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

Ribosomes are highly conserved ribonucleoprotein nanomachines that translate information in the genome to create the proteome in all cells. In yeast these complex particles contain four RNAs (>5400 nucleotides) and 79 different proteins. During the past 25 years, studies in yeast have led the way to understanding how these molecules are assembled into ribosomes in vivo. Assembly begins with transcription of ribosomal RNA in the nucleolus, where the RNA then undergoes complex pathways of folding, coupled with nucleotide modification, removal of spacer sequences, and binding to ribosomal proteins. More than 200 assembly factors and 76 small nucleolar RNAs transiently associate with assembling ribosomes, to enable their accurate and efficient construction. Following export of preribosomes from the nucleus to the cytoplasm, they undergo final stages of maturation before entering the pool of functioning ribosomes. Elaborate mechanisms exist to monitor the formation of correct structural and functional neighborhoods within ribosomes and to destroy preribosomes that fail to assemble properly. Studies of yeast ribosome biogenesis provide useful models for ribosomopathies, diseases in humans that result from failure to properly assemble ribosomes.

https://www.genetics.org/content/genetics/195/3/643.full.pdf

Ribosome Biogenesis in the Yeast Saccharomyces cerevisiae

pLink, software for data analysis of cross-linked proteins coupled with mass spectrometry, estimates false discovery rate and enables analysis of protein complexes without extensive purification. We have developed pLink, software for data analysis of cross-linked proteins coupled with mass-spectrometry analysis. pLink reliably estimates false discovery rate in cross-link identification and is compatible with multiple homo- or hetero-bifunctional cross-linkers. We validated the program with proteins of known structures, and we further tested it on protein complexes, crude immunoprecipitates and whole-cell lysates. We show that it is a robust tool for protein-structure and protein-protein–interaction studies.

Identification of cross-linked peptides from complex samples

Eukaryotic ribosomes are substantially larger and more complex than their bacterial counterparts. Although their core function is conserved, bacterial and eukaryotic protein synthesis differ considerably at the level of initiation. The eukaryotic small ribosomal subunit (40S) plays a central role in this process; it binds initiation factors that facilitate scanning of messenger RNAs and initiation of protein synthesis. We have determined the crystal structure of the Tetrahymena thermophila 40S ribosomal subunit in complex with eukaryotic initiation factor 1 (eIF1) at a resolution of 3.9 angstroms. The structure reveals the fold of the entire 18S ribosomal RNA and of all ribosomal proteins of the 40S subunit, and defines the interactions with eIF1. It provides insights into the eukaryotic-specific aspects of protein synthesis, including the function of eIF1 as well as signaling and regulation mediated by the ribosomal proteins RACK1 and rpS6e.

/pdf/crystal-structure-of-the-eukaryotic-40s-ribosomal-subunit-in-hy6orlmhoq.pdf

Crystal Structure of the Eukaryotic 40S Ribosomal Subunit in Complex with Initiation Factor 1

rRNAs are extensively modified during their transcription and subsequent maturation in the nucleolus, nucleus and cytoplasm. RNA modifications, which are installed either by snoRNA-guided or by stand-alone enzymes, generally stabilize the structure of the ribosome. However, they also cluster at functionally important sites of the ribosome, such as the peptidyltransferase center and the decoding site, where they facilitate efficient and accurate protein synthesis. The recent identification of sites of substoichiometric 2'-O-methylation and pseudouridylation has overturned the notion that all rRNA modifications are constitutively present on ribosomes, highlighting nucleotide modifications as an important source of ribosomal heterogeneity. While the mechanisms regulating partial modification and the functions of specialized ribosomes are largely unknown, changes in the rRNA modification pattern have been observed in response to environmental changes, during development, and in disease. This suggests that rRNA modifications may contribute to the translational control of gene expression.

https://tandfonline.com/doi/pdf/10.1080/15476286.2016.1259781

Tuning the ribosome: The influence of rRNA modification on eukaryotic ribosome biogenesis and function

