Direct Identification of Ubiquitination Sites on Ubiquitin-Conjugated

Maryland 20850, and Program in Molecular Cardiology, University of North Carolina,. Chapel Hill, North Carolina 27599. Received April 15, 2005. The st...
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Direct Identification of Ubiquitination Sites on Ubiquitin-Conjugated CHIP Using MALDI Mass Spectrometry Dongxia Wang,†,|,# Wanping Xu,‡,| Sara C. McGrath,† Cam Patterson,§ Len Neckers,‡ and Robert J. Cotter*,† Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, Urologic Oncology Branch National Cancer Institute, National Institutes of Health, Rockville, Maryland 20850, and Program in Molecular Cardiology, University of North Carolina, Chapel Hill, North Carolina 27599 Received April 15, 2005

The study of protein ubiquitination, a post-translational modification by ubiquitin, has emerged as one of the most active areas in biology because of the important role of this type of modification on the regulation of various cellular proteins. Advances in techniques for the determination and site mapping of protein ubiquitination can facilitate the elucidation of molecular mechanisms of this modification. We have recently described a novel method for identifying peptides containing ubiquitinated amino acid residues, based on the MALDI-MS/MS analysis of tryptic peptide derivatives. In particular, we have utilized N-terminal sulfonation of these peptides to provide a unique fragmentation pattern that leads to the direct identification and sequencing of ubiquitin modified peptides. Here we present an application of this new method on the characterization of ubiquitin conjugated C-terminal Hsc70interacting protein (CHIP), a recently identified U-box containing E3 enzyme. Three peptides bearing ubiquitination sites have been identified from the digest of ubiquitinated CHIP; one of these was a site on CHIP, while the other two were found on the ubiquitin molecules, demonstrating that sulfonation of tryptic peptides is a general and efficient method for characterizing protein ubiquitination. Keywords: ubiquitination • CHIP • MALDI mass spectrometry • N-terminal sulfonation

Introduction Ubiquitinationsthe covalent conjugation of the 76-amino acid globular protein ubiquitin to other proteinssis essential for the degradation, as well as functional regulation, of cellular proteins.1,2 The ubiquitination process occurs in three steps. First, ubiquitin, with its carboxy-terminal glycine, is conjugated to a ubiquitin-activating enzyme (also known as E1) via a thiolester bond in an ATP-dependent manner. Then, the ubiquitin is transferred to a ubiquitin-conjugating enzyme (E2), again involving the carboxyl terminal glycine. Finally, the E2conjugated ubiquitin is transferred to the -amino group of a lysine residue on a substrate protein, a process catalyzed by a ubiquitin ligase (E3). A novel family of E3 ligases, U-box containing E3s, was described recently.3 The predicted conformation of the U-box is similar to that of the RING finger. However, unlike the RING finger, which is stabilized by Zn2+ ions coordinated by cysteines and a histidine, the U-box structure is maintained by a system of salt-bridges and hydrogen bonds. Despite the structural * To whom correspondence should be addressed. E-mail: [email protected]. † Johns Hopkins University School of Medicine. ‡ National Institutes of Health. § University of North Carolina. | These authors contributed equally to this work. # Current address: Biotechnology Core Facility, National Center for Infectious Disease, Centers for Disease Control and Prevention, Atlanta, GA 30333.

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Scheme 1

similarity of the two families, these E3s may function in different mechanisms in catalyzing the transfer of ubiquitin from E2 to substrate. It was observed that U-box containing E3s were ubiquitinated themselves in in vitro ubiquitination assays.4 This suggests that U-box E3s may participate directly in the transfer of ubiquitin from E2 to substrate. CHIP (C-terminal Hsc70-interacting protein) is one of the six identified U-box containing E3s.5 This protein is thought to play a special role in the quality control of protein folding. Different from other U-box E3s, the CHIP protein associates with molecular chaperones. The N-terminal tetratricopeptide (TPR) domain of the CHIP protein mediates specific binding with the cellular chaperones Hsc70, Hsp70, and Hsp90 (Scheme 1). One of the major functions of these chaperones is to facilitate the folding and refolding of cellular proteins. When a protein fails to attain its native conformation, it either re-enters the folding process or is ubiquitinated and targeted to the proteasome for degradation. By binding to chaperones, CHIP may modulate the triage decision determining the fate of a client protein of the chaperone by modifying those proteins 10.1021/pr050104e CCC: $30.25

