Dynamic RNA Modifications in Gene Expression Regulation

Universal sample preparation method for proteome analysis

The BioPlex Network: A Systematic Exploration of the Human Interactome

According to Genome Sequencing Project statistics (http://www.ncbi.nlm.nih.gov/genomes/static/gpstat.html), as of Feb 16, 2012, complete gene sequences have become available for 2816 viruses, 1117 prokaryotes, and 36 eukaryotes.1–2 The availability of full genome sequences has greatly facilitated biological research in many fields, and has greatly contributed to the growth of proteomics.

Proteins are important because they are the direct bio-functional molecules in the living organisms. The term “proteomics” was coined from merging “protein” and “genomics” in the 1990s.3–4 As a post-genomic discipline, proteomics encompasses efforts to identify and quantify all the proteins of a proteome, including expression, cellular localization, interactions, post-translational modifications (PTMs), and turnover as a function of time, space and cell type, thus making the full investigation of a proteome more challenging than sequencing a genome. There are possibly 100,000 protein forms encoded by the approximate 20,235 genes of the human genome,5 and determining the explicit function of each form will be a challenge.

The progress of proteomics has been driven by the development of new technologies for peptide/protein separation, mass spectrometry analysis, isotope labeling for quantification, and bioinformatics data analysis. Mass spectrometry has emerged as a core tool for large-scale protein analysis. In the past decade, there has been a rapid advance in the resolution, mass accuracy, sensitivity and scan rate of mass spectrometers used to analyze proteins. In addition, hybrid mass analyzers have been introduced recently (e.g. Linear Ion Trap-Orbitrap series6–7) which have significantly improved proteomic analysis.

“Bottom-up” protein analysis refers to the characterization of proteins by analysis of peptides released from the protein through proteolysis. When bottom-up is performed on a mixture of proteins it is called shotgun proteomics,8–10 a name coined by the Yates lab because of its analogy to shotgun genomic sequencing.11 Shotgun proteomics provides an indirect measurement of proteins through peptides derived from proteolytic digestion of intact proteins. In a typical shotgun proteomics experiment, the peptide mixture is fractionated and subjected to LC-MS/MS analysis. Peptide identification is achieved by comparing the tandem mass spectra derived from peptide fragmentation with theoretical tandem mass spectra generated from in silico digestion of a protein database. Protein inference is accomplished by assigning peptide sequences to proteins. Because peptides can be either uniquely assigned to a single protein or shared by more than one protein, the identified proteins may be further scored and grouped based on their peptides. In contrast, another strategy, termed ‘top-down’ proteomics, is used to characterize intact proteins (Figure 1). The top-down approach has some potential advantages for PTM and protein isoform determination and has achieved notable success. Intact proteins have been measured up to 200 kDa,12 and a large scale study has identified more than 1,000 proteins by multi-dimensional separations from complex samples.13 However, the top-down method has significant limitations compared with shotgun proteomics due to difficulties with protein fractionation, protein ionization and fragmentation in the gas phase. By relying on the analysis of peptides, which are more easily fractionated, ionized and fragmented, shotgun proteomics can be more universally adopted for protein analysis. In fact, a hybrid of bottom-up and top-down methodologies and instrumentation has been introduced as middle-down proteomics.14 Essentially, middle-down proteomics analyzes larger peptide fragments than bottom-up proteomics, minimizing peptide redundancy between proteins. Additionally the large peptide fragments yield similar advantages as top-down proteomics, such as gaining further insight into post-translational modifications, without the analytical challenges of analyzing intact proteins. Shotgun proteomics has become a workhorse for the analysis of proteins and their modifications and will be increasingly combined with top-down methods in the future.



Figure 1

Proteomic strategies: bottom-up vs. top-down vs. middle-down. The bottom-up approach analyzes proteolytic peptides. The top-down method measures the intact proteins. The middle-down strategy analyzes larger peptides resulted from limited digestion or ...



In the past decade shotgun proteomics has been widely used by biologists for many different research experiments, advancing biological discoveries. Some applications include, but are not limited to, proteome profiling, protein quantification, protein modification, and protein-protein interaction. There have been several reviews nicely summarizing mass spectrometry history,15 protein quantification with mass spectrometry,16 its biological applications,5,17–26 and many recent advances in methodology.27–32 In this review, we try to provide a full and updated survey of shotgun proteomics, including the fundamental techniques and applications that laid the foundation along with those developed and greatly improved in the past several years.

