulation and function of the majority of cellular processes.

scholarly journals A Proteomics Signature of Mild Hypospadias: A Pilot Study

2020 ◽  
Vol 8 ◽  
Author(s):  
Coriness Piñeyro-Ruiz ◽  
Horacio Serrano ◽  
Inmaculada Jorge ◽  
Eric Miranda-Valentin ◽  
Marcos R. Pérez-Brayfield ◽  
...  
Keyword(s):  
High Throughput ◽  
Energy Production ◽  
Isotope Labeling ◽  
Cellular Processes ◽  
Proteomics Approach ◽  
Liquid Chromatography Tandem Mass ◽  
Congenital Condition ◽  
Group A ◽  
Control Samples

Background and Objective: Mild hypospadias is a birth congenital condition characterized by the relocation of the male urethral meatus from its typical anatomical position near the tip of the glans penis, to a lower ventral position up to the brim of the glans corona, which can also be accompanied by foreskin ventral deficiency. For the most part, a limited number of cases have known etiology. We have followed a high-throughput proteomics approach to study the proteome in mild hypospadias patients.Methods: Foreskin samples from patients with mild hypospadias were collected during urethroplasty, while control samples were collected during elective circumcision (n = 5/group). A high-throughput, quantitative proteomics approach based on multiplexed peptide stable isotope labeling (SIL) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to ascertain protein abundance changes in hypospadias patients when compared to control samples.Results: A total of 4,815 proteins were quantitated (2,522 with at least two unique peptides). One hundred and thirty-three proteins from patients with mild hypospadias showed significant abundance changes with respect to control samples, where 38 proteins were increased, and 95 proteins were decreased. Unbiased functional biological analysis revealed that both mitochondrial energy production and apoptotic signaling pathways were enriched in mild hypospadias.Conclusions: This first comprehensive proteomics characterization of mild hypospadias shows molecular changes associated with essential cellular processes related to energy production and apoptosis. Further evaluation of the proteome may expand the search of novel candidates in the etiology of mild hypospadias and could also lead to the identification of biomarkers for this congenital urogenital condition.

scholarly journals Metabolic Profile in Neonatal Pig Hearts

2021 ◽  
Vol 8 ◽  
Author(s):  
Pengsheng Li ◽  
Fan Li ◽  
Ling Tang ◽  
Wenjing Zhang ◽  
Yan Jin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Metabolic Profile ◽  
Protein Modification ◽  
High Cell ◽  
Metabolic Switch ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass ◽  
Neonatal Pig ◽  
Posttranslational Protein Modification ◽  
Mass Spectrometry Assay

We evaluated the metabolic profile in pig hearts at postnatal day 1, 3, 7, and 28 (P1, P3, P7, and P28, respectively) using a targeted liquid chromatography tandem mass spectrometry assay. Our data showed that there is a clear separation of the detected metabolites in P1 vs. P28 hearts. Active anabolisms of nucleotide and proteins were observed in P1 hearts when cardiomyocytes retain high cell cycle activity. However, the active posttranslational protein modification, metabolic switch from glucose to fatty acids, and the reduced ratio of collagen to total protein were observed in P28 hearts when cardiomyocytes withdraw from cell cycle.

scholarly journals RNAi expression tuning, microfluidic screening, and genome recombineering for improved protein production inSaccharomyces cerevisiae

2019 ◽  
Vol 116 (19) ◽  
pp. 9324-9332 ◽  
Author(s):  
Guokun Wang ◽  
Sara M. Björk ◽  
Mingtao Huang ◽  
Quanli Liu ◽  
Kate Campbell ◽  
...  
Keyword(s):  
Protein Secretion ◽  
High Throughput ◽  
Protein Modification ◽  
Protein Production ◽  
Enrichment Analysis ◽  
Cell Factory ◽  
Cellular Processes ◽  
Cellular Machinery ◽  
Specific Proteins

The cellular machinery that supports protein synthesis and secretion lies at the foundation of cell factory-centered protein production. Due to the complexity of such cellular machinery, the challenge in generating a superior cell factory is to fully exploit the production potential by finding beneficial targets for optimized strains, which ideally could be used for improved secretion of other proteins. We focused on an approach in the yeastSaccharomyces cerevisiaethat allows for attenuation of gene expression, using RNAi combined with high-throughput microfluidic single-cell screening for cells with improved protein secretion. Using direct experimental validation or enrichment analysis-assisted characterization of systematically introduced RNAi perturbations, we could identify targets that improve protein secretion. We found that genes with functions in cellular metabolism (YDC1,AAD4,ADE8, andSDH1), protein modification and degradation (VPS73,KTR2,CNL1, andSSA1), and cell cycle (CDC39), can all impact recombinant protein production when expressed at differentially down-regulated levels. By establishing a workflow that incorporates Cas9-mediated recombineering, we demonstrated how we could tune the expression of the identified gene targets for further improved protein production for specific proteins. Our findings offer a high throughput and semirational platform design, which will improve not only the production of a desired protein but even more importantly, shed additional light on connections between protein production and other cellular processes.

scholarly journals Proteomic Analysis of Plasma Membrane Proteins of Antler Stem Cells Using Label-Free LC–MS/MS

2018 ◽  
Vol 19 (11) ◽  
pp. 3477 ◽  
Author(s):  
Datao Wang ◽  
Hengxing Ba ◽  
Chenguang Li ◽  
Quanmin Zhao ◽  
Chunyi Li
Keyword(s):  
Stem Cells ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Cell Biology ◽  
Protein Modification ◽  
Label Free ◽  
Plasma Membrane Proteins ◽  
Cellular Processes ◽  
External Stimuli ◽  
Go Terms

