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complexity of pattern information present in the acquired images.

As an initial step, the authors sought to identify unique combinations of proteins that were found in a single pixel. They did so by using a threshold to decide whether each pixel was positive or negative for each protein, and then creating a binary vector of length equal to the number of proteins ana-lyzed (Fig. 1). They call these vectors combi-natorial molecular phenotypes (CMPs). Thus each pixel in an image can be described in terms of which unique combination of pro-teins it contains. Not surprisingly, they did not observe all possible combinations (for 48 proteins this would be 248 CMPs), but the number observed (in the hundreds of thousands for 48 proteins) suggests a higher degree of complexity of protein location than had previously been appreciated.

Even more significantly, the presence or absence of particular CMPs may reflect dif-ferent cell or tissue states. For example, the authors identified CMPs in skin images that can distinguish between patients with psoria-sis, patients with atopic dermatitis and healthy individuals.

Although the CMP analysis just scratches the surface of the information content in the MELC images, it reveals the value of this new technology both for top-down detection of altered states and for bottom-up understand-ing of protein location families. By permitting the acquisition of correlated images of a large number of proteins, the MELC technique can provide an important complement to cur-rent approaches. For fixed cells or tissue sec-tions, it provides the multiplexing that other methods lack. Other fluorescence microscopy methods, by contrast, can provide temporal information and high spatial resolution in live

cells (without requiring isolation of specific antibodies).

An additional complementary approach to determination of location is the predic-tion of location from sequence. The higher-complexity, higher-resolution information coming from automated microscopy will allow training of substantially improved prediction systems, which in turn can be used to guide determination experiments. I anticipate that all of these tools can be used in a data-driven planning process to learn everything we need to know about the locations of proteins.

1. Schubert, W. et al. Nat. Biotechnol. 24, 1270–1278 (2006).

2. Ideker, T., Galitski, T. & Hood, L. Annu. Rev. Genomics Hum. Genet. 2, 343–372 (2001).

3. Jarvik, J.W. et al. Biotechniques 33, 852–867 (2002).4. Huh, W-K. et al. Nature 425, 686–691 (2003).5. Taylor, D.L., Woo, E.S. & Giuliano, K.A. Curr. Opin.

Biotechnol. 12, 75–81 (2001).6. Price, J.H. et al. J. Cell. Biochem. 39 (Suppl.), 194–

210 (2002).7. Chen, X. & Murphy, R.F. Int. Rev. Cytol. 249, 193–227

(2006).8. Sigal, A. et al. Nat. Methods 3, 525–531 (2006).9. Chen, X., Velliste, M., Weinstein, S., Jarvik, J.W. &

Murphy, R.F. Proc. SPIE 4962, 298–306 (2003).10. Perlman, Z.E. et al. Science 306, 1194–1198

(2004).

Stain

Bleach Register,threshold,compare

Sample

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Protein 3

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010a

1

101b

0

010a

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011c

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Pixel n,n

Pixel1,1 1,2 1,3 ... n,n

Figure 1 Multi-epitope ligand cartography (MELC) technology. MELC technology starts with a fixed and mounted tissue or cell sample and subjects it to fully automated cycles of fluorescent staining, imaging and photobleaching. Each cycle can use an antibody specific for a different protein (or any other fluorescent probe) so that the result is a set of images of the distributions of many different proteins for the same field. After thresholding each of these images, each pixel can be represented as a vector of binary values (a row in the example table) indicating whether it is positive or negative for a particular protein. Each unique combination defines a combinatorial molecular phenotype (CMP), and the spatial pattern formed by all the pixels with the same CMP can be identified.

Complementary therapies for inflammationYi Wang

Monoclonal antibodies against the human C5a receptor offer new therapeutic prospects for complement inhibition.