Protein Analysis by Shotgun/Bottom-up Proteomics

Optimization of Codon Translation Rates via tRNA Modifications Maintains Proteome Integrity

Due to its compatibility and orthogonality to reversed phase (RP) liquid chromatography (LC) separation, ion exchange chromatography, and mainly strong cation exchange (SCX), has often been the first choice in multidimensional LC experiments in proteomics. Here, we have tested the ability of three strong anion exchanger (SAX) columns differing in their hydrophobicity to fractionate RAW264.7 macrophage cell lysate. IonPac AS24, a strong anion exchange material with ultralow hydrophobicity, demonstrated to be superior to other materials by fractionation and separation of tryptic peptides from both a mixture of 6 proteins as well as mouse cell lysate. The chromatography displayed very high orthogonality and high robustness depending on the hydrophilicity of column chemistry, which we termed hydrophilic strong anion exchange (hSAX). Mass spectrometry analysis of 34 SAX fractions from RAW264.7 macrophage cell lysate digest resulted in an identification of 9469 unique proteins and 126318 distinct peptides in one week of instrument time. Moreover, when compared to an optimized high pH/low pH RP separation approach, the method presented here raised the identification of proteins and peptides by 10 and 28%, respectively. This novel hSAX approach provides robust, reproducible, and highly orthogonal separation of complex protein digest samples for deep coverage proteome analysis.

Hydrophilic Strong Anion Exchange (hSAX) Chromatography for Highly Orthogonal Peptide Separation of Complex Proteomes

Maintenance of protein quality control has implications in various processes such as neurodegeneration and ageing. To investigate how environmental insults affect this process, we analysed the proteome of yeast continuously exposed to mild heat stress. In agreement with previous transcriptomics studies, amongst the most marked changes, we found up-regulation of cytoprotective factors; a shift from oxidative phosphorylation to fermentation; and down-regulation of translation. Importantly, we also identified a novel, post-translationally controlled, component of the heat shock response. The abundance of Ncs2p and Ncs6p, two members of the URM1 pathway responsible for the thiolation of wobble uridines in cytoplasmic tRNAs tK(UUU), tQ(UUG) and tE(UUC), is down-regulated in a proteasomal dependent fashion. Using random forests we show that this results in differential translation of transcripts with a biased content for the corresponding codons. We propose that the role of this pathway in promoting catabolic and inhibiting anabolic processes, affords cells with additional time and resources needed to attain proper protein folding under periods of stress.

/pdf/protein-degradation-and-dynamic-trna-thiolation-fine-tune-snevcv2n45.pdf

Protein degradation and dynamic tRNA thiolation fine-tune translation at elevated temperatures.

A bacterial strain, designated P15T, was isolated from the soil of an open hexachlorocyclohexane dumpsite. Comparative sequence analysis showed that strain P15T displayed high 16S rRNA gene sequence similarities (94.4–97.2 %) with members of the genus Pseudoxanthomonas. The isolate was most closely related to Pseudoxanthomonas mexicana AMX 26BT (97.2 % 16S rRNA gene sequence similarity) and Pseudoxanthomonas japonensis 12-3T (97.2 %). DNA–DNA relatedness studies showed unambiguously that strain P15T represented a novel species that was separate from P. mexicana DSM 17121T (7.7 %) and P. japonensis DSM 17109T (9.4 %). The predominant cellular fatty acids of strain P15T were iso-C16 : 0 (21.4 %), iso-C15 : 0 (16.1 %), summed feature 9 (comprising iso-C17 : 1ω9c and/or 10-methyl C16 : 0; 14.9 %), iso-C11 : 0 3-OH (8.3 %) and iso-C14 : 0 (7.0 %). The polar lipid profile of strain P15T showed the presence of large amounts of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol in addition to unknown glycolipids, phospholipids and an amino-group-containing polar lipid. Ubiquinone 8 was found as the major quinone. The polyamine profile showed the presence of spermidine. The DNA G+C content was 62.9±2 mol%. Strain P15T is described as representing a new member of the genus Pseudoxanthomonas, for which the name Pseudoxanthomonas indica sp. nov. is proposed. The type strain is P15T ( = MTCC 8596T = CCM 7430T).

Pseudoxanthomonas indica sp. nov., isolated from a hexachlorocyclohexane dumpsite.

The maximum tolerable clock jitter for high-speed ADCs is pessimistically predicted by Nyquist-rate input sinusoidal tests. We prove that the jitter can be greatly relaxed in the presence of lossy channels in wireline systems. We derive compact expressions that allow PLL designers to decide how much jitter can be tolerated for a given channel loss and symbol rate.

Performance Bounds of ADC-Based Receivers Due to Clock Jitter

The SSCS Student Circuit Contest is a worldwide competition open to both undergraduate and graduate students around the world. This year the contest changed its format by opening to a more creative format, where each participant has to design his own integrated circuit by starting from a predefined list of components. While the technology adopted can vary, some rules have to be respected: 

/pdf/sscs-circuit-analysis-and-design-contest-the-winners-of-the-39appy76.pdf

SSCS Circuit Analysis and Design Contest: The Winners of the 2022 Edition [Society News]

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