Deer antlers are unusual mammalian organs that can fully regenerate after annual shedding. Stem cells resident in the pedicle periosteum (PPCs) provide the main cell source for antler regeneration. Central to various cellular processes are plasma membrane proteins, but the expression of these proteins has not been well documented in antler regeneration. In the present study, plasma membrane proteins of PPCs and facial periosteal cells (FPCs) were analyzed using label-free liquid chromatography–mass spetrometry (LC–MS/MS). A total of 1739 proteins were identified. Of these proteins, 53 were found solely in the PPCs, 100 solely in the FPCs, and 1576 co-existed in both PPCs and FPCs; and 39 were significantly up-regulated in PPCs and 49 up-regulated in FPCs. In total, 226 gene ontology (GO) terms were significantly enriched from the differentially expressed proteins (DEPs). Five clusters of biological processes from these GO terms comprised responses to external stimuli, signal transduction, membrane transport, regulation of tissue regeneration, and protein modification processes. Further studies are required to demonstrate the relevancy of these DEPs in antler stem cell biology and antler regeneration.

scholarly journals Neuronal SUMOylation: Mechanisms, Physiology, and Roles in Neuronal Dysfunction

2014 ◽  
Vol 94 (4) ◽  
pp. 1249-1285 ◽  
Author(s):  
Jeremy M. Henley ◽  
Tim J. Craig ◽  
Kevin A. Wilkinson
Keyword(s):  
Protein Trafficking ◽  
Protein Modification ◽  
Synapse Formation ◽  
Neuronal Dysfunction ◽  
Cellular Physiology ◽  
Cellular Processes ◽  
Posttranslational Protein Modification ◽  
And Function ◽  
And Control ◽  
Protein Sumoylation

Protein SUMOylation is a critically important posttranslational protein modification that participates in nearly all aspects of cellular physiology. In the nearly 20 years since its discovery, SUMOylation has emerged as a major regulator of nuclear function, and more recently, it has become clear that SUMOylation has key roles in the regulation of protein trafficking and function outside of the nucleus. In neurons, SUMOylation participates in cellular processes ranging from neuronal differentiation and control of synapse formation to regulation of synaptic transmission and cell survival. It is a highly dynamic and usually transient modification that enhances or hinders interactions between proteins, and its consequences are extremely diverse. Hundreds of different proteins are SUMO substrates, and dysfunction of protein SUMOylation is implicated in a many different diseases. Here we briefly outline core aspects of the SUMO system and provide a detailed overview of the current understanding of the roles of SUMOylation in healthy and diseased neurons.

Characterization of frozen hydrated biological specimens by low-loss EELS

1992 ◽  
Vol 50 (2) ◽  
pp. 1572-1573
Author(s):  
Songquan Sun ◽  
Richard D. Leapman
Keyword(s):  
Water Content ◽  
Direct Method ◽  
Internal Standard ◽  
Dry Weight ◽  
Dark Field ◽  
Protein Solutions ◽  
Subcellular Compartment ◽  
Differential Shrinkage ◽  
Electron Energy Loss

Analyses of ultrathin cryosections are generally performed after freeze-drying because the presence of water renders the specimens highly susceptible to radiation damage. The water content of a subcellular compartment is an important quantity that must be known, for example, to convert the dry weight concentrations of ions to the physiologically more relevant molar concentrations. Water content can be determined indirectly from dark-field mass measurements provided that there is no differential shrinkage between compartments and that there exists a suitable internal standard. The potential advantage of a more direct method for measuring water has led us to explore the use of electron energy loss spectroscopy (EELS) for characterizing biological specimens in their frozen hydrated state.We have obtained preliminary EELS measurements from pure amorphous ice and from cryosectioned frozen protein solutions. The specimens were cryotransfered into a VG-HB501 field-emission STEM equipped with a 666 Gatan parallel-detection spectrometer and analyzed at approximately −160 C.

scholarly journals Development and validation of a liquid chromatography-tandem mass spectrometric method for the determination of lenalidomide in human plasma and its application on bioequivalence studies

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
R. Gopinath ◽  
S. T. Narenderan ◽  
M. Kumar ◽  
B. Babu
Keyword(s):  
Liquid Chromatography ◽  
Human Plasma ◽  
Internal Standard ◽  
Spectrometric Method ◽  
Tandem Mass ◽  
Mass Spectrometric Method ◽  
Mass Detection ◽  
Triple Quadrupole Mass Spectrometer ◽  
Liquid Chromatography Tandem Mass ◽  
Tandem Mass Spectrometric

AbstractA simple, sensitive, and specific liquid chromatography-tandem mass spectrophotometry (LC-MS/MS) method was developed and validated for the quantification of lenalidomide in human plasma. The separation was carried out on a symmetry, C18, 5-μm (50 × 4.6 mm) column as stationary phase and with an isocratic mobile phase of 0.1% formic acid in water-methanol in the ratio of (15:85, v/v) at a flow rate of 0.5 mL/min. Protonated ions formed by electrospray ionization in the positive mode were used to detect analyte and fluconazole (internal standard). The mass detection was made by monitoring the fragmentation of m/z 260.1/148.8 for lenalidomide and m/z 307.1/238.0 for internal standard on a triple quadrupole mass spectrometer. The developed method was validated over the concentration range of 10–1000 ng/mL for lenalidomide in human plasma with a correlation coefficient (r2) was 0.9930. The accuracy and precision values obtained from six different sets of quality control samples analyzed on separate occasions ranged from 99.41 to 106.97% and 2.88 to 4.22%, respectively. Mean extraction recoveries were 98.06% and 88.78% for the analyte and IS, respectively. The developed method was successfully applied for analyzing lenalidomide in human plasma samples.

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