supplementary material mass spectrometry based identification … · 2013. 5. 10. · supplementary...
Post on 29-Jan-2021
1 Views
Preview:
TRANSCRIPT
-
Supplementary Material
Mass Spectrometry–based Identification and Characterisation of Lysine and
Arginine Methylation in the Human Proteome
M. Bremanga,†, A. Cuomoa,†, A. M. Agrestaa,‡, M. Stugiewicza, V. Spadottoa and T.
Bonaldia,*
Supplementary Experimental
Estimation of incorporation efficiency
Incorporation of L-[13CD3]-Methionine (Met-4, heavy) was tested for HeLaS3 cells
cultivated in “heavy” medium after 9 replication cycles. Whole cell extracts from
HeLaS3 heavy-labeled were subjected to GeLCMS analysis (see Experimental
Section in the main text). Raw data from LTQ-Orbitrap Velos were than processed by
MaxQuant software 1.3.0.5, as described in the Experimental section, disabling the
re-quantify option1. Incorporation percentage was calculated by applying to non-
redundant peptide ratios, automatically extracted by a customized R script, using the
following formula: Incorporation rate (%) = ratio (H/L)/ ratio (H/L) +1 ×100.
Western blot analysis
The two different whole-cell protein extracts (prepared in Urea or Ripa buffer) were
separated by SDS-PAGE electrophoresis, blotted on the PVDF membrane and
incubated with tested antibodies. The following antibodies were used according to
the manufacturer’s instructions: AR-Kpan (Acris APO 9328PU-N, dilution 1:1000), LS-
Kpan (LifeSpan Biosciences LS-C60093/28979, dilution 1:1000), AC-Kme,di (AbCam
Ab23366 dilution 1:500), IC-Kme,di/tr (Immunechem ICP0501/ICP0601, dilution
1:2000), AC-Ktr (AbCam Ab76118, dilution 1:1000), CS-Rme-I (Cell Signaling
Technology D5A12, dilution 1:1000), CS-Rme-II (Cell Signaling Technology Me-R4-
100, dilution 1:1000), EG-Rsym (E. Guccione (IMCB) Rabbit 2 (SYM), dilution 1:1000)
MP-Rsym (Merck Millipore SYM10, dilution 1:1000) EG-Rasym (E. Guccione (IMCB)
Rabbit 1 (ASYM), dilution 1:1000), MP-Rasym (Merck Millipore ASYM24, dilution
1:1000). Vinculin was used as the loading control as well as Ponceau-stained core
histones, blotted on the PVDF membrane.
Electronic Supplementary Material (ESI) for Molecular BioSystemsThis journal is © The Royal Society of Chemistry 2013
-
Supplementary Figure and Table Legends
Figure S1. Monitoring cell growth in hmSILAC media and incorporation
analysis. (A) Growth curves of HeLaS3 cells, cultured in normal (blue) or hmSILAC
media (red). No differences in doubling time observed between the two cell
populations. (B) Full-MS spectrum of the methionine-containing peptide
LQIVEMPLAHK shows a H/L ratio of 9:1, reflecting the measured rate of
incorporation (green: heavy; black: light). The extracted ion chromatogram (XIC) for
both Heavy and Light forms of the same peptide is also reported on the right panel
(green: heavy; black: light). (C) Unimodal distribution of H/L ratios for tryptic peptides
from extracts of HeLaS3 grown in Met-4 SILAC medium after 9 doublings. The mode
of the distribution is 0.97 and describes the vast majority of peptides, indicating an
incorporation rate of 97% for methionine-containing peptides.
Figure S2. Detection of methylated proteins by anti-methyl-K/R antibodies. Left
panels: Examples of anti-methyl antibody specificity testing: HeLaS3 cells treated
with increased amounts of the methyltransferase inhibitor AdOx, for 24 and 48 hours
were resolved on SDS-PAGE and probed with different pan-methyl antibodies. Right
panels: Western blots with the same antibodies after a wash-out (WO) experiment,
where cells were treated with 40 µM AdOx for 72h, then the inhibitor was washed out
and cells allowed to grow for another 72hrs, then harvested and the extract probed
with anti-methyl antibodies.