 2005 American Chemical Society

Ubiquitination Sites on Ubiquitin-Conjugated CHIP

which have failed to fold properly, thus preventing them from re-entering the folding process and promoting their degradation by proteosomes. In addition, CHIP has been shown to mediate the ubiquitination and degradation of important cellular proteins regulating cell proliferation, apoptosis, stress response, and protein trafficking. CHIP was found to ubiquitinate the glucocorticoid receptor, the cystic fibrosis transmembrane conductance regulator, the receptor tyrosine kinase ErbB2, and the signal transduction protein Smad.6,7 Defects in the degradation of these proteins will cause diseases, such as cancer. Therefore, delineation of CHIP ubiquitination will not only help to elucidate the mechanism by which the U-box E3s catalyze the ubiquitination reaction, but also provide information that will enable us to understand the decision making process in protein folding, and provide a better understanding of the regulation of physiologically significant proteins. Direct determination and site mapping of protein ubiquitination can be achieved by mass spectrometry. Trypsin digestion of ubiquitin modified proteins produces diglycine branched peptide(s), in which the C-terminal Gly-Gly moiety of ubiquitin is attached to the -amino group of a modified lysine residue within a target peptide. Gygi and co-workers recently reported a proteomic approach to detecting protein ubiquitination, based on MS/MS analysis of tryptic peptides where the GlyGly branch is treated as a small modifier.8 Marshall and coworkers developed an accurate mass-based approach using Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry for the analysis of ubiquitinated proteins.9 We have recently developed a novel strategy for identifying ubiquitination sites with high efficiency and specificity using MS/MS and matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry.10 Rather than sequencing the native tryptic peptides, these were derivatized by N-terminal sulfonation, so that two sulfonic acid tags were introduced to the N-terminus of the peptide and its Gly-Gly branch, respectively, prior to fragmentation analysis. The MS/ MS spectra of the sulfonated branched peptide derivatives contain a unique fragment distribution that includes a signature portion that distinguishes these branched peptides from others, and a sequence portion that directly reveals the amino acid sequence of the modified peptide and the location of the ubiquitination site by a series of y type product ions. In the present work, we describe the application of this novel method on the identification of ubiquitination sites on ubiquitinconjugated CHIP modified through an in vitro reaction. Using MALDI-TOF MS/MS analysis of sulfonated tryptic peptides, one ubiquitination site on CHIP has been determined. In addition, two peptides bearing modified lysine residues at different locations on ubiquitin were detected, indicating the formation of polyubiquitin chains.

Experimental Section Materials. All chemicals used in this study were of analytical grade. 4-sulfophenyl isothiocyanate (SPITC), O-methyl isourea, sodium bicarbonate, ammonium bicarbonate, trifluoroacetic acid (TFA), ubiquitin and rabbit polyclonal anti-ubiquitin antibodies were purchased from Sigma (St. Louis, MO). Bovine pancreas modified trypsin was supplied by Roche Diagnostics Corporation (Indianapolis, IN). R-cyano-4-hydroxycinnamic acid was from Aldrich (Milwaukee, WI). Rabbit ubiquitin activating enzyme E1 and recombinant ubiquitin conjugating enzyme GST-UbcH5a were from Biomol (Plymouth Meeting, PA).

