Recent successes illustrate the role of mass spectrometry-based proteomics as an indispensable tool for molecular and cellular biology and for the emerging field of systems biology. These include the study of protein-protein interactions via affinity-based isolations on a small and proteome-wide scale, the mapping of numerous organelles, the concurrent description of the malaria parasite genome and proteome, and the generation of quantitative protein profiles from diverse species. The ability of mass spectrometry to identify and, increasingly, to precisely quantify thousands of proteins from complex samples can be expected to impact broadly on biology and medicine.

Mass spectrometry-based proteomics

https://cseweb.ucsd.edu/classes/sp10/cse283/notes/silac.pdf

Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.

The universality of calcium as an intracellular messenger depends on its enormous versatility. Cells have a calcium signalling toolkit with many components that can be mixed and matched to create a wide range of spatial and temporal signals. This versatility is exploited to control processes as diverse as fertilization, proliferation, development, learning and memory, contraction and secretion, and must be accomplished within the context of calcium being highly toxic. Exceeding its normal spatial and temporal boundaries can result in cell death through both necrosis and apoptosis.

The versatility and universality of calcium signalling

A network is a network — be it between words (those associated with ‘bright’ in this case) or protein structures. Many complex systems in nature and society can be described in terms of networks capturing the intricate web of connections among the units they are made of1,2,3,4. A key question is how to interpret the global organization of such networks as the coexistence of their structural subunits (communities) associated with more highly interconnected parts. Identifying these a priori unknown building blocks (such as functionally related proteins5,6, industrial sectors7 and groups of people8,9) is crucial to the understanding of the structural and functional properties of networks. The existing deterministic methods used for large networks find separated communities, whereas most of the actual networks are made of highly overlapping cohesive groups of nodes. Here we introduce an approach to analysing the main statistical features of the interwoven sets of overlapping communities that makes a step towards uncovering the modular structure of complex systems. After defining a set of new characteristic quantities for the statistics of communities, we apply an efficient technique for exploring overlapping communities on a large scale. We find that overlaps are significant, and the distributions we introduce reveal universal features of networks. Our studies of collaboration, word-association and protein interaction graphs show that the web of communities has non-trivial correlations and specific scaling properties.

/pdf/uncovering-the-overlapping-community-structure-of-complex-1f119qtsda.pdf

Uncovering the overlapping community structure of complex networks in nature and society

Most cellular processes are carried out by multiprotein complexes. The identification and analysis of their components provides insight into how the ensemble of expressed proteins (proteome) is organized into functional units. We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae. We processed 1,739 genes, including 1,143 human orthologues of relevance to human biology, and purified 589 protein assemblies. Bioinformatic analysis of these assemblies defined 232 distinct multiprotein complexes and proposed new cellular roles for 344 proteins, including 231 proteins with no previous functional annotation. Comparison of yeast and human complexes showed that conservation across species extends from single proteins to their molecular environment. Our analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions. This higher-order map contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.

/pdf/functional-organization-of-the-yeast-proteome-by-systematic-10g3dn6mw1.pdf

Functional organization of the yeast proteome by systematic analysis of protein complexes

Nuclear pre-mRNA splicing occurs via a two-step transesterification reaction that is catalyzed by a multicomponent complex termed the spliceosome (25). The spliceosome is built by the ordered association of the U1, U2, U4, U5, and U6 small nuclear ribonucleoprotein particles (snRNPs) and extrinsic protein factors with the pre-mRNA (reviewed in references 19, 25, 40, and 46). Each individual snRNP contains a unique set of particle-specific proteins and seven common Sm proteins, while the U6 snRNP contains a unique set of Sm-like (Lsm) proteins. The U5, U4, and U6 snRNPs form a trimeric complex in which the U4 and U6 snRNAs are extensively base paired.

