A Proteomics Signature of Mild Hypospadias: A Pilot Study

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.

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An acoustic platform for single-cell, high-throughput measurements of the viscoelastic properties of cells

10.1101/2020.09.07.286898 ◽

2020 ◽

Author(s):

Valentin Romanov ◽

Giulia Silvani ◽

Huiyu Zhu ◽

Charles D Cox ◽

Boris Martinac

Keyword(s):

Mechanical Properties ◽

Single Cell ◽

High Throughput ◽

Single Cells ◽

Adherent Cells ◽

Dependent Manner ◽

Cellular Processes ◽

Membrane Cytoskeleton ◽

Change Temperature

ABSTRACTCellular processes including adhesion, migration and differentiation are governed by the distinct mechanical properties of each cell. Importantly, the mechanical properties of individual cells can vary depending on local physical and biochemical cues in a time-dependent manner resulting in significant inter-cell heterogeneity. While several different methods have been developed to interrogate the mechanical properties of single cells, throughput to capture this heterogeneity remains an issue. While new high-throughput techniques are slowly emerging, they are primarily aimed at characterizing cells in suspension, whereas high-throughput measurements of adherent cells have proven to be more challenging. Here, we demonstrate single-cell, high-throughput characterization of adherent cells using acoustic force spectroscopy. We demonstrate that cells undergo marked changes in viscoelasticity as a function of temperature, the measurements of which are facilitated by a closed microfluidic culturing environment that can rapidly change temperature between 21 °C and 37 °C. In addition, we show quantitative differences in cells exposed to different pharmacological treatments specifically targeting the membrane-cytoskeleton interface. Further, we utilize the high-throughput format of the AFS to rapidly probe, in excess of 1000 cells, three different cell-lines expressing different levels of a mechanosensitive protein, Piezo1, demonstrating the ability to differentiate between cells based on protein expression levels.

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Comprehensive Characterization of Posttranslational Modifications of Histones Using a Novel High‐Throughput Middle‐Down Proteomics Approach

The FASEB Journal ◽

10.1096/fasebj.27.1_supplement.772.5 ◽

2013 ◽

Vol 27 (S1) ◽

Author(s):

Sonja Hess ◽

Anastasia Kalli ◽

Annie Moradian ◽

Michael J Sweredoski

Keyword(s):

High Throughput ◽

Posttranslational Modifications ◽

Proteomics Approach ◽

Comprehensive Characterization

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NMR Characterization of Conformational Interconversions of Lys48-Linked Ubiquitin Chains

International Journal of Molecular Sciences ◽

10.3390/ijms21155351 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5351

Author(s):

Methanee Hiranyakorn ◽

Saeko Yanaka ◽

Tadashi Satoh ◽

Thunchanok Wilasri ◽

Benchawan Jityuti ◽

...

Keyword(s):

Isotope Labeling ◽

Hydrophobic Surface ◽

Enzymatic Reactions ◽

Multidomain Proteins ◽

Solvent Exposure ◽

Hydrophobic Surfaces ◽

Cellular Processes ◽

Nmr Characterization

Ubiquitin (Ub) molecules can be enzymatically connected through a specific isopeptide linkage, thereby mediating various cellular processes by binding to Ub-interacting proteins through their hydrophobic surfaces. The Lys48-linked Ub chains, which serve as tags for proteasomal degradation, undergo conformational interconversions between open and closed states, in which the hydrophobic surfaces are exposed and shielded, respectively. Here, we provide a quantitative view of such dynamic processes of Lys48-linked triUb and tetraUb in solution. The native and cyclic forms of Ub chains are prepared with isotope labeling by in vitro enzymatic reactions. Our comparative NMR analyses using monomeric Ub and cyclic diUb as reference molecules enabled the quantification of populations of the open and closed states for each Ub unit of the native Ub chains. The data indicate that the most distal Ub unit in the Ub chains is the most apt to expose its hydrophobic surface, suggesting its preferential involvement in interactions with the Ub-recognizing proteins. We also demonstrate that a mutational modification of the distal end of the Ub chain can remotely affect the solvent exposure of the hydrophobic surfaces of the other Ub units, suggesting that Ub chains could be unique design frameworks for the creation of allosterically controllable multidomain proteins.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1820561116 ◽

2019 ◽

Vol 116 (19) ◽

pp. 9324-9332 ◽

Cited By ~ 13

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.

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Expanding the CITE-seq tool-kit: Detection of proteins, transcriptomes, clonotypes and CRISPR perturbations with multiplexing, in a single assay

10.1101/466466 ◽

2018 ◽

Cited By ~ 1

Author(s):

Eleni Mimitou ◽

Anthony Cheng ◽

Antonino Montalbano ◽

Stephanie Hao ◽

Marlon Stoeckius ◽

...

Keyword(s):

Single Cell ◽

High Throughput ◽

Single Cells ◽

Surface Markers ◽

Protein Levels ◽

Cellular Processes ◽

Cell Transcriptome ◽

Single Cell Transcriptome ◽

Lymphoma Sample

ABSTRACTRapid technological progress in the recent years has allowed the high-throughput interrogation of different types of biomolecules from single cells. Combining several of these readouts into integrated multi-omic assays is essential to comprehensively understand and model cellular processes. Here, we report the development of Expanded CRISPR-compatible Cellular Indexing of Transcriptomes and Epitopes by sequencing (ECCITE-seq) for the high-throughput characterization of at least five modalities of information from each single cell: transcriptome, immune receptor clonotypes, surface markers, sample identity and sgRNAs. We demonstrate the use of ECCITE-seq to directly and efficiently capture sgRNA molecules and measure their effects on gene expression and protein levels, opening the possibility of performing high throughput single cell CRISPR screens with multimodal readout using existing libraries and commonly used vectors. Finally, by utilizing the combined phenotyping of clonotype and cell surface markers in immune cells, we apply ECCITE to study a lymphoma sample to discriminate cells and define molecular signatures of malignant cells within a heterogeneous population.

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Identification of Potential Universal Vaccine Candidates against Group A Streptococcus by Using High Throughput in Silico and Proteomics Approach

Journal of Proteome Research ◽

10.1021/pr3005265 ◽

2012 ◽

Vol 12 (1) ◽

pp. 336-346 ◽

Cited By ~ 11

Author(s):

Abhinay Sharma ◽

Deepak Kumar Arya ◽

Vivek Sagar ◽

René Bergmann ◽

Gursharan Singh Chhatwal ◽

...

Keyword(s):

High Throughput ◽

In Silico ◽

Group A Streptococcus ◽

Universal Vaccine ◽

Vaccine Candidates ◽

Proteomics Approach ◽

Group A

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

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2013.210 ◽

2013 ◽

Vol 34 (3) ◽

pp. 425-432 ◽

Cited By ~ 21

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.

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