Some RNAs, called guide RNAs, use their encoded sequence information to help direct a protein to where it is required. One of these RNA-protein (RNP) complexes, known as the box C/D RNP, catalyses the site-specific modification of RNAs with a 2′-O-methylation group. The structure of the full box C/D RNP complex, including three protein subunits, the guide RNA and either of two substrate RNAs, has now been determined. The structure reveals how the guide and target RNAs are aligned, and how the methyltransferase subunit, fibrillarin, facilitates positioning of the target ribose into the active site. A complex of RNA and protein known as the box C/D RNP catalyses the site-specific modification of RNAs with a 2′-O-methylation group. The structure of the full complex has now been solved, including the guide RNA and either of two substrate RNAs. This structure reveals how the guide and target RNAs are aligned, and how the methyltransferase subunit, fibrillarin, facilitates placement of the target ribose into the active site. Box C/D RNA protein complexes (RNPs) direct site-specific 2′-O-methylation of RNA and ribosome assembly1,2,3,4. The guide RNA in C/D RNP forms base pairs with complementary substrates and selects the modification site using a molecular ruler5,6,7. Despite many studies of C/D RNP structure8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25, the fundamental questions of how C/D RNAs assemble into RNPs and how they guide modification remain unresolved. Here we report the crystal structure of an entire catalytically active archaeal C/D RNP consisting of a bipartite C/D RNA associated with two substrates and two copies each of Nop5, L7Ae and fibrillarin at 3.15-A resolution. The substrate pairs with the second through the eleventh nucleotide of the 12-nucleotide guide, and the resultant duplex is bracketed in a channel with flexible ends. The methyltransferase fibrillarin binds to an undistorted A-form structure of the guide–substrate duplex and specifically loads the target ribose into the active site. Because interaction with the RNA duplex alone does not determine the site specificity, fibrillarin is further positioned by non-specific and specific protein interactions. Compared with the structure of the inactive C/D RNP, extensive domain movements are induced by substrate loading. Our results reveal the organization of a monomeric C/D RNP and the mechanism underlying its site-specific methylation activity.

Structural basis for site-specific ribose methylation by box C/D RNA protein complexes

†MitoNEET is a protein of unknown function present in the mitochondrial membrane that was recently shown to bind specifically the antidiabetic drug pioglizatone. Here, we report the crystal structure of the soluble domain (residues 32–108) of human mitoNEET at 1.8-A resolution. The structure reveals an intertwined homodimer, and each subunit was observed to bind a [2Fe-2S] cluster. The [2Fe-2S] ligation pattern of three cysteines and one histidine differs from the known pattern of four cysteines in most cases or two cysteines and two histidines as observed in Rieske proteins. The [2Fe-2S] cluster is packed in a modular structure formed by 17 consecutive residues. The cluster-binding motif is conserved in at least seven distinct groups of proteins from bacteria, archaea, and eukaryotes, which show a consensus sequence of (hb)-C-X1-C-X2-(S/T)-X3-P-(hb)-C-D-X2-H, where hb represents a hydrophobic residue; we term this a CCCH-type [2Fe-2S] binding motif. The nine conserved residues in the motif contribute to iron ligation and structure stabilization. UV-visible absorption spectra indicated that mitoNEET can exist in oxidized and reduced states. Our study suggests an electron transfer function for mitoNEET and for other proteins containing the CCCH motif. 2Fe-2S thiazolidinediones mitochondria

https://www.pnas.org/content/pnas/104/37/14640.full.pdf

Crystal structure of human mitoNEET reveals distinct groups of iron–sulfur proteins

Box C/D guide RNAs are abundant noncoding RNAs that primarily function to direct the 2′-O-methylation of specific nucleotides by base-pairing with substrate RNAs. In archaea, a bipartite C/D RNA assembles with L7Ae, Nop5, and the methyltransferase fibrillarin into a modification enzyme with unique substrate specificity. Here, we determined the crystal structure of an archaeal C/D RNA–protein complex (RNP) composed of all 3 core proteins and an engineered half-guide RNA at 4 Å resolution, as well as 2 protein substructures at higher resolution. The RNP structure reveals that the C-terminal domains of Nop5 in the dimeric complex provide symmetric anchoring sites for 2 L7Ae-associated kink-turn motifs of the C/D RNA. A prominent protrusion in Nop5 seems to be important for guide RNA organization and function and for discriminating the structurally related U4 snRNA. Multiple conformations of the N-terminal domain of Nop5 and its associated fibrillarin in different structures indicate the inherent flexibility of the catalytic module, suggesting that a swinging motion of the catalytic module is part of the enzyme mechanism. We also built a model of a native C/D RNP with substrate and fibrillarin in an active conformation. Our results provide insight into the overall organization and mechanism of action of C/D RNA–guided RNA methyltransferases.

/pdf/structural-organization-of-box-c-d-rna-guided-rna-2j4l4478ns.pdf

Structural organization of box C/D RNA-guided RNA methyltransferase

http://www.jbc.org/content/286/24/21937.full.pdf

Jinzhong Lin

Papers

Identification of cross-linked peptides from complex samples

Structural basis for site-specific ribose methylation by box C/D RNA protein complexes

Crystal structure of human mitoNEET reveals distinct groups of iron–sulfur proteins

Structural organization of box C/D RNA-guided RNA methyltransferase

Structure of the autocatalytic cysteine protease domain of potyvirus helper-component proteinase.