Safety of human therapeutic Abs is generally assessed in nonhuman primates. Whereas IgG1 shows identical FcγR interaction and effector function profile in both species, fundamental differences in the IgG2 and IgG4 Ab subclasses were found between the two species. Granulocytes, the main effector cells against IgG2- and IgG4-opsonized bacteria and parasites, do not express FcγRIIIb, but show higher levels of FcγRII in cynomolgus monkey. In humans, IgG2 and IgG4 adapted a silent Fc region with weak binding to FcγR and effector functions, whereas, in contrast, cynomolgus monkey IgG2 and IgG4 display strong effector function as well as differences in IgG4 Fab arm exchange. To balance this shift toward activation, the cynomolgus inhibitory FcγRIIb shows strongly increased affinity for IgG2. In view of these findings, in vitro and in vivo results for human IgG2 and IgG4 obtained in the cynomolgus monkey have to be cautiously interpreted, whereas effector function-related effects of human IgG1 Abs are expected to be predictable for humans.

The nucleosome, the fundamental structural unit of chromatin, contains an octamer of core histones H3, H4, H2A, and H2B. Incorporation of histone variants alters the functional properties of chromatin. To understand the global dynamics of chromatin structure and function, analysis of histone variants incorporated into the nucleosome and their covalent modifications is required. Here we report the first global mass spectrometric analysis of histone H2A and H2B variants derived from Jurkat cells. A combination of mass spectrometric techniques, HPLC separations, and enzymatic digestions using endoproteinase Glu-C, endoproteinase Arg-C, and trypsin were used to identify histone H2A and H2B subtypes and their modifications. We identified nine histone H2A and 11 histone H2B subtypes, among them proteins that only had been postulated at the gene level. The two main H2A variants, H2AO and H2AC, as well as H2AL were either acetylated at Lys-5 or phosphorylated at Ser-1. For the replacement histone H2AZ, acetylation at Lys-4 and Lys-7 was found. Within the eukaryotic cell nucleus the genetic information is organized in a highly conserved structural polymer, termed chromatin, that supports and controls crucial functions of the genome. The fundamental unit of eukaryotic chromatin, the nucleosome, consists of 146 base pairs of genomic DNA wrapped around an octamer of the core histone proteins H2A, H2B, H3, and H4. The amino-terminal tails of each of the four core histones are subject to several types of covalent modifications, including acetylation, methylation, and phosphorylation. These modifications affect lysines (acetylation, mono-, di-, and trimethylation), serines and threonines (phosphoryla-

The N-terminal tails of the four core histones are subject to several types of covalent post-translational modifications that have specific roles in regulating chromatin structure and function. Here we present an extensive analysis of the core histone modifications occurring through the cell cycle. Our MS experiments characterized the modification patterns of histones from HeLa cells arrested in phase G 1 , S, and G 2 /M. For all core histones, the modifications in the G 1 and S phases were largely identical but drastically different during mitosis. Modification changes between S and G 2 /M phases were quantified using the SILAC (stable isotope labeling by amino acids in cell culture) approach. Most striking was the mitotic phosphorylation on histone H3 and H4, whereas phosphorylation on H2A was constant during the cell cycle. A loss of acetylation was observed on all histones in G 2 /M-arrested cells. The pattern of cycle-dependent methylation was more complex: during G 2 /M, H3 Lys 27 and Lys 36were decreased, whereas H4 Lys 20 was increased. Our results show that mitosis was the period of the cell cycle during which many modifications exhibit dynamic changes.

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Activity-based proteomics is a methodology that is used to quantify the catalytically active subfraction of enzymes present in complex mixtures such as lysates or living cells. To apply this approach for in-cell selectivity profiling of inhibitors of serine proteases, we designed a novel activity-based probe (ABP). This ABP consists of (i) a fluorophosphonate-reactive group, directing the probe toward serine hydrolases or proteases and (ii) an alkyne functionality that can be specifically detected at a later stage with an azide-functionalized reporter group through a Cu(I)-catalyzed coupling reaction ("click chemistry"). This novel ABP was shown to label the active site of several serine proteases with greater efficiency than a previously reported fluorophosphonate probe. More importantly, our probe was cell-permeable and achieved labeling of enzymes within living cells with efficiency similar to that observed for the corresponding lysate fraction. Several endogenous serine hydrolases whose activities were detected upon in-cell labeling were identified by two-dimensional gel and MS analyses. As a proof of principle, cell-permeable inhibitors of an endogenous serine protease (prolyl endopeptidase) were assessed for their potency and specificity in competing for the in situ labeling of the selected enzyme. Altogether these results open new perspectives for safety profiling studies in uncovering potential cellular "side effects" of drugs (unanticipated off-target inhibition or activation) that may be overlooked by standard selectivity profiling methods. Molecular & Cellular Proteomics 7:1241-1253, 2008.Activity-based proteomics, in comparison with classic genomics and proteomics approaches, has been specially devised to enable the detection of active enzymes. Such a methodology is of particular relevance for example in the protease field where only a subfraction of the total enzyme pool (having undergone successive translocation, post-translational modifications, and proteolytic activation and having escaped binding of endogenous inhibitors) effectively participates in the cellular processes. To profile enzymatic activities in biological samples, activity-based proteomics relies on small reactive marker molecules called activity-based probes (ABPs), 1 which covalently and specifically label the accessible active sites of catalytic enzymes (1-3). So far, several directed and non-directed ABPs have been described that allow the monitoring of more than 20 enzyme classes (for extensive reviews, see Refs. 2-5).The detection of the population of active enzymes is of primary relevance for biological and in particular for pharmaceutical research because it could lead to the discovery of new targets for drug development. Indeed by comparing the activity profile of enzymes under physiological versus pathological conditions (e.g. of normal cells versus parasite-infected cells (6) or versus cancer cells (7-11)), several groups have identified up-regulated active enzymes potentially involved in the development and/or the mainte...