Tryptic peptide mapping of ubiquitin and derivatives using reverse-phase high performance liquid chromatography

Cox, M J; Shapira, Raymond; Wilkinson, Keith D.

doi:10.1016/0003-2697(86)90535-x

Cited by 37 publications

(23 citation statements)

References 14 publications

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“…Most mutations are to Ala; Ala residues and one Ser are mutated to Gly (Figure 2B below). We subcloned the 54 mutant genes to a bacterial vector and expressed the mutant proteins in Escherichia coli , then purified them and treated with trypsin under native conditions to release Ub's C‐terminal GlyGly moiety (Cox et al , 1986). (To avoid anticipated inhibition of trypsin cleavage in Ub‐R74A, we constructed a synthetic Ub 74 gene for this mutant.)…”

Section: Resultsmentioning

confidence: 99%

Molecular determinants of polyubiquitin linkage selection by an HECT ubiquitin ligase

Wang

Cheng

Peng

et al. 2006

EMBO J

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Ubiquitin (Ub)‐protein ligases (E3s) frequently modify their substrates with multiple Ub molecules in the form of a polyubiquitin (poly‐Ub) chain. Although structurally distinct poly‐Ub chains (linked through different Ub lysine (Lys) residues) can confer different fates on target proteins, little is known about how E3s select the Lys residue to be used in chain synthesis. Here, we used a combination of mutagenesis, biochemistry, and mass spectrometry to map determinants of linkage choice in chain assembly catalyzed by KIAA10, an HECT (Homologous to E6AP C‐Terminus) domain E3 that synthesizes K29‐ and K48‐linked chains. Focusing on the Ub molecule that contributes the Lys residue for chain formation, we found that specific surface residues adjacent to K48 and K29 are critical for the usage of the respective Lys residues in chain synthesis. This direct mechanism of linkage choice bears similarities to the mechanism of substrate site selection in sumoylation catalyzed by Ubc9, but is distinct from the mechanism of chain linkage selection used by the Mms2/Ubc13 (Ub E2 variant (UEV)/E2) complex.

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Section: Resultsmentioning

confidence: 99%

Molecular determinants of polyubiquitin linkage selection by an HECT ubiquitin ligase

Wang

Cheng

Peng

et al. 2006

EMBO J

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“…Inspection of trace c further suggests that ethyleneimine did not react significantly with other potential nucleophilic side chains, such as Met 1 and His 68 ; modification of these side chains would probably be manifested as shifts in peaks 9 and 11, respectively (27). Mass spectrometric analysis of alkylated diubiquitin (bearing a proximal Asp 77 residue, see below) confirmed this inference; the observed mass of 17,232.7 Ϯ 12.4 Da compared favorably with the calculated mass of 17,245.8 Da.…”

Section: Resultsmentioning

confidence: 99%

“…Trace a in Fig. 3 shows the map of wild type ubiquitin; peptides 4 -11 are clearly resolved (27). The K48C mutation abolishes a tryptic cleavage site (top , Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Protein iodination with chloramine T was carried out as described (4). Tryptic digestion of ubiquitin derivatives (5 mg/ml total ubiquitin and 5% w/w trypsin) and reversed phase high performance liquid chromatography separation of peptides were carried out as described (27,28).…”

Section: Methodsmentioning

confidence: 99%

“…In some experiments aliquots were withdrawn to monitor the level of ubiquitinsubstrate conjugates by SDS-polyacrylamide gel electrophoresis and autoradiography (see "Experimental Procedures" and Fig. 4 (27). Lines and boldface numbers below the sequence indicate peptides visible in the peptide maps; lines above sequence indicate unresolved peptides and peptide 12 in K48C-ubiquitin (see "Results and Discussion").…”

Section: Table Imentioning

confidence: 99%

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Inhibition of the 26 S Proteasome by Polyubiquitin Chains Synthesized to Have Defined Lengths

Piotrowski¹,

Beal²,

Hoffman³

et al. 1997

Journal of Biological Chemistry

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210

211

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Ubiquitin is a covalent signal that targets cellular proteins to the 26 S proteasome. Multiple ubiquitins can be ligated together through the formation of isopeptide bonds between Lys 48 and Gly 76 of successive ubiquitins. Such a polyubiquitin chain constitutes a highly effective proteolytic targeting signal, but its mode of interaction with the proteasome is not well understood. Experiments to address this issue have been limited by difficulties in preparing useful quantities of polyubiquitin chains of uniform length. We report a simple method for large scale synthesis of Lys 48 -linked polyubiquitin chains of defined length. In the first round of synthesis, two ubiquitin derivatives (K48C-ubiquitin and Asp 77 -ubiquitin) were used as substrates for the well characterized ubiquitin-conjugating enzyme E2-25K. Diubiquitin blocked at the nascent proximal and distal chain termini was obtained in quantitative yield. Appropriately deblocked chains were then combined to synthesize higher order chains (tetramer and octamer in the present study). Deblocking was achieved either enzymatically (proximal terminus) or by chemical alkylation (distal terminus). Chains synthesized by this method were used to obtain the first quantitative information concerning the influence of polyubiquitin chain length on binding to the 26 S proteasome; this was done through comparison of different length (unanchored) polyubiquitin chains as inhibitors of ubiquitin-conjugate degradation. K 0.5 was found to decrease ϳ90-fold, from 430 to 4.8 M, as the chain was lengthened from two to eight ubiquitins. The implications of these results for the molecular basis of chain recognition are discussed.

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Femtomole Peptide Mapping by Derivatization, High-Performance Liquid Chromatography, and Fluorescence Detection

Liu

Krull

Cohen

2001

Analytical Biochemistry

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Tryptic peptide mapping of ubiquitin and derivatives using reverse-phase high performance liquid chromatography

Cited by 37 publications

References 14 publications

Molecular determinants of polyubiquitin linkage selection by an HECT ubiquitin ligase

Molecular determinants of polyubiquitin linkage selection by an HECT ubiquitin ligase

Inhibition of the 26 S Proteasome by Polyubiquitin Chains Synthesized to Have Defined Lengths

Femtomole Peptide Mapping by Derivatization, High-Performance Liquid Chromatography, and Fluorescence Detection

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