Figure S3. Comparison of anti-pan-methyl-K antibody performance and In-Gel
Intensity distribution. (A) Venn Diagram describing the contribution of each anti-an-
methyl-K antibody used to the definition of the K-methylome, with no single antibody
outperforming the others (B) Intensity density distribution comparing arginine-and
lysine methylated peptides to Met-0/Met-4 containing (un-methylated) peptides in the
In-Gel nucleosol experiment. Un-methylated peptides from proteins with an identified
methylation site have been excluded.
Figure S4. Detection of lysine-methylated sites by In-Gel and OFFGEL
approaches. (A) Comparison of number of methyl-K sites obtained by the two
separation approaches. (B) The amino acid composition of the subset of lysine-
methylated peptides uniquely identified in the two techniques, excluding the
methylated arginine.
Electronic Supplementary Material (ESI) for Molecular BioSystemsThis journal is © The Royal Society of Chemistry 2013
-
Figure S5. Histones methylated peptides pairs encoded by hmSILAC-specific
delta masses. A, B, C upper panels: Zoomed MS spectrum for light and heavy
methyl doublets corresponding to KSAPATGGVK (27-36) peptide from Histone H3, is
identified in mono-, di- and tri-methylated form, respectively. A, B, C lower panels:
HCD MSMS spectra for the light and heavy peptides are reported. In each case the
mass shifts due to the presence of the methyl group is confirmed in the characteristic
b- and y- ion series. D, E upper panels: Zoomed MS spectrum for light and heavy
methyl doublets corresponding to EIAQDFKTDLR (73-83) peptide from Histone H3
protein, identified in mono- and di-methylated form, respectively.. D, E lower panel: -
b and -y ion series are displaced by 4 Da and 8 Da, respectively.
Figure S6. Contribution of each experimental strategy to the Total Modified
Sites annotated
Table S1. List of methylated peptides and sites
The spreadsheet named “Key for Table” includes a description of each sheet (“All
Sites Non Redundant”, “All Histones”, “All Non Histones”, “Ambiguous Sites”) as well
as an explanation of the included columns and abbreviations.
Supplementary References
1. J. Cox, I. Matic, M. Hilger, N. Nagaraj, M. Selbach, J. V. Olsen and M. Mann,
Nat Protoc, 2009, 4, 698-705.
Electronic Supplementary Material (ESI) for Molecular BioSystemsThis journal is © The Royal Society of Chemistry 2013
-
53.5
A
427 429m/z
Rel
ativ
e A
bund
ance
53.7Time (min)
0
50
100
L
H
C
B
0.0 0.2 0.4 0.6 0.8 1.0
05
1015
20 Incorporation Rate Estimation
N: 151
Den
sity
Methionine-containingPeptides: 0.97
“Only Heavy” 47kDa Heat Shock ProteinLQIVEMPLAHK [428.7892]2+
0
50
100
0
1
2
3
4
5
6
0 5 0 1 0 0 1 5 0
Growth curve of HeLaS3 in normal ( ) and Met-0 hmSILAC ( )
Time (hours)
Cum
ulat
ive
Num
ber
HeL
aS3
Cel
ls (x
106 )
XIC
Bremang M. et al., 2013 Figure S1
Incorporation Rate
Electronic Supplementary Material (ESI) for Molecular BioSystemsThis journal is © The Royal Society of Chemistry 2013
-
- 10 4020 10 4020170
72
433426
17
AdOx (µM) -
Vinc
170
72
433426
17
Vinc
170
72
433426
17
Vinc
170
