Toward a General Chemical Method for Rapidly Mapping Multi-Protein

efficiently cross-link several interacting proteins. However, this methodology has not yet been used to map the architecture of large multi-protein co...
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Toward a General Chemical Method for Rapidly Mapping Multi-Protein Complexes Carilee Denison and Thomas Kodadek* Center for Biomedical Inventions and the Departments of Internal Medicine and Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Texas 75390-8573 Received September 9, 2003

Ru(II)(bpy2)32+Cl2, ammonium persulfate, and visible light irradiation has been shown to rapidly and efficiently cross-link several interacting proteins. However, this methodology has not yet been used to map the architecture of large multi-protein complexes. In this study, this chemistry is applied to the crystallographically characterized yeast proteasome. The data obtained demonstrate both the method’s increased generality and fidelity in comparison to traditional bifunctional cross-linking reagents, while also highlighting the future need for developing better analytical techniques to separate cross-linked products. Keywords: cross-linking • proteasome • PICUP

Introduction Most biological processes are mediated by large multiprotein complexes. A key step toward gaining a full understanding of how these “protein machines” function is to characterize the protein complement of the complex and then to map the network of protein-protein interactions between them. Recently, tandem affinity purification strategies coupled to sensitive analysis by mass spectrometry has emerged as a powerful approach to characterizing the proteins present in a given complex,1,2 particularly in yeast and other organisms where tagged proteins are readily substituted for the native analogue. But elucidating the architecture of multi-protein complexes (i.e., the network of protein-protein contacts) remains an unsolved problem. Most commonly used biological tools to study protein-protein interactions, such as the twohybrid system and related schemes, are not well-suited to this task, because they require that the “bait protein” be employed as an artificial fusion outside of the native complex. This is perhaps one of the reasons for the high false positive rate in these assays. Chemical cross-linking would appear to be an attractive tool for the elucidation of protein machine architecture. Crosslinking experiments could be carried out with affinity-purified complexes to probe contacts under native conditions. Given sufficiently rapid chemistry, it might also be possible to use cross-linking to obtain dynamic information regarding how the contacts within a multi-protein complex might change over the course of a catalytic cycle. Nonetheless, chemical cross-linking remains an underutilized technique. This is due largely to two limitations. The first involves the propensity of common bivalent cross-linking reagents to provide false positives and negatives. The second is the requirement for large numbers * To whom correspondence should be addressed. Phone: 214-648-1239. Fax: 214-648-1415. E-mail: [email protected]. 10.1021/pr034071j CCC: $27.50

 2004 American Chemical Society

of antibodies in order to characterize the many products that result from cross-linking a complex containing several proteins.3-5 Most traditional protein cross-linking reagents consist of two reactive moieties linked by a flexible chain. The reactive groups are most often lysine- or cysteine-reactive species such as activated esters, R,β-unsaturated carbonyl compounds, etc. Thus, in order for a cross-linked product to form, two relatively slow reactions must occur, but obviously they do not occur simultaneously. This means that there is a steady-state buildup of chemically modified proteins that may be much less stable than their native forms, particularly in cases where charged surface lysines are transformed into neutral amides. This can lead to structural destabilization of the complex and non-native interactions between partially unfolded proteins that are rendered covalent by reaction at the other end of the bivalent cross-linker. Clearly, such false positives cause serious complications in building a model for a complex of unknown architecture from cross-linking data. On the other hand, the relatively modest reactivity of commonly used electrophilic moieties in cross-linkers (a necessity because the chemistry is carried out in aqueous solution) means that many interactions are not detected by common cross-linkers. These drawbacks of current cross-linking techniques are illustrated well by an architectural study of the human 20S proteasome6 performed prior to the publication of the highresolution crystal structure.7,8 The 20S proteasome contains 14 unique proteins (two copies of each) arranged in a barrel-like structure comprised of four stacked heptameric rings, as shown in Figure 1. In the cross-linking study,4 the purified complex was treated with dithiobis(succinimidylpropionate) (DSP) a bivalent, lysine-reactive cross-linking agent that allows the cross-link to be dissolved at a later stage by reduction of the disulfide bond in the linker. Other experiments employed a second cross-linking reagent (ethyleneglycol bis(sulfosuccinJournal of Proteome Research 2004, 3, 417-425

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Figure 1. Structure of the 20S proteasome. The X-ray crystallography data obtained by Huber and co-workers8 was utilized to generate this picture of the 20S complex. This structure displays the four heptameric rings that comprise the 20S complex. The two outer rings of this complex are composed of seven different R-proteins, whereas the inner two rings consist of seven different β-proteins. The identity of each of these individual proteins is as follows: R1(bright yellow), R2(royal blue), R3(green), R4(red), R5(aqua blue), R6(magenta), R7(grey), β1(orange), β2(purple), β3(pink), β4(pale yellow), β5(mauve), β6(sky blue), and β7(blue-green).