As one of the most potent mediators of inflam-mation, the complement component C5a is a promising target for anti-inflammatory drugs. However, a drawback of using animal models to develop antagonists of complement activa-tion is that interspecies differences in receptors and ligands may complicate drug development and delay the evaluation of their efficacy in humans. In this issue, Lee et al.1 generate mice

in which the native C5a receptor has been replaced by its human counterpart and use these animals to produce high-affinity mono-clonal antibodies (mAbs) capable of prevent-ing and reversing inflammation in a mouse model of rheumatoid arthritis. This strategy may find broader application in raising anti-bodies to cell-surface targets and facilitating more meaningful preclinical trials.

The activation of complement is central to defense against infection and harmful stimuli. However, as activated complement components do not discriminate between self and non-self, inappropriate complement

Yi Wang is at Alexion Pharmaceuticals, Inc., 352 Knotter Drive, Cheshire, Connecticut 06410,USA e-mail: wangy@alxn.com

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activation can cause tissue inflammation in conditions such as rheumatoid arthritis, asthma, psoriasis, sepsis, inflammatory bowel disease, immune complex—mediated renal disease, age-related macular degeneration and ischemia-reperfusion injury. However, the attractiveness of complement components as therapeutic targets must be weighed against the risks of disrupting this critical aspect of the immune response.

The complement cascade can be activated through the classical, alternative and lectin pathways, all of which converge at the com-plement proteins C3 and C5. In addition, a recently identified pathway through the coagulation system acts on C5 but not on C3 (ref. 2; Fig. 1). C3 and C5 are cleaved by their respective multisubunit convertases. Of the two products generated by C5 cleavage, C5a is a potent anaphylactic, chemotactic mediator that induces a broad spectrum of proinflam-matory activities, whereas C5b initiates assem-bly of the C5b-9 membrane attack complex. Although C3a shares many properties with C5a, it is much less potent. C3b contributes to the formation of the C5 convertase and is also critical in the opsonization of immune complexes and invading microorganisms.

In considering the optimal site for comple-ment blockade, components that are common to each of the activation pathways may rep-resent the most effective therapeutic targets. Methods of targeting these convergence points comprise blocking C3 or C5 convertase activity or blocking signaling by the C3a receptor or C5a receptor. However, a limitation of blocking C3a receptor activity is that this will not obstruct complement activities downstream of C3b, a component of the C5 convertase. Furthermore,

C5 can still be activated by the coagulation path-way despite inhibition of C3a receptor signaling or the C3 convertase. Additionally, inhibition of the C3 convertase blocks the generation of C3b, which is critical for innate defense against infection and clearance of immune complexes. In contrast, blocking further downstream at C5, the only convergence point of all four comple-ment activation pathways, still permits the generation of C3b and seems a more promising therapeutic strategy.

Antibodies against human C5aR generated using the knock-in strategy of Lee et al. had IC50 values at least half those of the best mAbs raised by a more conventional strategy involv-ing use of C5aR-transfected cells as the immu-nogen. Presumably, the improvement was due to better antigen presentation resulting from the higher density of C5aR expression on the neutrophils of the knock-in mice. The anti-human C5aR mAbs showed improved therapeutic efficacy, completely reversing pre-existing joint inflammation in a mouse model of rheumatoid arthritis. This complete inhibi-tion of disease contrasts with results obtained by blocking C5 convertase in another mouse model of joint inflammation3.

mAbs against human4 and mouse5 C5 do not bind C5a, but block C5 convertase and prevent the generation of both C5a and C5b-9 (refs. 4,6). Systemic administration of the mAb against mouse C5 prevented the progression of inflammation to other joints and significantly reduced pre-existing inflammation3. However, it did not completely ameliorate inflammation, as was shown for the C5aR antibodies. Lee et al. propose that as anti-C5aR mAb did not block activity of C5L2, a decoy C5a receptor that is co-expressed with C5aR7, the uninterrupted

C5a-C5L2 signaling and subsequent immune-regulatory effect7 may contribute to the rapid reversal of pre-existing joint inflammation.

