(a) Structure showing the human PKM2 monomer and tetramer (PDB: 3SRH).
Cysteine peptides forming persulfides are highlighted (yellow spheres; A, B,
C, D). Black: intersubunit contact site; letters in square brackets:
sequence of misscleaved peptide. (b) Purified PKM2 from rabbit muscle
was incubated with increasing concentrations of Na2S4,
Na2S, NaSH and GYY4137. PKM2 activity
(μmol/min/mg) was measured in a coupled enzyme assay with LDH as
second enzyme monitoring the consumption of NADH at 340 nm.
(c) The activity of PKM2 was followed as decline in absorption at
340 nm. After 1 min 5 mM pyruvate was
added to bypass the reaction catalysed by PKM2. (d) The experiment
was carried out as described in (b) except that 1 mM DTT
was added in parallel to treatment with 200 μM
Na2S4. Data are
means +/− SD,
**p < 0.01 Na2S4 vs
Na2S4 + DTT,
***p < 0.001 Ctrl as well as DTT vs
Na2S4. (e) Workflow to confirm that
persulfides are formed at PKM2. PKM2 was incubated with
100 μM and 500 μM
Na2S4 or kept untreated. Induced persulfides were
modified with iodoTMT similar to qPerS-SID. After digestion with trypsin the
persulfide peptides were enriched using an anti-iodoTMT resin and subjected
to TCEP elution followed by IAM blocking as described for the proteomic
approach. In parallel, direct labelled persulfides (S-iodoTMT) were eluted
using iodoTMT elution buffer. The eluted peptides were subjected to LC-MS/MS
measurement and the peptides were identified using PEAKS 7.0. (f)
Spectra counts of iodoTMT labelled cysteine peptides (iodoTMT), persulfide
peptides identified according to the qPerS-SID protocol (TCEP elution, IAM)
and iodoTMT labelled persulfide peptides (TMT elution, S-iodoTMT).