imidyl-succinate); (sulfo-EGS)). Products formed after a long incubation were analyzed by a tedious electrophoresis-based protocol and Western blotting was employed to identify the proteins in each cross-linked product. This approach was made possible by the fact that antibodies were available against each of the fourteen unique proteins in the 20S proteasome. A model of the 20S architecture was published based on these data. Unfortunately, the subsequent publication of the crystal structure of the 20S complex revealed that 36% of the cross-linked species reported were false positives. Not surprisingly, this had led to a flawed model for the complex in which four of the fourteen 20S subunits were positioned incorrectly.4,9 This example is not an unusual case, as pointed out in a recent review.3 For example, cross-linking studies have been reported for both the RNA polymerase II and Arp2/3 complexes. Subsequent crystallographic studies of these species showed that 41% and 14% of the reported contacts within RNA polymerase II and Arp2/3, respectively, were false positives.10-12 While it is true that this rate of false positives is better than that observed using two-hybrid analysis,3 it is obvious that major improvements are required before chemistry can be used with confidence to probe the organization of structurally uncharacterized multi-protein complexes. Several years ago, our laboratory reported the development of a new type of protein cross-linking reaction that was 418

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intended to begin to address this issue.13 It was shown that several closely associated proteins could be linked covalently when photolyzed for only 0.5 s in the presence of tris(2,2′bipyridyl)ruthenium (II) dication (Ru(II)(bpy)32+) and ammonium persulfate (APS). These conditions are known to result in the photooxidation of Ru(II)(bpy)32+ to Ru(III)(bpy)33+. This intermediate is proposed to oxidize tyrosine or tryptophan residues on a protein, resulting in the production of radical species that couple rapidly to appropriate nearby functional groups such as other aromatics, nucleophilic side chains, etc. Thus, this reagent employs fundamentally different chemistry than traditional cross-linkers. The ruthenium complex acts as a catalyst for direct cross-linking of closely associated functional groups in the interacting proteins, resulting in a so-called “zeroangstrom” or “traceless” cross-link. One would imagine that this fact, combined with the speed of the reaction, should limit the number of artifactual cross-linked products generated by this chemistry, an idea supported by the results currently available. However, the Ru(II)(bpy)32+/APS/light-mediated chemistry has yet to be tested in the more complex setting of a large multi-protein complex. In this work, therefore, we report the application of this technology to mapping the protein-protein contacts present in the yeast 20S proteasome. Since, as mentioned above, standard cross-linking produced a flawed model of this complex, the 20S complex provides a good test of the utility of any new protein cross-linking chemistry. The results show that the ruthenium-mediated cross-linking reaction performs well in the sense that no evidence was obtained for false positives and the majority of the proteins in the complex were observed in cross-linked products. However, this work also reveals the severe limitations imposed by deficiencies in the separation and mass spectrometry-based analysis of cross-linked products, which must be improved before this approach can be employed to construct useful models for the architecture of multi-protein complexes.

Results and Discussion Defining the Conditions for 20S Cross-Linking. The crystal structure of the Saccharomyces cerevisiae 20S complex reveals four heptameric rings that stack on top of each other to form a barrel-shaped structure. The building blocks of this cylindrical structure are two copies each of 14 different proteins. The two outer rings of this complex consist of seven different R subunits, whereas the inner two rings of the complex are comprised of seven different β subunits (categorized by sequence similarity). To examine the utility of Ru(bpy)32+/APS/light-mediated crosslinking in this system, it was first necessary to work out optimized conditions. To do so, several cross-linking experiments were carried out under various conditions and the results were analyzed by one-dimensional SDS-PAGE and Western blotting using antibodies against various proteasomal proteins. Figure 2 demonstrates such an experiment using an antibody raised against the β4 protein (Pre1). In this experiment, 1.5 µM immunopurified 20S complex was irradiated with visible light (>400 nm) in the presence of 0.13 mM Ru(bpy)32+and 2.5 mM APS. Lane 1 represents a control sample that was not irradiated and Lane 2 represents an identical sample that had been irradiated with visible light (>400 nm) for 0.5 s. Good yields of β4-containing products are clearly visible on the Western blot. Analysis of 20S Cross-Linked Products by MALDI-TOF Mass Spectrometry. We then proceeded to analyze, in a more global sense, the products produced in the Ru(bpy)32+/APS/lightmediated cross-linking of the 20S complex under the optimized

Rapidly Mapping Multi-Protein Complexes

research articles then excised from the gel, digested with trypsin, and the masses of the resultant peptides were measured by MALDI-TOF mass spectrometry and identified by comparison to the database. In theory, if proteasomal protein A is cross-linked to proteasomal protein B, peptides derived from both of these proteins would be apparent in the mass spectrometer. Thus, if peptides from both proteins are found in a band with the appropriate apparent molecular mass for a dimeric product, we could conclude that protein A and protein B were in direct contact in the native complex. It is important to note that in this approach we are not searching for the cross-linked peptides themselves. Because the chemical structures of the cross-linked products are unknown and because this radical-based reaction probably produces a heterogeneous mixture of cross-linked products at the atomic level, it would be difficult to find these species in a large mixture of peptides.

