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

The activity of the c-Src protein tyrosine kinase is regulated by phosphorylation of a tyrosine residue (Tyr-527) in the C-terminal tail of the molecule. Phosphorylation of Tyr-527 promotes association of the tail with the SH2 domain and a concomitant reduction of the enzymatic activity of Src. We asked the question whether regulation by C-terminal phosphorylation was accompanied by a change in the quaternary structure of the enzyme or if it occurred within a monomeric form of Src. For this purpose we purified to homogeneity a chicken Src form lacking the unique domain from Schizosaccharomyces pombe cells. The cells were engineered to express Src along with Csk, a protein kinase able to phosphorylate Tyr-527 efficiently. Mass spectrometric analysis showed that purified Src was homogeneously phosphorylated at Tyr-527. The enzyme was in the regulated form, because it could be activated by a phosphorylated peptide able to bind the SH2 domain with high affinity. Using gel filtration chromatography, dynamic light scattering, and ultracentrifugation, we found that the regulated form of Src was a monomer. We have obtained crystals diffracting to 2.4 A with space group P2(1)2(1)2(1) and one molecule per asymmetric unit, in agreement with the monomeric state. These results indicate that the structural rearrangements of regulated Src are of an intramolecular nature.

https://www.pnas.org/content/pnas/94/8/3590.full.pdf

Src regulated by c-terminal phosphorylation is monomeric

Discovery of 5-(2-amino-[1,2,4]triazolo[1,5-a]pyridin-7-yl)-N-(tert-butyl)pyridine-3-sulfonamide (CZC24758), as a potent, orally bioavailable and selective inhibitor of PI3K for the treatment of inflammatory disease.

The utility of parent ion scans for the selective detection of small molecules which yield indicative fragments in tandem mass spectrometry is well recognized. This work established the feasibility of parent (or precursor) ion experiments with large molecules such as proteins and oligonucleotides. Tandem mass spectrometry of proteins yields substantial and specific immonium ion fragmentation and fragments characteristic of post-translational modifications. Phosphoproteins are specifically detected by parent ion scans of the phospho group (PO3-) in the negative ion mode. The same ion allows mass measurements of oligonucleotides with improved signal-to-noise ratio. Scans for the parents of the oxonium ion of hexosamine distinguish glycoproteins from other proteins in mixtures. Parent ion scans of intact proteins is a novel concept which may find applications in the determination of post-translational modifications, in protein charting and in the study of the fragmentation behavior of large molecules. © 1997 by John Wiley & Sons, Ltd.

Parent Ion Scans of Large Molecules

http://digital.csic.es/bitstream/10261/141159/1/Avino1996BMCcarbamate.pdf

New carbamate supports for the preparation of 3'-amino-modified oligonucleotides.

The invention relates to compounds of formula (I) Said compounds are useful as protein kinase inhibitors, especially inhibitors of P13K, for the treatment or prophylaxis of immunological, inflammatory, autoimmune, or allergic disorders. The invention also relates to pharmaceutical compositions including said compounds, the preparation of such compounds as well as the production of and use as medicaments.

Gitte Neubauer

Papers

Src regulated by c-terminal phosphorylation is monomeric

Discovery of 5-(2-amino-[1,2,4]triazolo[1,5-a]pyridin-7-yl)-N-(tert-butyl)pyridine-3-sulfonamide (CZC24758), as a potent, orally bioavailable and selective inhibitor of PI3K for the treatment of inflammatory disease.

Parent Ion Scans of Large Molecules

New carbamate supports for the preparation of 3'-amino-modified oligonucleotides.

Amino triazoles as pi3k inhibitors