Evaluation of the mirn23a Cluster through an iTRAQ-based Quantitative Proteomic Approach

Ludwig, Katelyn R.; Dahl, Richard; Hummon, Amanda B.

doi:10.1021/acs.jproteome.5b01101

Cited by 11 publications

(8 citation statements)

References 49 publications

(94 reference statements)

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“…Global identification and quantification of proteins and peptides has become a critical tool for the analysis of biological systems. − Mass spectrometry-based studies using ultra performance liquid chromatography (UPLC) − or capillary zone electrophoresis (CZE) − coupled to a mass spectrometer are often performed to identify thousands of proteins in a sample of interest. Large-scale proteomic studies often rely on digestion of proteins into peptides using the enzyme trypsin, and the corresponding peptide ion intensities can be exploited for quantification .…”

Section: Introductionmentioning

confidence: 99%

Comparison of In-Solution, FASP, and S-Trap Based Digestion Methods for Bottom-Up Proteomic Studies

2018

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Bottom-up proteomic strategies rely on efficient digestion of proteins into peptides for mass spectrometry analysis. In-solution and filter-based strategies are commonly used for proteomic analysis. In recent years, filter-aided sample preparation (FASP) has become the dominant filter-based method due to its ability to remove SDS prior to mass spectrometry analysis. However, the time-consuming nature of FASP protocols have led to the development of new filter-based strategies. Suspension traps (S-Traps) were recently reported as an alternative to FASP and in-solution strategies as they allow for high concentrations of SDS in a fraction of the time of a typical FASP protocol. In this study, we compare the yields from in-solution, FASP, and S-Trap based digestions of proteins extracted in SDS and urea-based lysis buffers. We performed label-free quantification to analyze the differences in the portions of the proteome identified using each method. Overall, our results show that each digestion method had a high degree of reproducibility within the method type. However, S-Traps outperformed FASP and in-solution digestions by providing the most efficient digestion with the greatest number of unique protein identifications. This is the first work to provide a direct quantitative comparison of two filter-based digestion methods and a traditional in-solution approach to provide information regarding the most efficient proteomic preparation.

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Section: Introductionmentioning

confidence: 99%

Comparison of In-Solution, FASP, and S-Trap Based Digestion Methods for Bottom-Up Proteomic Studies

2018

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“…Furthermore, a crosstalk between proteome and non-coding RNAs was proposed by proteome and miRNome data during ongoing endothelial senescence 52 and during telomere shortening in cancer cells 53 . Possible targets for miR-23a, miR-24-2, and miR-27a were identified by mass spectrometry in pre B-lymphoblasts 54 . On proteome-wide scale, ectopic expression of hsa-mir-34a caused moderate changes in protein translation 44 .…”

Section: Discussionmentioning

confidence: 99%

Functional omics analyses reveal only minor effects of microRNAs on human somatic stem cell differentiation