Figure 2. Probing cross-linking of the 20S proteasome with the β4 (Pre1) antibody. Both a 20S non-cross-linked (Lane 1) and cross-linked sample (Lane 2) were separated by SDS-PAGE. Western blot analysis was performed with an antibody raised against one of the β-proteins of the 20S, β4.

Figure 3. Mass spectrometry- based protocol for analyzing crosslinked products. Cross-linked products are separated by SDSPAGE, cut from the gel, and trypsinized. The masses of the resulting peptides are measured by MALDI-TOF mass spectrometry and submitted to database search programs. These programs render the identity of the proteins present in a particular cross-linked product by recognizing non-cross-linked peptides. Because the masses of cross-linked peptides are not present in databases, they cannot be used in this identification process.

conditions. Our laboratory only had antibodies raised against three of the 14 unique 20S proteins, a fairly typical situation. Therefore, we decided to utilize a mass-spectrometry-based protocol that should be adaptable to any multi-protein complex as long as the sequences of its protein components are known. The procedure used to analyze cross-linked species is outlined in Figure 3. After Ru(bpy)32+/APS/light -mediated cross-linking, the protein mixture was separated by SDS-PAGE. Each band representing one or more cross-linked products was

Panels A and B of Figure 4 illustrate the first step of this protocol for the 20S complex. The proteins present in samples that had or had not been treated with Ru(bpy)32+/APS/light were separated by one-dimensional SDS-PAGE and stained. In Figure 4A, these proteins were visualized by silver staining because this is a sensitive method of protein detection and therefore provided a reasonable estimation of the total number of cross-linked products present. Clearly, a multitude of interesting cross-linked products were generated for this complex, including many products that exhibited the electrophoretic migration expected of multiply cross-linked products. With this result in hand, the 20S cross-linking reaction was scaled up in order to facilitate the subsequent mass spectrometry-based analysis steps. Figure 4B shows the result of this large-scale reaction which was visualized by staining with colloidal coomassie dye. Although less sensitive than silver staining, this staining method was chosen since colloidal coomassie dye has been reported to be more compatible with tryptic digest/mass spectrometry protocols.14,15 Although these cross-linking bands were less distinctive than those seen by silver staining, 25 different bands were discerned that were not present in a control sample which had not been cross-linked. Each of the 25 bands were cut from the gel, trypsinized, and the resultant peptides were analyzed by mass spectrometry. Recognizable peptides were identified in all 25 samples. The data revealed two types of cross-linked products, “simple” species containing only two proteins and complex species containing more than two proteins. There were two types of the latter product. Of course, the products that migrated slowly through the gel were anticipated to have more than two proteins as a result of two or more cross-linking events. However, some of the bands with the electrophoretic migration expected of a dimeric product also exhibited peptides from more than two proteins, presumably representing the comigration of two different dimeric products. Figure 4C,D depicts both types of products. When the masses of the trypsinized peptides of cross-linked band A were submitted to the database, the β7 and R6 proteins were identified. This product was expected because β7 and R6 clearly contact one another in the crystal structure. No other peptides representing proteasomal protein-derived peptides could be detected in this sample. Band B, on the other hand, migrates at a position on the gel corresponding to the molecular weight of a 1:1 cross-linked product, but peptides derived from four different proteins were identified in the mass spectrum (R1, R2, R5, and R6). From the structure, R1 is known to interact Journal of Proteome Research • Vol. 3, No. 3, 2004 419

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Figure 4. Analysis of two types of 20S cross-linked products by mass spectrometry. A. Silver stained visualization of 20S cross-linked products. Both a non-cross-linked and a cross-linked 20S sample were separated by SDS-PAGE. After silver staining analysis, a multitude of cross-linked products were visible. B. Colloidal coomassie stained visualization of 20S cross-linked products. To identify the crosslinked products, the cross-linking reaction of panel A was scaled up to aid subsequent analysis steps. The products were visualized by colloidal Coomassie staining. C. Identification of Bands containing single cross-linked products. Band A from panel B was digested with trypsin and the masses of the resulting peaks were measured by MALDI-TOF mass spectrometry. These peaks matched to the β7 and R6 proteins in subsequent database searches indicating a β7-R6 cross-link. This interaction is in accordance with the crystal structure. D. Identification of bands containing multiple cross-linked products. Band B from panel B was analyzed in the same manner. This band runs at a position predicted for a 1:1 cross-linked product. However, peaks corresponding to the R1, R2, R5, and R6 proteins were identified suggesting R1-R2 and R5-R6 cross-links that migrate at identical positions in SDS-PAGE.