Comparison of results from preclinical asthma trials6,8 involving inhibition of C5a signaling or inhibition of the generation of C5a and C5b-9 corroborates the notion that antibodies against C5 and C5aR can have different effects. T-helper type 2 (Th2) cyto-kines, such as IL-4, IL-5 and IL-13, help to recruit inflammatory cells into the airways. The observation that blockade of C5a signal-ing using an anti-C5aR mAb reduced airway inflammation and increased levels of intra-pulmonary Th2 cytokines8 was interpreted to indicate that C5a has both anti- and proin-flammatory components in asthma, making C5 inhibition unsuitable as a therapeutic stra-tegy8. In contrast, another study showed that blockade of C5a and C5b-9 generation using a mAb against mouse C5 ameliorated intrapul-monary inflammation without any significant influence on Th2 cytokine profiles6. The latter data suggest that activated C5 components are potent proinflammatory mediators and that terminal complement blockade, such as results from anti-C5 mAb treatment, indeed offers promise for asthma therapy6.

The key difference between the two studies may be the presence of C5b-9 and C5a after anti-C5aR mAb treatment. Uninterrupted C5a-C5L2 signaling during C5aR blockade may explain the elevated Th2 cytokine profile seen in the first study. Also, the presence of C5b-9, which possesses potent proinflammatory activ-ities in addition to its well-known property of lysing target cells9, might account at least par-tially for the different conclusions reached in the two asthma studies. Comparison of the

Classical Lectin

C3 convertase C5 convertase

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Microbial infections, proteolytic allergens, immune complexes & other stimuli

Figure 1 A simplified scheme of the complement activation cascade shows that C3 and C5 are critical convergence points of four activation pathways and indicates potential targets of anti-inflammatory drugs. Inhibitors targeting C5 may impact activation of all four pathways and offer advantages over C3 inhibitors, most notably the generation of C3b, a key component of the innate immune response. Inhibitors of C5aR signaling seem to have a different mechanism of action and associated risk-benefit profile compared with those that block cleavage of C5 to generate C5a and C5b-9.

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different effects of antibodies against C5 and C5aR shows that a better understanding of the roles of both C5a and C5b-9 and the develop-ment of more relevant experimental models are critical for selecting the appropriate comple-ment target for particular indications.

Eculizumab, a mAb against human C54, has recently shown promise in the clinic, and its long-term administration seems to be safe and well tolerated10. Together with new methods of creating mAbs, this will undoubtedly encour-age the development of new therapeutics targeting the complement cascade. However, choices between different approaches to block-ing C5 activity need to weigh the benefit to risk profile for each disease indication. As the complement cascade can be activated both systemically and locally, such strategies may be further refined by local rather than sys-temic complement inhibition, particularly in diseases such as rheumatoid arthritis or aller-gic asthma, that are associated with significant local elevations of C5 activity.

Careful consideration is required when selecting mAbs designed to target specific components of the complement cascade. Inhibition of each target entails distinct ben-efits and consequences. Choosing the best target from the growing number of choices, coupled with the proper delivery method, will be critical for the success of complement inhibitors as therapeutics.

1. Lee, H. et al. Nat. Biotechnol. 24, 1279–1284 (2006).

2. Huber-Lang, M. et al. Nat Med. 12, 682–687 (2006).

3. Wang, Y., Rollins, S.A., Madri, J.A. & Matis, L.A. Proc. Natl. Acad. Sci. USA 92, 8955–8959 (1995).

4. Thomas, T.C. et al. Mol. Immunol. 33, 1389–1401 (1996).

5. Frei, Y., Lambris, J.D. & Stockinger, B. Mol. Cell Probes 1, 141–149 (1987).

6. Peng, T. et al. J. Clin. Invest. 115, 1590–1600 (2005).

7. Gerard, N.P. et al. J. Biol. Chem. 280, 39677–39680 (2005).

8. Kohl, J. et al. J. Clin. Invest. 116, 783–796 (2006).9. Dobrina, A. et al. Blood 99,185–192 (2002).10. Hillmen, P. et al. N. Engl. J. Med. 355, 1233–1243

(2006).

Automated phosphorylationsite mappingOle N Jensen

Improved computational methods facilitate automated assignment of protein phosphorylation sites by mass spectrometry.

