reSeArCH Letter
c)
10080Relative intensity (%)0406020200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000
m/zb 2y 3y 4
b 5y 6 y 7y 8
y 9b 10 -NH 3b 12 b 13b 14 -H 2 Ob 15b 16b 4y 10y 11y 12y 13y 14b 14b 15 -H 2 Ob 16 -H 2 O b
17b 20 -H 2 Ob
20b 22 -H 2 Ob
22 b 23 b^24 -H^2 O
y 25-QVGRHGEGTESEFSVYLPEDVALVPVK-
OCNH
HOOC COOHy 25 y 17 y 16 y 15 y 14 y 13 y 12 y 11 y 10 y 9 y 8 y 7 y 6 y 5 y 4 y 3 y 2b 2 b 4 b 5 b 6 b 10 b 11 b 12 b 13 b 14 b 16 b 17 b 18 b 20 b 21 b 22 b 2310080Relative intensity (%)0406020400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000
m/zy 2y 3y 4 b 5y 6 y^7
y 8
y 9b 10b 12b 12 -H 2 Ob 15b 16b 4y 10y 11y 12y 13 y^14b 14b 15 -H 2 Ob^16 -H^2 O
b 17b 20 -H 2 O
b 20b 22 -H 2 O
b 21b 23b 24 -H 2 Oy 25-QVGRHGEGTESEFSVYLPEDVALVPVK-
Di-Gluy 25 y 17 y 16 y 15 y 14 y 13 y 12 y 11 y 10 y 9 y 8 y 7 y 6 y 5 y 4 y 3 y 2b 2 b 4 b 5 b 6 b 10 b 11 b 12 b 13 b 14 b 16 b 17 b 18 b 20 b 21 b 22 b 23b 9b 13b 14 -H 2 Ob 22 b 24b 23 -H 2 Ob 7 b 8 b 19 b 24-4 -2 02412345
SidJ
Calmodulin
SdeA QVGRHGEGTESEFSVYLPEDVALVPVKGlu...TESEFSV....GluDi-Glu
QVGRHGEGTESEFS-
VYLPEDVALVPVK * **low abundance missed
cleavage fragmentslog2 fold-change +/- ATP-log10 p-valuea)
b)
Extended Data Fig. 4 | In vitro glutamylation of SdeA. a, Samples of
in vitro glutamylation reactions that contained SdeA and SidJ, with or
without ATP, were TMT-labelled and analysed by quantitative mass
spectrometry. Mono- and di-glutamylation of the catalytic E860 of SdeA
was enriched in samples that contained ATP. In vitro glutamylation was
performed in n = 3 biologically independent experiments. Samples
were labelled with TMT six-plex reagent and analysed in one liquid
chromatography–mass spectrometry run. Significant differences between
samples were detected by a two-sided Student’s t-test. b, Annotated mass
spectra for mono-glutamylation of E860 of SdeA, from SidJ glutamylase
in vitro reactions. c, Annotated mass spectra for di-glutamylation of
E860 of SdeA, from SidJ glutamylase in vitro reactions.