with R2, and R5 interacts with R6. Hence, the results obtained from this band most likely reflect comigrating R1-R2 and R5R6 cross-linked products (which have predicted molecular masses of 55 kDa and 54 kDa, respectively). Because all of the 20S proteins are between 22 and 35 kDa, the problem of electrophoretic overlap is particularly severe with this complex. Unfortunately, experiments using two-dimensional electrophoresis were not productive, as few distinct spots representing cross-linked products were observed (data not shown). This may reflect the aforementioned chemical heterogeneity of the products. Another notable fact was that a number of individual interactions were detected in multiple bands migrating at very similar positions on the gel. For example, in the region of the gel that 1:1 20S cross-linked products are predicted to run, the R1-R2 interaction was detected in two consecutive bands (bands 6 and 7; Table 1). This is presumably due to the production of two chemically distinct products involving the 420

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same two proteins that migrate differently through the gel. The detailed chemistry that leads to cross-linking is not well understood, but the reaction is thought to be initiated by the formation of tyrosine or tryptophan radicals. These highly reactive species could couple to other moieties via a variety of distinct pathways, leading to the production of chemically heterogeneous product mixtures in some cases. A related possibility is the formation of intramolecular cross-links within a protein in addition to cross-linking to another protein. It would not be surprising if the electrophoretic mobility of the products were altered by such intramolecular cross-linking events. A summary of the proteins present in each cross-linked product is presented in Table 1. Using the crystal structure as a guide, we were able to rationalize all of the data and infer the presence of the cross-links shown in Figure 5. Panel A represents the intra-ring contacts detected and Panel B outlines the inter-ring interactions.

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Rapidly Mapping Multi-Protein Complexes Table 1. Analysis of 20S Cross-Linked Products by Mass Spectrometrya band

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

22 23

24

25

protein

β7 β7 β7 β7 β7 R1 R1 R5 R1 R4 R6 R1 R5 R1 R6 R4 R4 R1 R1 R4 R3 R1 β7 β5 β7 β4 β4 β7 β4 R1 R6 R4 R1 R4 R1 R3 β7 R4 R1 β4 R1 β4 R3 R6 R2 R1 R3 R1 R6 β4 R3 R1

peptides

MOWSE

protein

peptides

MOWSE

assignment(s)

4 4 4 7 5 7 7 4 6 4 5 5 4 6 3 3 5 7 8 5 2 8 5 3 5 2 3 5 5 6 4 3 8 5 5 3 6 5 6 5 6 3 5 5 4 7 5 7 5 3 6 6

1.9 × 1.9 × 103 1.9 × 103 4.1 × 103 2.1 × 104 1.6 × 104 1.6 × 104 2.1 × 103 1.3 × 104 2.7 × 102 5.5 × 103 2.3 × 103 2.1 × 103 3.1 × 103 8.2 × 102 2.7 × 102 4.0 × 104 1.5 × 104 7.1 × 104 6.8 × 103 8.0 × 102 2.8 × 104 8.5 × 103 9.6 × 102 4.5 × 104 8.1 × 102 2.5 × 102 1.1 × 103 2.4 × 103 1.1 × 104 2.0 × 103 4.2 × 102 2.2 × 105 2.1 × 103 2.1 × 103 2.9 × 102 1.1 × 103 3.1 × 103 4.0 × 102 5.8 × 102 1.3 × 104 1.9 × 103 5.2 × 103 7.6 × 103 7.2 × 102 6.1 × 104 2.5 × 103 4.4 × 104 1.1 × 104 2.5 × 102 1.2 × 104 1.1 × 104

n/a n/a β4 β4 R6 R2 R2 R6 R3 R5 n/a R3 R6 R3 R5 n/a R3 R6 R7 R5 β2 R7 β1 R2 β2 β5 β5 n/a β5 R3 R7 n/a R3 R5 R6 R4 R7 R5 R3 n/a R3 n/a R4 R7 β7 β4 R4 R7 β7 β5 R4 R6

n/a n/a 5 5 4 4 4 3 2 4 n/a 3 6 3 3 n/a 4 3 5 2 3 4 4 2 4 2 6 n/a 5 3 4 n/a 3 3 4 3 6 6 7 n/a 5 n/a 7 6 5 3 6 4 4 6 6 6

