The irradiation of oxygen-free, face-centered-cubic C 60 films with visible or ultraviolet light phototransformed C 60 into a different solid phase. Results from laser desorption mass spectroscopy, scanning electron microscopy, Raman and infrared spectroscopy, optical absorption, and luminescence indicate that a photopolymerization of the fullerene molecules within the film occurred.

Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.

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Although hundreds of heparan sulfate binding proteins have been identified, and implicated in a myriad of physiological and pathological processes, very little information is known about ligand requirements for binding and mediating biological activities by these proteins. This difficulty results from a lack of technology for establishing structure-activity-relationships, which in turn is due to the structural complexity of natural heparan sulfate (HS) and difficulties of preparing well-defined HSoligosaccharides. To address this deficiency, we have developed a modular approach for the parallel combinatorial synthesis of HS oligosaccharides that utilizes a relatively small number of selectively protected disaccharide building blocks, which can easily be converted into glycosyl donors and acceptors. The utility of the modular building blocks has been demonstrated by the preparation of a library of twelve oligosaccharides, which has been employed to probe structural features of HS for inhibiting the protease, BACE-1. The complex variations in activity with structural changes support the view that important functional information is embedded in HS sequences. Furthermore, the most active derivative provides an attractive lead compound for the preparation of more potent compounds, which may find use as a therapeutic agent for Alzheimer's disease.

The first application of electron detachment dissociation (EDD) to carbohydrates is presented. The structural characterization of glycosaminoglycan (GAG) oligosaccharides by mass spectrometry is a longstanding problem because of the lability of these acidic, polysulfated carbohydrates. Doubly-charged negative ions of four GAG tetrasaccharides are examined by EDD, collisionally activated dissociation (CAD), and infrared multiphoton dissociation (IRMPD). EDD is found to produce information-rich mass spectra with both cross ring and glycosidic cleavage product ions. In contrast, most of the product ions produced by CAD and IRMPD result from glycosidic cleavage. EDD shows great potential as a tool for locating the sites of sulfation and other modifications in glycosaminoglycan oligosaccharides. (J Am Soc Mass Spectrom 2007, 18, 234 -244)

Proteoglycans are complex glycoconjugates that regulate critical biological pathways in all higher organisms. Bikunin, the simplest proteoglycan having a single glycosaminoglycan chain, is a serine protease inhibitor used to treat acute pancreatitis. Unlike the template driven synthesis of nucleic acids and proteins, Golgi synthesized glycosaminoglycans are not believed to have predictable or deterministic sequence. Bikunin peptidoglycosaminoglycans were prepared and fractionated to obtain a collection of size similar and charge similar chains. Fourier transform mass spectral analysis identified a small number of parent molecular-ions corresponding to mono-compositional peptidoglycosaminoglycans. Fragmentation using collision induced dissociation surprisingly afforded a single sequence for each mono-compositional parent-ion, unequivocally demonstrating the presence of a defined sequence. The common biosynthetic pathway for all proteoglycans suggests that even more structurally complex proteoglycans, such as heparan sulfate, may have defined sequences, requiring a readjustment of our understanding of information storage in complex glycans.

The effect of ion space-charge on mass accuracy in Fourier transform ion cyclotron resonance mass spectrometry is examined. Matrix-assisted laser desorption/ionization is used to form a population of high-molecular-weight polymer ions with a wide mass distribution. The density of the ions in the analyzer cell is varied using ion remeasurement and suspended trapping techniques to allow the effect of ion space charge to be examined independently of other experimental influences. Observed cyclotron frequency exhibits a linear correlation with ion population. Mass errors of 100 ppm or more in externally calibrated mass spectra result when ion number is not taken into account. By matching the total ion intensities of calibrant and analyte mass spectra, the protonated ion of insulin B-chain, 3494.6513 Da, is measured with an accuracy of 0.07 ppm (average of 10 measurements, σ = 2.3 ppm, average absolute error 1.6 ppm) using a polymer sample as an external calibrant. Alternatively, the correction for space charge can be made by using a calibration equation that accounts for the total ion intensity of the mass spectrum. A calibration procedure is proposed and is tested with the measurement of the mass of insulin B-chain. A mass accuracy of 2.0 ppm (average of 20 measurements, σ = 4.2 ppm, average absolute error 3.5 ppm) is achieved. Space-charge-induced mass errors are more significant for samples with many components, such as a polymer, than for single-component samples such as purified peptides or proteins.

The process of Fe-S cluster biogenesis is highly conserved in nature and centers around two key proteins, termed IscS and IscU in prokaryotes, which are found in almost all organisms. 1 IscS is a homodimeric, pyridoxal phosphate-dependent L-cysteine desulfurase that catalyzes the conversion of cysteine to alanine and elemental sulfur via the formation of a persulfide intermediate on a conserved cysteine residue. 2 The ubiquitous role of IscSlike enzymes in sulfur trafficking, in general, 3 and Fe-S cluster biosynthesis, in particular, 4 is now firmly established. IscU is a homodimeric protein that forms a 1:1 complex with IscS, 5 and gene knockout studies in yeast 6 and bacteria 4d have demonstrated a crucial role for IscU in general Fe-S cluster biosynthesis. Moreover, IscU contains three conserved cysteines and has been shown to provide a scaffold for IscS-directed sequential assembly of [Fe 2 S 2 ] 2+ and [Fe 4 S 4 ] 2+ clusters. 7 These clusters are likely to be inserted, intact, into apo Fe-S proteins in a process that has yet to be fully characterized. In this communication, we address the mechanism of IscS-directed Fe-S cluster assembly on the IscU scaffold. Mass spectrometry has been used to demonstrate direct transfer of sulfane sulfur, S 0 , from the cysteine persulfide on IscS to the cysteine residues on IscU. The available evidence indicates that this reaction constitutes the first step in Fe-S cluster biosynthesis on an IscU scaffold.A comparison of the mass spectra of Azotobacter Vinelandii IscU obtained under a variety of reaction conditions is shown in Figure 1. Reaction mixtures were purified by reverse-phase HPLC, and discrete fractions were introduced by an electrospray ionization (ESI) source for analysis using a single quadrupole mass spectometer (see Supporting Information for experimental details). 8 The conditions used for repurification are denaturing, and the observed charged-state distributions, centered around m/z ≈ 1000 amu, are characteristic of highly denatured proteins. Hence, all observed molecular ions must be covalently associated.The mass spectrum of IscU after DTT treatment ( Figure 1A) comprises the monomer molecular ion peak at 13 744 Da (theoretical mass based on primary sequence, 13 875 Da, minus 131 Da, corresponding to cleavage of the N-terminal methionine), and features at 13 774 and 13 805 Da. Since the two higher mass components occur at +30 and +61 Da, respectively, and are dramatically decreased in intensity on addition of excess potassium cyanide ( Figure 1E), they are attributed to the addition of one and two sulfur atoms, respectively, in DTT-inaccessible polysulfides or persulfides. Direct evidence for multiple sulfur transfers from IscS to IscU is provided by the mass spectrum of IscU in a reaction mixture involving stoichiometric IscS and IscU with an excess of the substrate, cysteine ( Figure 1B). The major peaks at 13 805 and 13 836 Da correspond to the addition of two and three sulfur atoms, respectively, and peaks corresponding to the addition of one sulfu...