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

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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High-throughput identification of C/D box snoRNA targets with CLIP and RiboMeth-seq.

10.1101/037259 ◽

2016 ◽

Cited By ~ 2

Author(s):

Rafal Gumienny ◽

Dominik Jedlinski ◽

Georges Martin ◽

Arnau Vina-Villaseca ◽

Mihaela Zavolan

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Expression Patterns ◽

Ribonucleoprotein Complexes ◽

Short Rnas ◽

Genome Wide ◽

The Many ◽

Nucleolar Rnas ◽

Comprehensive Characterization

Identification of long and short RNAs, their processing and expression patterns have been greatly facilitated by high-throughput sequencing. Frequently, these RNAs act as guides for ribonucleoprotein complexes that regulate the expression or processing of target RNAs. However, to determine the targets of the many newly discovered regulatory RNAs in high-throughput remains a challenge. To globally assign guide small nucleolar RNAs to site of 2'-O-ribose methylation in human cells, we here developed novel computational methods for the analysis of data that was generated with protocols designed to capture direct small RNA-target interactions and to identify the sites of 2'-O-ribose methylation genome-wide. We thereby determined that many "orphan" snoRNAs appear to guide 2'-O-ribose methylation at sites that are targeted by other snoRNAs and that snoRNAs can be reliably captured in interaction with many mRNAs, in which a subsequent 2'-O-methylation cannot be detected. Our study provides a reliable approach to the comprehensive characterization of snoRNA-target interactions in species beyond those in which these interactions have been traditionally studied and contribute to the rapidly developing field of "epitranscriptomics".

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