Top-Down 193-nm Ultraviolet Photodissociation Mass Spectrometry for Simultaneous Determination of Polyubiquitin Chain Length and Topology

Abstract

Protein ubiquitin modifications present a vexing analytical challenge, because of the dynamic changes in the site of modification on the substrate, the number of ubiquitin moieties attached, and the diversity of linkage patterns in which they are attached. Presented here is a method to confidently assign size and linkage type of polyubiquitin modifications. The method combines intact mass measurement to determine the number of ubiquitin moieties in the chain with backbone fragmentation by 193-nm ultraviolet photodissociation (UVPD) to determine the linkage pattern. UVPD fragmentation of proteins leads to reproducible backbone cleavage at almost every inter-residue position, and in polyubiquitin chains, the N-terminally derived fragments from each constituent monomer are identical, up to the site of conjugation. The N-terminal ubiquitin fragment ions are superimposed to create a diagnostic pattern that allows easy recognition of the dominant chain linkages. The method is demonstrated by achieving almost-complete fragmentation of monoubiquitin and then, subsequently, fragmentation of dimeric, tetrameric, and longer Lys48- and Lys63-linked ubiquitin chains. The utility of the method for the analysis of mixed linkage chains is confirmed for mixtures of Lys48 and Lys63 tetramers with known relative concentrations and for an in vitro-formulated ubiquitin chain attached to a substrate protein.

Ubiquitin chain diversity at a glance

“…Our knowledge regarding ubiquitin chain length, the exact topology of ubiquitin signals and ubiquitin dynamics in vivo is still very limited. But continuous advancement of methodology will help to answer these questions (Cannon et al, 2015). In recent years, each layer of the ubiquitin system has attracted attention as potential novel targets for molecular therapy.…”

Substrate Ubiquitination Controls the Unfolding Ability of the Proteasome

“…pET3a-ubiquitin, GST-E2-25K, GST-Ubc13, and GST-Mms2 were gifts from Andreas Matouschek (76). Human UbcH5 and UbcH7 were cloned into pGEX6p-1 (GE Healthcare) from a cDNA library.…”

“…Synthesis and Purification of Unanchored Polyubiquitin Chains-Unanchored K48 polyubiquitin chains were synthesized as described previously (76,79) by incubating 0.1 M mammalian E1, 25 M E2-25K, and 20 mg ml Ϫ1 of ubiquitin in K48 buffer (50 mM Tris-Cl, 5 mM MgCl 2 , 0.1 mM DTT, 0.6 units ml Ϫ1 of pyrophosphatase, 10 mM creatine phosphate, 0.002 mg ml Ϫ1 of creatine phosphokinase, and 2.5 mM ATP, pH 8.0) at 37°C overnight. K63 chains were synthesized as described previously (76,79) by incubating 0.1 M mammalian E1, 4 M Ubc13 (E2), 4 M Mms2, and 20 mg ml Ϫ1 of ubiquitin in K63 buffer (50 mM Tris-Cl, 5 mM MgCl 2 , 0.1 mM DTT, 0.6 unit ml Ϫ1 of pyrophosphatase, 10 mM creatine phosphate, 0.002 mg ml Ϫ1 of creatine phosphokinase, and 2.5 mM ATP, pH 7.6) at 37°C overnight.…”

“…K63 chains were synthesized as described previously (76,79) by incubating 0.1 M mammalian E1, 4 M Ubc13 (E2), 4 M Mms2, and 20 mg ml Ϫ1 of ubiquitin in K63 buffer (50 mM Tris-Cl, 5 mM MgCl 2 , 0.1 mM DTT, 0.6 unit ml Ϫ1 of pyrophosphatase, 10 mM creatine phosphate, 0.002 mg ml Ϫ1 of creatine phosphokinase, and 2.5 mM ATP, pH 7.6) at 37°C overnight. Both K48 and K63 synthesis reactions were treated with 5 mM DTT, 1 mM EDTA, and protease inhibitor for 20 min at room temperature and then purified as previously described (76,79). Briefly, the ubiquitination reaction was applied to a HiTrap-Q column (GE Healthcare) and the flow-through was collected.…”

Top-Down Analysis of Branched Proteins Using Mass Spectrometry

“…Top-down MS/MS spectra of homo polymers of ubiquitin, 12 isomeric ubiquitin trimers 13 and isomeric ubiquitin tetramers 14 have been reported recently, in which UVPD or CID-assisted ETD was used to activate the heavy ions. However, examples of top-down analysis of branched proteins, proteins modified by ubiquitin or Rub1/NEDD8 have not been reported.…”