research articles Preparation of Ubiquitin Conjugated Protein. Histidine tagged CHIP was prepared from bacteria as described previously.11 The ubiquitination reaction mixture contained 4 µM CHIP, 0.1 µM purified rabbit E1, 8 µM GST-UbcH5a (E2), 2.5 mg/mL ubiquitin in 20 mM MOPS, pH 7.2, 100 mM KCl, 5 mM MgCl2, 5 mM ATP, 10 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. (The purities of CHIP, E1 and GSTUbcH5a were approximately 90%, 98% and 95%, respectively, visually estimated on SDS-PAGE or specified on the manufacturer’s labels.) The reaction was carried out at 30 °C for 4 h. After incubation, CHIP protein was extracted from the mixture with Cobalt beads. An aliquot of the reaction mixture was resolved on 4-20% SDS-PAGE, transferred to PVDF membrane, and probed with anti-CHIP or anti-ubiquitin antibodies to check the ubiquitination of the CHIP protein. The remaining reaction mixture was subjected to metal affinity purification by Talon resin (BD Biosciences, San Jose, CA) following manufacturer’s instruction. Guanidination. Intact proteins (∼300 pmol) bound on Talon resin were first derivatized by guanidination reaction using a modified method of Reilly et al.12 50 µL of freshly prepared O-methyl isourea solution (6 M in 10% NH4OH, pH ≈ 11.0) and 50 µL of H2O were added to the Eppendorf tube containing protein bound beads. The reaction was conducted at 60 °C for 30 min. After reaction, the mixture was centrifuged and the supernatant was removed. The beads were washed three times (100 µL each time) with 25 mM ammonium bicarbonate. Trypsin Digestion. The beads were resuspended by 100 µL of 25 mM ammonium bicarbonate (pH ) 8.0). To this was added 2 µL of trypsin (1 µg/µL) solution. The reaction was carried out at 37 °C for 16 h. The mixture was then centrifuged and the supernatant was transferred to a new test tube. The beads were then washed three times with ddH2O (100 µL each time), the supernatants were collected and transferred to the tube containing reaction solution. HPLC Fractionation. Digested peptide mixtures were fractionated by RP - HPLC on a microbore C18 column (2.1 × 250 mm, Grace Vydac, Hesperia, CA) on Waters 600E Multisolvent Delivery system (Waters, Milford, MA). Solvent A (0.1% trifluroacetic acid in water) and solvent B (0.1% trifluroacetic acid in acetonitrile) were used, and the peptides were eluted by increasing the concentration of solvent B from 5 to 20 in 10 min and then from 20 to 60 in 60 min at a flow rate of 0.2 mL/min. The eluants were monitored at 214 nm, and the fractions were collected at 1 tube per min. The collected fractions were evaporated to dryness by SpeedVac and were resuspended in 5 µL of water. 0.5 µL of each of these fractions was spotted on the MALDI target for mass measurement as described below. Sulfonation. The N-terminal sulfonation reaction was conducted as previously described.10 In brief, 1 µL of the solution taken from the fractions containing peptides was mixed with 3 µL of freshly prepared SPITC (10 mg/mL in 20 mM NaHCO3, pH 9.0). The pH values of the solutions were monitored by pH paper (EM Science, Gibbstown, NJ) and were adjusted to above 7.5 by adding more 20 mM NaHCO3 if necessary. The sulfonation reaction was performed at 55 °C for 30 min and was terminated by 1 µL of 1% TFA. The mixture was subjected to purification with micropipet tip (C18 OMIX, Varian, Lake Forest, CA) followed by eluting with 10 µL of 75% acetonitrile/0.1%TFA. The eluant was dried to 0.5 ∼ 1 µL by SpeedVac and deposited on the MALDI target. Journal of Proteome Research • Vol. 4, No. 5, 2005 1555

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of nonconjugated CHIP after reaction indicates a low modification efficiency that increases the complexity for mapping the modification sites. The extra band immediately above the unmodified CHIP (lane 2) has a molecular weight approximately 8 kDa higher than that of unmodified CHIP, suggesting that the majority of the ubiquitin conjugates might be monoubiquitinated, but it could not be determined from this gel whether this band corresponds to a homogenic species with one specific modification site or to heterogenic conjugates with different mono-ubiquitination sites. The observation of high molecular mass bands in lane 4, implies the formation of multiple heterogenic conjugates or polyubiquitin chains with various chain lengths.