The spliceosome is a highly dynamic molecular machine, forming anew on each pre-mRNA substrate in an ordered manner (4, 40). First, the U1 snRNP binds the pre-mRNA by interacting with the 5′ splice site (34, 38), leading to the formation of commitment complexes in yeast (33, 38) and the E complex in mammals (24). The U2 snRNP, which interacts with the pre-mRNA branchpoint region, is then added in an ATP-dependent step, resulting in the formation of the prespliceosome (reviewed in reference 25). Finally, U4, U5, and U6 snRNPs, preassembled in a single particle, join the prespliceosome to form the mature spliceosome, in which the pre-mRNA splicing reaction occurs. Several rearrangements then take place that activate the spliceosome for the two catalytic steps of splicing. For example, U1 and U4 are destabilized and the spliceosome is activated for catalysis (4). The first splicing reaction creates two intermediates, a cleaved upstream exon and an intron-downstream exon in a branched “lariat” configuration. The second splicing reaction results in the ligation of the exons and excision of the lariat-intron. The spliceosome then disassembles, and the products of the reaction are released.

As mentioned above, at the molecular level the assembly pathway involves rearrangements of RNA-RNA interactions, many of which must be disrupted to allow the formation of others (27, 40). After the 5′ splice site is recognized by U1 through base pairing (37, 39, 49), this interaction must be replaced by a mutually exclusive interaction with U6 (15, 18, 21, 36, 44). Similarly, the base-pairing interaction between U4 and U6 must be disrupted to allow the U2-U6 interaction, which comprises a major element of the spliceosome's catalytic core (23). These rearrangements are thought to be critical for activating the spliceosome for the catalytic steps of splicing. Notably, the DEAD-box ATPase Prp28p was identified as a potential mediator of the U1-to-U6 switch, perhaps by unwinding either the U1-5′ splice site duplex or the U4-U6 duplex (41). In addition, a mutation in U4 snRNA, which blocks U4-U6 unwinding as well as U1 release, can be suppressed by a specific mutation in the U5 snRNP protein Prp8p (20).

These and other data indicate that snRNP proteins play a key role in triggering the activation of the spliceosome and in spliceosome assembly (8). Some of these proteins were identified genetically in yeast, while others were characterized biochemically in various systems. Using these tools, the yeast U1 snRNP and the [U4/U5.U6] tri-snRNP recently have been extensively characterized (11, 12, 26, 32, 42). In contrast, our knowledge of the yeast U2 snRNP is less comprehensive, since a distinct U2 snRNP equivalent to the human U2 snRNP could only be partially purified from yeast (6). This led to the identification of a novel essential splicing factor, Rse1p/Snu154p, the yeast ortholog of the human protein SAP130, which is one of the subunits of the heteromeric U2 snRNP-associated splicing factor SF3b (6, 11). The human U2 snRNP contains nine characterized specific proteins besides the seven common Sm proteins (2). Sequence analysis revealed that homologs of all these proteins exist in yeast (5, 35, 43). Yib9p and Lea1p are the orthologs of metazoan U2B" and U2A′, respectively (5, 43). Cus2p is also a yeast U2 snRNP-associated protein but apparently does not have a human counterpart that associates with the human U2 snRNP (48). Based on several criteria, the PRP9, PRP11, and PRP21 gene products are homologs of the mammalian SF3a subunits; Hsh49p, Cus1p, and ySAP155, together with Rse1p (Snu154p), are homologs of the mammalian SF3b subunits (14, 28, 30, 31, 45, 47).