n/a n/a 4.1 × 103 2.4 × 103 1.9 × 103 1.9 × 103 4.8 × 102 8.2 × 102 8.4 × 102 8.4 × 102 n/a 2.9 × 102 7.6 × 102 2.9 × 102 1.7 × 102 n/a 9.6 × 102 7.0 × 102 5.6 × 102 7.0 × 102 2.6 × 102 9.5 × 102 2.4 × 102 6.7 × 102 1.6 × 102 8.1 × 102 1.8 × 102 n/a 1.2 × 103 2.9 × 102 1.9 × 103 n/a 2.9 × 102 5.0 × 102 1.9 × 103 2.7 × 102 2.5 × 103 5.1 × 104 4.0 × 104 n/a 2.3 × 103 n/a 1.7 × 106 2.3 × 102 5.8 × 102 3.1 × 102 1.7 × 106 1.6 × 102 1.6 × 103 6.3 × 103 1.3 × 105 1.2 × 104

β7 β7 β7;β4 β7;β4 β7-R6 R1-R2 R1-R2 R5-R6 R1-R3 R4-R5;R3-R4 R5-R6 R1-R3 R5-R6 R1-R3 R5-R6 R3-R4;R4-R5 R3-R4;R3-R6 R1-R3;R1-R622 R1-R7;R1-R3 R4-R5,R3-R4 R3-β2 R1-R7 β7-β1 β5-R222 β7-β2 β4-β5 β4-β5 β7-β7 β4-β5 R1-R3;R1-R7 R6-R7 R3-R4 R1-R3 R4-R5;R3-R4 R1-R6;R1-R3 R3-R4 β7-R7 R4-R5 R1-R3;R3-R4 β4-β4 R1-R3 β4-β4 R3-R4 R6-R7;β7-R7 R2-R3;R6-β7 R1-R7,β4-β4 R3-R4 R1-R7;R1-R3 R6-β7;R6-R7 β4-β5;β7-R7 R3-R4;R3-R6 R1-R6;R1-R3

103

a The proteins identified in each cross-linking band are listed, as are the number of peptides identified for each protein and the MOWSE score of that match. Finally, the protein-protein interaction(s) assigned to each band is tabulated.

Fidelity and Generality of the Ru(II)(bpy)32+/APS/LightMediated Reaction. The information represented in Table 1 and Figure 5 bears on two questions relevant to the utility of the Ru(II)(bpy)32+/APS/light-mediated cross-linking reaction in the analysis of multi-protein complexes. The first is whether artifactual products are produced, representing covalent trapping of non-native interactions. As mentioned in the Introduction, this was a major problem in an earlier analysis of the 20S structure using a bivalent cross-linking reagent.4,9 We find no evidence for such products. All of the products inferred from the mass spectrometry analysis of the peptides present in each band can be rationalized by contacts seen in the crystal structure. However, we cannot rule out completely the possibility that some artifactual products might have been present in the bands containing several proteins. For example, band B

contained peptides derived from R1, R2, R5, and R6, consistent with the expected production of comigrating R1-R2 and R5R6 products, but we cannot unambiguously eliminate the possibility that, for example, R1-R5 or R2-R6 products were present in this band. A technical issue that merits attention is that a number of the proteasomal proteins present in the cross-linked bands were identified with relatively low certainty. This is indicated by the MOWSE (MOlecular Weight SEarch) scores found in Table 1. These values are a calculation of the probability that the assignment is a random match based on the differences between the masses of a theoretical digestion and the experimental values measured.16 In this process, scores are calculated with a complex algorithm for each individual peptide match, multiplied together, and, ultimately, reported as the -10*log Journal of Proteome Research • Vol. 3, No. 3, 2004 421

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Figure 5. Summary of 20S cross-linked products. A. Intra-ring interactions. All R-subunit intra-ring interactions were detected in this study, and two β-subunit intra-ring interactions were detected. No false positives were seen. B. Inter-ring contacts. A number of inter-ring interactions were also observed for the 20S complex. As seen for the intra-ring interactions, no false positives were detected for inter-ring associations. C. Number of times each protein appeared in a cross-linked product. All 20S proteins except β3 and β6 were present in at least one cross-linked product. In general, it was clear that R-subunits were better substrates for this photochemistry than β-subunits.