Figure 1. Verification of CHIP ubiquitination. Samples from in vitro ubiquitination assays with (lanes 2 and 4) or without (lanes 1 and 3) E1 were separated by SDS-PAGE and transferred to nitrocellulose membrane. Duplicated blots were probed with antibodies against either CHIP (lanes 1 and 2) or ubiquitin (lanes 3 and 4), and exposed to film aligned side-by-side.

Mass Spectrometry. All MS and MS/MS spectra were acquired in the positive ion mode using a Kratos Analytical (Manchester, UK) AXIMA-CFR MALDI-TOF high performance mass spectrometer equipped with a pulsed extraction source, a 337-nm pulsed nitrogen laser and a curved-field reflectron. The acceleration voltage was 20 kV. External calibration was performed using a mixture of angiotensin II (m/z 1046.5), ACTH 18-39 (m/z 2465.2) and Insulin chain B (m/z 3494.7). The matrix solution was prepared by dissolving R-cyano-4-hydroxycinnamic acid (10 mg/mL) in 50% acetonitrile containing 0.1% TFA. A thin layer of the matrix was applied onto the sample plate first. This was followed by the addition of 0.5 µL of sample and 0.5 µL of matrix solution and was allowed to dry at room temperature. The MS spectra were obtained in the linear mode and each sample spot was scanned by 50 profiles and 5 shots per profile. The MS/MS spectra were acquired in reflectron mode. For the purpose of screening, the MS/MS spectra of individual peptides detected were acquired from the accumulation of 5 profiles at 10 shots per profile, while the fine MS/MS spectra of candidate peptides were obtained with the acquisition parameters of 100500 profiles at 10 shots per profile. The spectra obtained were interpreted manually or by database searching using the MASCOT algorithm (http://www.matrixscience.com).

Results and Discussion N-terminal His-tagged CHIP was prepared from bacteria and modified by ubiquitin through an in vitro ubiquitination reaction in the presence of CHIP (the E3 enzyme), ubiquitin, E1, and E2 enzymes. The reaction product was first examined by Western blotting using anti-CHIP or anti-Ubiquitin antibody. Two blots were exposed together and the results are shown in Figure 1. In comparison with the control sample (lane 1, 3), new products were formed after the ubiquitination reaction and are observed as extra bands above the unmodified CHIP and ubiquitin proteins (lane 2, 4), suggesting the formation of ubiquitin conjugated proteins. The existence of a large quantity 1556

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As noted above, in our approach to identify protein ubiquitination,10 N-terminal sulfonation of the diglycine branched peptides, derived from trypsin digestion of a ubiquitin modified protein, generates unique MALDI MS/MS spectra in which several high-mass fragments serve as the signature for this modification while a series of y type ions enhances peptide sequencing and ubiquitination-site mapping. Interestingly, the very abundant signature fragments can be observed clearly in far fewer scans than are required to produce a full sequence spectrum with good signal/noise, and so can be used to rapidly survey the tryptic peptides for those containing modification sites. Figure 2 shows the MALDI MS/MS spectra of a synthetic diglycine peptide derivative after sulfonation. The three spectra were obtained by signal averaging various numbers of scans applied on the sample spot. In the spectrum (Figure 2a) of 1 profile (10 laser shots), several high-mass fragments were observed with good signal/noise. These fragments represent the signature ions resulting from the preferential cleavage of one or two of the sulfonation tags and the first N-terminal amino acid from both the peptide and the diglycine branch as previously described.10 In contrast, low mass fragments were buried in noise peaks and not as easily detected. In the spectra of 1-10 and 1-100 profiles (Figure 2b,c), however, the lower mass sequence fragments became clear and the overall signalto-noise ratio was improved significantly as more spectra were signal averaged. This approach is particularly useful when peptide mixtures rather than synthetic or purified peptide were analyzed and far more peptides must be screened. For a normal linear tryptic peptide, the dominant fragment peak would correspond to the loss of a single N-terminal tag, while a doubly tagged peptide containing Gly-Gly branch would show the loss of two tags in the signature portion of the spectra. Once the doubly tagged peptides are identified, MS/MS spectra with longer acquisition time can be used to generate a complete fragmentation spectrum for amino acid sequencing. Because our goal in this particular research is to identify diglycine branched peptides, it is not necessary to obtain sequencing information from unrelated peptides that are usually present in very high proportion. Similar to precursor screening or marker ion screening methods developed in other mass spectrometers,13 this approach should save instrument time and spectrum interpretation effort significantly. N-terminal sulfonation will also occur on lysine-terminated peptides derived from trypsin digestion. To protect -amino group on the side chain of an unprotected lysine residue from interacting with sulfonation reagent, a widely used method is to carry out a guanidination reaction on the peptide prior to sulfonation.12,14 An added benefit of this additional step is its enhancement on the ionization efficiency by the conversion of a lysine residue to homoarginine.15 We demonstrated previ-