Mass spectrometry (MS) allows the localiza-tion of phosphorylated amino acid residues in proteins. Precise annotation of phosphory-lation sites is not trivial, however, because of the inherent complexity of most phospho-peptide mass spectra. In this issue, Beausoleil et al.1 report computational techniques for automated phosphorylation site assignment in large-scale proteomic experiments. Their methods promise to alleviate an important bottleneck in phosphoproteomics and to facil-itate faster and more comprehensive analysis of regulatory networks in cells and tissues.

Protein phosphorylation is arguably the most important post-translational modifica-tion, as it plays decisive roles during cellular development, growth and differentiation and in numerous pathological conditions. Functional

genomic screens for investigating cell signaling processes and protein phosphorylation promise to generate detailed insights into the molecular networks that maintain the integrity and propa-gation of living organisms2,3. MS enables amino acid sequencing of proteins4 and is one of the key analytical technologies in functional phos-phoproteomic research for the identification, quantification and characterization of phos-phorylated proteins5–8.

Experimental tools for large-scale analysis of the phosphoproteome by MS have improved considerably in recent years, but dedicated computational techniques for reliable and automatic interpretation of thousands of phosphopeptide mass spectra have been lack-ing. One of the main challenges is to identify the precise phosphorylation site in phospho-peptides that contain multiple candidate sites, that is, two or more threonine, serine or tyro-sine residues within their sequence. Although the necessary information is often contained in the phosphopeptide mass spectra, this information is not used efficiently by current

algorithms such as Sequest and Mascot. Precise annotation of phosphorylation sites is there-fore often achieved by manual interpretation and validation of each individual phospho-peptide tandem mass spectroscopy (MS/MS) spectrum. This manual step is a major bottle-neck in large-scale phosphoproteomics.

Beausoleil et al. consider several stages of the data analysis pipeline. First, they demonstrate that a target/decoy database search strategy can estimate the degree of false-positive phos-phopeptide assignments and enhance the per-formance of phosphopeptide identification processes. They investigated several database search constraints, such as the requirement for tryptic cleavage specificity of peptides (hydroly-sis C-terminal to arginine or lysine residue) and for high mass accuracy (50 p.p.m. or better). They show that phosphopeptides are identifi-able with high sensitivity at a false-positive rate of approximately 1% when stringent criteria are used for trypsin cleavage, solution charge state and phosphopeptide mass accuracy.

Second, they present a probabilistic approach and scoring scheme for precise annotation of phosphorylation sites via MS/MS spectra of identified phosphopeptides. The algorithm considers several candidate ‘solutions’ to the phosphorylation-site annotation problem and scores the experimental data—that is, the most prominent peaks in the phosphopeptide MS/MS spectrum—against each candidate phos-phopeptide solution. The numeric difference between the two highest-scoring phosphopep-tide solutions is then converted into an Ascore. The authors argue that the algorithm provides an objective score that is suitable for automatic evaluation of large-scale phosphoproteomic datasets. For example, an Ascore of 20 repre-sents a phosphorylation-site assignment that has 99% certainty. The excellent performance of this approach was validated using libraries of known phosphopeptides, and it was com-pared to those of two widely used database search and scoring algorithms, Mascot and Sequest, that are not efficiently optimized for phosphopeptide analysis.

To further demonstrate the utility and per-formance of the probabilistic Ascore method, the authors performed a phosphoproteomic experiment on nocodazole-arrested HeLa cells using MS/MS (Fig. 1). Automated data analysis with the Ascore method resulted in the identification and annotation of a total of 1,761 phosphorylation sites (1,289 phos-phopeptides from 491 proteins). The majority (62%) of these phosphorylation sites gener-ated an Ascore of >19, and therefore repre-sented high-certainty assignments.

The findings of Beausoleil et al. highlight the value of high-quality MS data for localization

Ole N. Jensen is at the Department of Biochemistry & Molecular Biology, University of Southern Denmark, DK-5230Odense M, Denmark. e-mail: jenseno@bmb.sdu.dk

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