72
433426
17
Vinc
- 40 WO
24h 48h
LS-Kpan
AC-Kme,di
AR-Kpan
IC-Kme,di/tri
Bremang M. et al., 2013 Figure S2Electronic Supplementary Material (ESI) for Molecular BioSystemsThis journal is © The Royal Society of Chemistry 2013
-
Bremang M. et al., 2013 Figure S3
A
B
6 7 8 9 10 11
0.0
0.2
0.4
0.6
0.8
Den
sity
log10(Intensity)
Met-0/Met-4 Methyl-R Methyl-K
p < 10-3
NucleosolIn-Gel
AR-Kpan
LS-Kpan
AC-Kme,di
IC-Kme,di/trAC-Ktr
6
3
3
1010
42
1
1
1
2
1
1
4
22
Electronic Supplementary Material (ESI) for Molecular BioSystemsThis journal is © The Royal Society of Chemistry 2013
-
A
B
Bremang M. et al., 2013 Figure S4
In-GelOFFGEL
3
K-Sites
15 31
Lysine-methylated Peptides
Amino Acid Frequency0 10.5
In-Gel
OFFGELP VNFDCYSEM LTA Q K R I G
Electronic Supplementary Material (ESI) for Molecular BioSystemsThis journal is © The Royal Society of Chemistry 2013
-
465.4 466.4 467.6 468.8m/z
0
50
100R
elat
ive
Abu
ndan
ceA
H3F3AK(me1)SAPATGGVK (27-36)
L[465.2743]2+
H[467.2854]2+
L
H
Kme1
Kme1
2.011Th
Bremang M. et al., 2013 Figure S5Electronic Supplementary Material (ESI) for Molecular BioSystemsThis journal is © The Royal Society of Chemistry 2013
-
B
H3F3AK(me2)SAPATGGVK (27-36)L
[472.2821]2+H
[476.3043]2+
L
H
Kme2
Kme2
Rel
ativ
e A
bund
ance
472.5 473.5 474.5 475.5 476.5 477.5m/z
0
50
100
4.022Th
Bremang M. et al., 2013 Figure S5Electronic Supplementary Material (ESI) for Molecular BioSystemsThis journal is © The Royal Society of Chemistry 2013
-
H3F3AK(me3)SAPATGGVK (27-36)
L[479.2900]2+
H[485.3232]2+
L
H
Kme3
Kme3
m/z480.0 481.0 482.0 483.0 484.0 485.0 486.0
0
50
100R
elat
ive
Abu
ndan
ce
6.033Th
Bremang M. et al., 2013 Figure S5
C
Electronic Supplementary Material (ESI) for Molecular BioSystemsThis journal is © The Royal Society of Chemistry 2013
-
675.6 676.4 677.2 678.0 678.8m/z
0
50
100
Rel
ativ
e A
bund
ance
H3F3AEIAQDFK(me1)TDLR (73-83)
L[675.3566]2+
H[677.3677]2+
2.011Th
D
Kme1
Kme1
Bremang M. et al., 2013 Figure S5Electronic Supplementary Material (ESI) for Molecular BioSystemsThis journal is © The Royal Society of Chemistry 2013
-
L[682.3649]2+
H[686.3865]2+
L
H
E
Kme2
Kme2
683.0 684.0 685.0 686.0 687.0m/z
0
50
100R
elat
ive
Abu
ndan
ce
H3F3AEIAQDFK(me2)TDLR (73-83)
4.022Th
Kme2
L
Bremang M. et al., 2013 Figure S5Electronic Supplementary Material (ESI) for Molecular BioSystemsThis journal is © The Royal Society of Chemistry 2013
-
Bremang M. et al., 2013 Figure S6
63
56100
73
918 4
IP In-Gel
OFFGEL
25 17
1
13
2
14
In-GelIP
OFFGEL
B
A Methyl-K Sites
Methyl-R Sites
Electronic Supplementary Material (ESI) for Molecular BioSystemsThis journal is © The Royal Society of Chemistry 2013
Bremang_et_al_MolBiosys_2013_Supplementary_ACCEPTED_MBedit_TBeditBremang_et_al_MolBiosys_2013_Figure_S1Bremang_et_al_MolBiosys_2013_Figure_S2Bremang_et_al_MolBiosys_2013_Figure_S3Bremang_et_al_MolBiosys_2013_Figure_S4Bremang_et_al_MolBiosys_2013_Figure_S5ABremang_et_al_MolBiosys_2013_Figure_S5BBremang_et_al_MolBiosys_2013_Figure_S5CBremang_et_al_MolBiosys_2013_Figure_S5DBremang_et_al_MolBiosys_2013_Figure_S5EBremang_et_al_MolBiosys_2013_Figure_S6
top related