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Figure 2. MS/MS spectra of the sulfonation derivative of a synthetic, diglycine branch-containing peptide. The spectra were acquired with various numbers of scans applied on the sample spot.

ously that guanidination of a lysine-terminated, diglycine branched peptide did not impact the unique fragmentation pattern of its sulfonation derivative,10 and applied this method to present investigation, where it was anticipated that the elevated ionization will compensate the losses of sample and/ or the addition of impurities introduced by an additional chemical reaction. The guanidination reaction generally modifies the amino groups of lysine residues rather than the N-terminal amine of a peptide, except in the case when the N-terminal amino acid is glycine, presumably due to the less hindered effect of this simple residue.12 In most published reports, the guanidination reaction was performed on the mixture of peptides after proteolytic digestion. However, it is not practical in the present investigation because of the formation of a diglycine branch. To avoid unwanted blockage of the N-terminus of the Gly-Gly branch on a target peptide by the guanidino group, we carried out the guanidination reaction on intact ubiquitinated CHIP conjugates prior to

trypsin digestion. In this way, the diglycine branches can be utilized for subsequent derivatization by the sulfonation reagent. In addition, a target peptide(s) containing both modification sites and an N-terminal glycine residue should be detected if it exists. Moreover, modification on lysine residues within proteins might affect their stability and/or conformation16 and thus increase the efficiency of subsequent trypsin digestion. On the basis of the above considerations, a procedure for the analysis of ubiquitin-CHIP conjugates was developed as illustrated in Scheme 2. The ubiquitin-CHIP conjugates were purified from the reaction mixture by metal affinity cobalt beads through the polyhistidine tags attached to the Nterminus of the CHIP protein. The guanidination reaction was performed on the intact proteins retained on solid beads so that excess reagents could be removed easily after the reaction and less peptide loss could be anticipated. Peptide mixtures produced from trypsin digestion of guanidinated proteins were Journal of Proteome Research • Vol. 4, No. 5, 2005 1557

research articles Scheme 2

separated by reverse-phase HPLC chromatography. This was followed by N-terminal sulfonation using SPITC on the fractions that contain tryptic peptides confirmed by MS measurement. Tryptic peptides were not derivatized before chromatographic separation to avoid the coelution of unreacted sulfonation reagent in the peptide-containing fractions, a phenomenon observed before (data not shown). Following separation, the peptide derivatives were subjected to mass spectrometric analysis. MALDI-MS/MS screening was first conducted on all detected individual peptides. Although we have shown that a single profile (10 scans) was sufficient to generate a clear signature fragmentation pattern for a synthetic peptide (Figure 2a), we set 5 profiles as the acquisition parameter to avoid a false negative result. For the peptide(s) that displayed the doubly tagged signatures the acquisition parameter was generally set at 100 to 1000 profiles or until the best quality spectrum was obtained. Among hundreds of tryptic peptide derivatives, three were identified as doubly tagged, diglycine branch-containing, peptides upon MS/MS screening. The sequences of these peptides were then obtained using signal averaged MS/MS analysis. Figure 3a shows the tandem mass spectrum of one of such peptide whose protonated molecular ion was observed at m/z 1891.8. Consistent with the results obtained from synthetic