of the probability. Therefore, a larger score is indicative of a better match. Despite the relatively low values (in the 102-103 range) seen in some cases, we are nonetheless confident in the assignments provided. In questionable cases, two peptides for any suspect proteins were utilized to calibrate the mass spectrum. If this resulted in a well-calibrated spectrum, as indicated by the masses of the internal trypsin and keratin peaks, then these proteins were concluded to be probable matches. The most likely source of the low MOWSE scores is the simple fact that all of the 20S proteins are relatively small and therefore produce fewer tryptic fragments than larger proteins. It seems likely that the low MOWSE scores observed for some of the cross-linked products reflect this property rather than any problem with the cross-linking chemistry per se, such as massive covalent modification of the protein, leading to few recognizable tryptic products. To probe this point, we also carried out a thorough analysis of the intact, immunopurified 26S proteasome. This complex includes the 19S proteasome regulatory complex as well as the 20S core complex. As displayed in Figure 6, the proteins in the 19S exhibit a more typical range of masses. The bands derived from an untreated sample of immunopurified 26S proteasome were analyzed by mass spectrometry. As expected, the 20S protein bands provided lower MOWSE scores than most of the higher molecular mass 19S proteins although none of the proteins had been exposed to the ruthenium cross-linking agent or APS. 422

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The second major issue addressed by the data is the generality of the cross-linking chemistry. In other words, how many of the 14 unique 20S proteins would be found in crosslinked products? As shown in panel C of Figure 4, all but two of the 20S proteins (β3 and β6) appeared in at least one crosslinked product. However, it was clear that some proteins were detected in cross-linked species more often than others. Such trends were not due to variations in the ability of the peptides of different proteins to fly in the mass spectrometer, as demonstrated by the analysis of the unmodified 26S proteasome. Rather, it apparently reflects the fact that some proteins are better substrates for this oxidative reaction than others. In general, most R subunits were better substrates than the β-proteins. For this reason, one R-β interaction (R2-β2) and several β-β interactions (β2-β3, β5-β3′, β6-β3′, and β6-β2′) expected from the crystal structure were not detected in this study. Although the reason for this has not been further studied, it seems probable that this is attributable to the tyrosine rich N-terminal tails of the R-proteins which are known to cover the ports leading into the interior chamber of the 20S.8,17 Tyrosine is easily oxidized by the Ru(III) intermediate. Therefore, although many cross-linked products were produced using the oxidative cross-linking reaction, the data suggest that in order to achieve coverage of most or all of the protein contacts in a large complex, complementary experiments using other types of cross-linking chemistries would be required. We are currently experimenting with other reagents, in particular diimide-based species that also support “zero-Å” cross-linking, in the context of the 20S complex to evaluate their fidelity and generality.

Conclusions This study represents the first use of Ru(II)(bpy)32+/APS/ visible light-mediated cross-linking chemistry in the context of a large multi-protein complex, the 20S proteasome core particle. The results were mixed. On one hand, it was encouraging that no evidence was found for the production of false positives, i.e., cross-linked species representing non-native contacts between proteins. In addition, the Ru(II)(bpy)32+/APS/ visible light-mediated chemistry trapped all but two of the 14 20S proteins in cross-linked products, representing reasonable generality. On the other hand, not all of the expected contacts in the complex could be detected, suggesting that complementary data sets obtained using cross-linkers that operate via different chemistry will be required to construct complete contact maps of large complexes. In general however, it seems reasonable to conclude that the Ru(II)(bpy)32+/APS/visible lightmediated chemistry performed quite well in this complex application. The major limitation of this protocol clearly lies in the analysis of the cross-linked products. We chose to rely on mass spectrometry rather than Western blotting techniques to characterize products, since in most cases investigators will not have good antibodies against every protein in a large complex. Unfortunately, standard one-dimensional SDS-PAGE did not provide sufficient resolving power to separate all of the crosslinked products, an issue exacerbated in the case of the 20S by the fact that most of the substituent proteins have similar masses. Although not reported in detail here, we have expended a great deal of effort in an attempt to circumvent this problem. As mentioned above, two-dimensional gels proved disappointing. Two-dimensional (2D) gel electrophoresis is notorious for

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Figure 6. Comparison of MOWSE scores obtained for proteins of both the 19S and 20S proteasome. A. Separation of the 26S proteasome by two-dimensional electrophoresis. The proteins identified by mass spectrometry from this gel are numbered. B. Separation of the 26S proteasome by one-dimensional electrophoresis. As seen for the two-dimensional analysis, protein bands that were isolated and characterized by mass spectrometry are numbered. C. Proteins identified by mass spectrometry from the gels in panels A and B. The identity of the proteins identified from the one-dimensional and two-dimensional separations of the 26S complex are indicated in this panel, along with both the number of peptides identified for each protein and the MOWSE score with which this assignment was made. In general, higher MOWSE scores were obtained for the larger proteins of the 19S complex than were seen for the smaller proteins of the 20S.