Although considerable progress has been made in identifying the critical RNA-RNA and RNA-protein interactions required for splicing, little is understood about the factors mediating the structural dynamics. While characterizing the yeast [U4/U6.U5] tri-snRNP proteins, we identified several U1 and U2 snRNP-specific proteins in our tri-snRNP preparations, which probably were due to the small amounts of copurifying U1 and U2 snRNPs (11). Here, we describe the characterization of one of these proteins with a predicted molecular mass of 17 kDa encoded by open reading frame YIR005w. This protein, named Snu17p, contains an RNA recognition motif (RRM). We show that Snu17p is specifically associated with the U2 snRNP and is an intrinsic component of prespliceosomes and spliceosomes, even after the first catalytic step of splicing has occurred. Deletion of the SNU17 gene leads to a slow-growth phenotype and to a splicing defect in vivo and in vitro. We demonstrate that the in vitro splicing defect is due to the lack of Snu17p since it can be specifically reversed by addition of recombinant protein. Analysis of spliceosomal complexes shows that in extracts lacking Snu17p an unusual complex accumulates that contains U1, U2, U4, U5, and U6 snRNPs. Thus, deletion of Snu17p traps the prespliceosome and allows the [U4/U6.U5] tri-snRNP to bind to this intermediate. However, progression to a more mature form of the spliceosome is inhibited.

A Novel Yeast U2 snRNP Protein, Snu17p, Is Required for the First Catalytic Step of Splicing and for Progression of Spliceosome Assembly

SAR studies around a series of triazolopyridines as potent and selective PI3Kγ inhibitors

A Selective Inhibitor Reveals Pi3Kgamma Dependence of T(H)17 Cell Differentiation.

The catalytic domain of chicken Src including the C-terminal tail (Src-CD), has been expressed in Schizosaccharomyces pombe and purified to homogeneity. The expressed protein is a mixture of unphosphorylated (80%) and mono-phosphorylated (20%) species, that can be separated from each other by Mono Q chromatography. By a novel mass spectrometric method that utilizes parent ion scans of unseparated peptide mixtures, we found that the mono-phosphorylated form is phosphorylated either at Tyr416 or at Tyr436. The stability of Src-CD is comparable to the wild-type protein. Src-CD auto-phosphorylates and efficiently phosphorylates substrate peptides and proteins. Auto-phosphorylation occurs by an intermolecular mechanism and is completely inhibited by an excess of substrate peptide. Kinetic measurements for two exogenous substrates, the Src substrate peptide (AEEEIYGEFEAKKKK) and denatured enolase, showed that the overall activity (kcat) of the Src-CD molecule is about 10 times higher than that of wild-type Src. The kcat, values for phosphorylation of the Src substrate peptide are similar for the unphosphorylated and monophosphorylated Src-CD (50 min−1), but the apparent Km values differ significantly (approximately 3 μM and 10 μM, respectively). Therefore, at low substrate concentrations in vitro the mono-phosphorylated form is more active, in agreement with the importance of Tyr416 for in vivo activity. The apparent Km values of the mono-phosphorylated Src-CD and wild-type Src for the Src substrate peptide and enolase are similar, indicating that, under these conditions, the kinase domain is mainly responsible for substrate binding.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1996.0756h.x

The purification and characterization of the catalytic domain of Src expressed in Schizosaccharomyces pombe. Comparison of unphosphorylated and tyrosine phosphorylated species.

The addition of submillimolar levels of sodium acetate to the mobile phase during an LC/ESI-MS experiment encourages the formation of sodium replacement ions in addition to the normally observed protonated species. The spacing of these sodiated species allows unambiguous determination of the charge state of the ions and, hence, their actual mass. Such information is very useful when unknown or modified peptides are being analyzed. At the levels reported here (250 μM), the sodium salt seems to cause no deterioration in chromatographic performance and does not unduly foul the mss spectrometer

Gitte Neubauer

Papers

A Novel Yeast U2 snRNP Protein, Snu17p, Is Required for the First Catalytic Step of Splicing and for Progression of Spliceosome Assembly

SAR studies around a series of triazolopyridines as potent and selective PI3Kγ inhibitors

A Selective Inhibitor Reveals Pi3Kgamma Dependence of T(H)17 Cell Differentiation.

The purification and characterization of the catalytic domain of Src expressed in Schizosaccharomyces pombe. Comparison of unphosphorylated and tyrosine phosphorylated species.

Identifying Charge States of Peptides in Liquid Chromatography/Electrospray Ionization Mass Spectrometry