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peptides previously described,10 a group of high-mass fragments (P1, P2, P1p, P2p, P1′, P2′, P1′′, and y11) and a series of y type ions were observed. The former corresponded to losses of one or two tags and the tag plus N-terminal residues, and not only provide a unique signature for diglycine branched peptides but also provided sequence information for the identification of the N-terminal residues. In this case, the mass difference (m/z 113.2) between the peak P1 (m/z 1676.4) and the peak P1′′ (m/z 1563.2) implies that the N-terminal residue of the peptide could be either Leu or Ile, while the N-terminal residue, glycine, of the diglycine branch was revealed by the mass difference (m/z 57.1) of the peaks of P1 (m/z 1676.4) and P1′ (m/z 1619.3). On the basis of the series of y-ions (y1 through y11) and the protein sequence of ubiquitin, the amino acid sequence of this peptide was identified as modified Ub43-54, LIFAGK(GG-)QLEDGR. The ubiquitin modified residue K48 was easily located directly from the mass difference between peaks y6 and y7. These data reveals that ubiquitin was modified by another ubiquitin molecule, indicating the formation of polyubiquitin chain through ubiquitin’s K48 residue, which could not be concluded by conventional Western Blotting (Figure 1). Figure 4 shows the MS/MS spectrum of the second peptide identified by the sulfonation method. Surprisingly, this modified peptide, Ub55-72: TLSDYNIQK(GG-)ESTLHLVLR, is digested from a different region of ubiquitin where the modification site is the K63 residue. It has been known from biochemistry and/ or biology investigations that polyubiquitination through K48 and K63 usually lead to different cellular outcomes.1 Our results here imply either the coexistence of two types of polyubiquitin chains, Poly-K48 and Poly-K63, or the formation of heteropolymers including both modification sites in the same chain. Whether these data are artifacts arising from the in vitro conditions used, or whether they have biological significance remains for further investigation. The third peptide did not generate a high quality MS/MS spectrum (Figure 5a) even though it was acquired with 1000 profiles, presumably due to the low amount of the peptide on the sample spot. While the signature region of the spectrum is very clear, the sequence region is not as good as that on the other spectra and it is not easy to determine the y ions

Figure 3. MS/MS spectrum of the sulfonation derivative of a diglycine branched peptide, Ub43-54: LIFAGK(GG-)QLEDGR, digested from ubiquitin conjugated CHIP 1558

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Figure 4. MS/MS spectrum of the sulfonation derivative of a diglycine branched peptide, Ub55-72: TLSDYNIQK(GG-)ESTLHLVLR, digested from ubiquitin conjugated CHIP.

Figure 5. (a) MS/MS spectra of the sulfonation derivatives of a diglycine branched peptide, CHIP13-35: LGAGGGSPEK(GG-)SPSAQELK(Gu-)EQGNR. Gu- represents guanidinated lysine residue, or homoarginine residue. (b) MASCOT search result based on the masses of the fragment ions produced from the peptide derivative.

manually. In this situation, the masses of the fragments lower than m/z 2350 Da were input into MASCOT17 algorithm to

perform database searching against NCBInr human protein database. To increase searching specificity, all ions input were Journal of Proteome Research • Vol. 4, No. 5, 2005 1559

research articles defined as y ions. As shown in Figure 5b, MASCOT searching resulted in a high score (56) for the peptide, CHIP13-35: LGAGGGSPEK(GG-)SPSAQELK(guanidinated)EQGNR, derived from CHIP protein. As expected, most of the product ions within sequencing region are y ions while the majority of remaining ions are z ions, presumably resulting from high laser energy and long acquisition.