its inability to separate high molecular weight proteins.18 Almost all of the cross-linked products detected in the 2D gel migrated at a position in the gel corresponding to a 1:1 species. Very few higher order bands were visible in this analysis and even a significant fraction of the 1:1 products that were visible in the one-dimensional analysis were not seen in the twodimensional analysis (unpublished observations). This may have been due to these species being unable to enter the iso-

electric focusing (IEF) gel or to transfer from the IEF gel to the denaturing gel. Alternatively, some of the bands in the onedimensional gel may have smeared in the IEF dimension as a result of the suspected chemical heterogeneity. In any event, we found 2D electrophoresis to be an unsatisfactory solution to the analysis problem. Another option that we pursued was the use of gradient or zonal one-dimensional gels. The 20S cross-linked products Journal of Proteome Research • Vol. 3, No. 3, 2004 423

research articles separated in this manner were visualized by silver-staining (data not shown). Although the observed products were not characterized by mass spectrometry, it was clear that indeed better separation was achieved for many of the cross-linked species. However, this was not the case for many of the bands. Running significantly longer zonal gels (approximately two feet in height) further enhanced this effect, but, again, did not fractionate all cross-linked products sufficiently. Clearly, a major hurdle in the future development of this technique will be to improve the analytical protocols downstream of the cross-linking steps. If we had not already known the structure of the 20S complex, it would not have been possible to build a model of the protein contacts within it from the data obtained here. A fundamentally different alternative strategy that is under investigation is to attempt to identify the cross-linked peptides themselves using a labeling scheme developed by Anderson and co-workers19 and then sequence these species by MS/MS. The results of these efforts will be reported in due course.

Experimental Section 20S Crystal Structure. Crystallographic data points were obtained from pdb file 1RYP found at www.rcsb.org.8 A picture was generated with the Swiss pdb viewer program and then rendered in POVRAY. Proteasome Purification and Cross-Linking Experiments. Affinity purified 20S proteasome was isolated from a FLAG(His)6-tagged β4 (Pre1) S. cerevisiae strain (RJD) as reported previously.20,21 Small-scale cross-linking reactions contained 10 µg of 20S complex in PBS Buffer (15 mM sodium phosphate (pH 7.5) and 150 mM NaCl). Immediately before irradiation, 0.13 mM Ru(bpy)3Cl2 and 2.5 mM APS were added, and the mixture was photolyzed for 0.5 s. The samples were quenched with loading buffer, separated on a 9% SDS-PAGE gel and visualized by silver staining or Western blot analysis with a mouse antibody raised against the β4 protein (1:3000 dilution). The large scale cross-linking reaction of the 20S complex was the same, except 30 µg of 20S protein was utilized in the experiment and the results were visualized with colloidal Coomassie dye (Invitrogen). Trypsin Digests. Appropriate colloidal stained bands were transferred to 0.65 mL siliconized eppendorf tubes and destained by vortexing in a 25 mM NH4HCO3 (pH 8.0)/ 50% methanol solution for 10 min. The solution was removed, and this destaining step was repeated two more times. Next, the clear gel piece was soaked in 10% acetic acid/ 50% methanol for 1 h at 4 °C. After removing the liquid, the gel was soaked in the same solution for an additional 8 h, changing the solution after the 4 h mark. The gel pieces were then soaked in water for 2 h, changing the solution at the 1 h mark. The gel pieces were washed in a 50 mM NH4HCO3 (pH 8.0) solution for 5 min. and crushed into fine pieces with gel-crushing sticks (Biospec Products). Sequencing grade modified trypsin (Promega) in 50 mM NH4HCO3 was added to each tube to submerge all gel pieces. These samples were incubated at 37 °C for 2 h. After this incubation, one volume of acetonitrile was added to each tube, and the samples were vortexed for 3 min. to extract the peptides. The supernatant of each sample was transferred to a new eppendorf tube. Next, the gel pieces were dried in a speed vacuum and rehydrated by vortexing the samples in 50 mM NH4HCO3 (pH 8.0) for 3 min. The above acetonitrile extraction step was repeated, and then the entire drying, rehydration, and 424

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extraction procedure was performed one final time. All supernatants for a given sample were combined and speed vacuumed to dryness. MALDI-TOF Analysis. To prepare the matrix solution, 10 mg of recrystallized R-cyano-4-hydroxycinnamic acid (CHCA; Sigma) was dissolved in 1 mL of 50% acetonitrile/0.3% TFA. Peptide samples were dissolved in 10 µL of 0.1% TFA and desalted on a Zip Tip C18 column (Millipore). The peptides were then eluted directly onto the MALDI plate with 2 µL of the above matrix mixture. Mass spectra were obtained on a MALDI-TOF Voyager 5.0 Mass Spectrometer (PE Biosystems). All spectra were calibrated either internally with multiple trypsin and/or keratin peaks or externally with a calibration mixture (PE Biosystems). The resulting peptide mass fingerprints were submitted to the MS-Fit program found at the http://prospector.ucsf.edu web site. These searches were run with no bias of species, molecular weight or pI. MOWSE scores reported in Figure 3 were obtained from searches run at a 100 ppm mass tolerance. If the MOWSE score was relatively low for a particular hit, then two peptides for this protein were used to calibrate a new spectrum for the sample, and the identification process was repeated.