Schira‐Heinen

Czapla

Hendricks

et al. 2020

Sci Rep

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The contribution of microRNA-mediated posttranscriptional regulation on the final proteome in differentiating cells remains elusive. Here, we evaluated the impact of microRNAs (miRNAs) on the proteome of human umbilical cord blood-derived unrestricted somatic stem cells (USSC) during retinoic acid (RA) differentiation by a systemic approach using next generation sequencing analysing mRNA and miRNA expression and quantitative mass spectrometry-based proteome analyses. Interestingly, regulation of mRNAs and their dedicated proteins highly correlated during RA-incubation. Additionally, RA-induced USSC demonstrated a clear separation from native USSC thereby shifting from a proliferating to a metabolic phenotype. Bioinformatic integration of up-and downregulated miRNAs and proteins initially implied a strong impact of the miRNome on the XXL-USSC proteome. However, quantitative proteome analysis of the miRNA contribution on the final proteome after ectopic overexpression of downregulated miR-27a-5p and miR-221-5p or inhibition of upregulated miR-34a-5p, respectively, followed by RA-induction revealed only minor proportions of differentially abundant proteins. In addition, only small overlaps of these regulated proteins with inversely abundant proteins in non-transfected RA-treated USSC were observed. Hence, mRNA transcription rather than miRNAmediated regulation is the driving force for protein regulation upon RA-incubation, strongly suggesting that miRNAs are fine-tuning regulators rather than active primary switches during RA-induction of USSC. MicroRNAs (miRNAs) are small non-coding RNAs which primarily bind to the 3′UTR of target mRNAs in a sequence-specific manner resulting in translational repression or destabilization and degradation of the targeted mRNA 1. In animals, primary miRNA transcripts are trimmed in a two-step process involving double strand-specific nucleases Drosha and Dicer. The mature single-stranded miRNA is then incorporated into the RNA-induced silencing complex (RISC) where functional binding to the mRNA target sequence takes place 1. As a most important feature, miRNA-mediated regulation is characterised by a bidirectional target gene redundancy. This allows a single miRNA to target the 3′ UTRs of hundreds of mRNAs in parallel 2. Vice versa, binding sites for several miRNAs can be found on a single 3′ UTR. In consequence of these properties, miRNAs primarily can act as network regulators being able to affect large numbers of regulatory targets in parallel 2,3 .

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“…Despite the large number of predicted potential interactions, many predicted conserved miRNA target sequences are known to not significantly regulate expression levels of their targets in cells (Baek et al, 2008 ). Proteomic analysis of the same miRNAs in different cellular backgrounds reveals cell-specific differences (Ludwig et al, 2016 ; Piragasam et al, 2020 ) and changes in protein abundances that may counter interactions shown by miRNA target predictions (Piragasam et al, 2020 ). These proteomic analyses are holistic in that they reveal both direct and indirect impacts on protein abundance through miRNAs.…”

Section: Microrna Target Prediction Of J-proteinsmentioning

confidence: 99%

Deciphering Network Crosstalk: The Current Status and Potential of miRNA Regulatory Networks on the HSP40 Molecular Chaperone Network

2021

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Molecular chaperone networks fulfill complex roles in protein homeostasis and are essential for maintaining cell health. Hsp40s (commonly referred to as J-proteins) have critical roles in development and are associated with a variety of human diseases, yet little is known regarding the J-proteins with respect to the post-transcriptional mechanisms that regulate their expression. With relatively small alterations in their abundance and stoichiometry altering their activity, post-transcriptional regulation potentially has significant impact on the functions of J-proteins. MicroRNAs (miRNAs) are a large group of non-coding RNAs that form a complex regulatory network impacting gene expression. Here we review and investigate the current knowledge and potential intersection of miRNA regulatory networks with the J-Protein chaperone network. Analysis of datasets from the current version of TargetScan revealed a great number of predicted microRNAs targeting J-proteins compared to the limited reports of interactions to date. There are likely unstudied regulatory interactions that influence chaperone biology contained within our analysis. We go on to present some criteria for prioritizing candidate interactions including potential cooperative targeting of J-Proteins by multiple miRNAs. In summary, we offer a view on the scope of regulation of J-Proteins through miRNAs with the aim of guiding future investigations by identifying key regulatory nodes within these two complex cellular networks.

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Evaluation of the mirn23a Cluster through an iTRAQ-based Quantitative Proteomic Approach

Cited by 11 publications

References 49 publications

Comparison of In-Solution, FASP, and S-Trap Based Digestion Methods for Bottom-Up Proteomic Studies

Comparison of In-Solution, FASP, and S-Trap Based Digestion Methods for Bottom-Up Proteomic Studies

Functional omics analyses reveal only minor effects of microRNAs on human somatic stem cell differentiation

Deciphering Network Crosstalk: The Current Status and Potential of miRNA Regulatory Networks on the HSP40 Molecular Chaperone Network

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