Conclusions Using our recently developed strategy, we analyzed ubiquitin-CHIP conjugates by MALDI MS/MS analysis of tryptic peptide derivatives following N-terminal sulfonation. Three peptides were identified to contain a diglycine branch through MS/MS screening on signature ions. Further analysis allowed us to identify one ubiquitination site as the K23 residue of CHIP protein and two residues of ubiqutin, K48 and K63, involved in the formation of polyubiquitination chains. While this approach has been applied to a relatively simple system with a single ubiquitination site, we anticipate that it can support the investigation of more complex ubiquitinated proteins containing several sites and ubiquitin heterogeneity.

Acknowledgment. This work was supported by a contract BAAHL-02-04 from the National Heart Lung and Blood Institute (Jennifer Van Eyk, PI) and a grant (No. U54RR020839) from the National Institutes of Health (Jef Boeke, PI). Mass spectral analyses were carried out at the Middle Atlantic Mass Spectrometry Laboratory. References (1) Pickart, C. M. Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 2001, 70, 503-533. (2) Weissman, A. M. Themes and variations on ubiquitylation. Nat. Rev. Mol. Cell Biol. 2001, 2, 169-178. (3) Aravind, L.; Koonin, E. V. The U box is a modified RING Fingers A common domain in ubiquitination. Curr. Biol. 2000, 10, R132134.

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Wang et al. (4) Hatakeyama, S.; Yada, M.; Matsumoto, M.; Ishida, N.; Nakayama, K. I. U box proteins as a new family of ubiquitin-protein ligases. J. Biol. Chem. 2001, 276, 33111-33120. (5) Ballinger, C. A. et al. Identification of CHIP, a novel tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions. Mol. Cell Biol. 1999, 19, 4535-4545. (6) Murata, S.; Chiba, T.; Tanaka, K. CHIP: a quality-control E3 ligase collaborating with molecular chaperones. Int. J. Biochem. Cell Biol. 2003, 35, 572-578. (7) Li, L. et al. CHIP mediates degradation of Smad proteins and potentially regulates Smad-induced transcription. Mol. Cell Biol. 2004, 24, 856-864. (8) Peng, J. et al. A proteomics approach to understanding protein ubiquitination. Nat. Biotechnol. 2003, 21, 921-926. (9) Cooper, H. J. et al. Identification of sites of ubiquitination in proteins: A Fourier transform ion cyclotron resonance mass spectrometry approach. Anal. Chem. 2004, 76, 6982-6988. (10) Wang, D.; Cotter, R. J. A Novel Approach for the Determination and Identification of Protein Ubiquitination by MALDI-TOF Mass Spectrometry. Anal. Chem. 2005, 77, 1458-1466. (11) Jiang, J. et al. CHIP is a U-box-dependent E3 ubiquitin ligase: identification of Hsc70 as a target for ubiquitylation. J. Biol. Chem. 2001, 276, 42938-42944. (12) Beardsley, R. L.; Reilly, J. P. Optimization of guanidination procedures for MALDI mass mapping. Anal. Chem. 2002, 74, 1884-1890. (13) Mann, M.; Jensen, O. N. Proteomic analysis of post-translational modifications. Nat. Biotechnol. 2003, 21, 255-261. (14) Keough, T.; Lacey, M. P.; Youngquist, R. S. Derivatization procedures to facilitate de novo sequencing of lysine-terminated tryptic peptides using postsource decay matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun. Mass Spectrom. 2000, 14, 2348-2356. (15) Brancia, F. L.; Oliver, S. G.; Gaskell, S. J. Improved matrix-assisted laser desorption/ionization mass spectrometric analysis of tryptic hydrolysates of proteins following guanidination of lysinecontaining peptides. Rapid Commun. Mass Spectrom. 2000, 14, 2070-2073. (16) Cupo, P.; El-Deiry, W. S.; Whitney, P. L.; Awad, W. M., Jr. Stability of acetylated and superguanidinated chymotrypsinogens. Arch. Biochem. Biophys. 1982, 216, 600-604. (17) Website of MASCOT algorithm is http://www.matrixscience.com.

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