Acknowledgment. We would like to thank Dr. David Fancy for his assistance in generating the crystal structure in Figure 1 and both Dr. Kathlynn Brown and Dr. Yingming Zhao for helpful suggestions concerning MALDI-TOF analysis. This work was supported by grants from the National Institutes of Health (GM 58075) and the Welch Foundation (I-1299). References (1) Gavin, A. C.; Bosche, M.; Krause, R.; Grandi, P.; Marzioch, M.; Bauer, A.; Schultz, J.; Rick, J. M.; Michon, A.; Cruciat, C.; Remor, M.; Hofert, C.; Schelder, M.; Brajenovic, M.; Ruffner, H.; Merino, A.; Klein, K.; Hudak, M.; Dickson, D.; Rudi, T.; Gnau, V.; Bauch, A.; Bastuck, S.; Huhse, B.; Leutwein, C.; Heurtier, M.; Copley, R. R.; Edelmann, A.; Querfurth, E.; Rybin, V.; Drewes, G.; Raida, M.; Bouwmeester, T.; Bork, P.; Seraphin, B.; Kuster, B.; Neubauer, G.; Superti-Furga, G. Nature 2002, 415, 141-147. (2) Ho, Y.; Gruhler, A.; Heilbut, A.; Bader, G. D.; Moore, L.; Adams, S.; Miller, A.; Taylor, P.; Bennett, K.; Boutilier, K.; Yang, L.; Wolting, C.; Donaldson, I.; Schandorff, S.; Shewnarane, J.; Vo, M.; Taggart, J.; Goudreault, M.; Muskat, B.; Alfarano, C.; Dewar, D.; Lin, Z.; Michalickova, K.; Willems, A. R.; Sassi, H.; Nielsen, P. A.; Rasmussen, K. J.; Andersen, J. R.; Johansen, L. E.; Hansen, L. H.; Jespersen, H.; Podtelejnikov, A.; Nielsen, E.; Crawford, J.; Poulsen, V.; Sorensen, B. D.; Matthiesen, J.; Hendrickson, R. C.; Gleeson, F.; Pawson, T.; Moran, M. F.; Durocher, D.; Mann, M.; Hogue, C. W. V.; Figeys, D.; Tyers M. Nature 2002, 415, 180-183. (3) Edwards, A. M.; Kus, B.; Jansen, R.; Greenbaum, D.; Greenblatt, J.; Gerstein, M. Trends Genet. 2002, 18, 529-536. (4) Kopp, F., Hendil, K. B.; Dahlmann, B.; Kristensen, P.; Sobek, A.; Uerkvitz, W. Proceed. Natl. Acad. Sci. 1997, 94, 2939-2944. (5) Stoffler, G.; Redl, B.; Walleczek, J.; Stoffler-Meilicke. M. Methods Enzymol. 1988, 164, 64-76. (6) Baumeister, A.; Walz, J.; Zuhl, F.; Seemuller, E. Cell 1998 92, 367380. (7) Lowe, J.; Stock, D.; Jap, B.; Zwickl, P.; Baumeister, W.; Huber, R. Science 1995, 268, 533-539. (8) Groll, M.; Ditzel, L.; Lowe, J.; Stock, D.; Bochtler, M.; Bartunik, H. D.; Huber, R. Nature 1997, 386, 463-471. (9) Dahlmann, B.; Kopp, F.; Kristensen, P.; Hendil, K. B. Arch. Biochem. Biophys. 1999, 363, 296-300. (10) Ishiguro, A.; Kimura, M.; Yasui, K.; Iwata, A.; Ueda, S.; Ishihama, A. J. Mol. Biol. 1998, 279, 703-712. (11) Mullins, R. D.; Stafford, W. F.; Pollard, T. D. J. Cell Biol. 1997, 136, 331-343. (12) Mullins, R. D.; Pollard, T. D. Curr. Opin. Struct. Biol. 1999, 9, 244249. (13) Fancy, D. A.; Kodadek, T. Proc. Natl. Acad. Sci. 1999, 96, 60206024. (14) Lauber, W. M.; Carrol, J. A.; Dufield, D. R.; Kiesel, J. R.; Radabaugh, M. R.; Malone, J. P. Electrophoresis 2001, 22, 906-918.

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