supplementary material mass spectrometry based identification … · 2013. 5. 10. · supplementary...

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.


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.


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

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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

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Growth curve of HeLaS3 in normal ( ) and Met-0 hmSILAC ( )

Time (hours)

Cum

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Num

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Bremang M. et al., 2013 Figure S1

Incorporation Rate


- 10 4020 10 4020170

72

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AdOx (µM) -

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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


A

B

6 7 8 9 10 11

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465.4 466.4 467.6 468.8m/z

0

50

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H3F3AK(me1)SAPATGGVK (27-36)

L[465.2743]2+

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B

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Rel

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472.5 473.5 474.5 475.5 476.5 477.5m/z

0

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H3F3AK(me3)SAPATGGVK (27-36)

L[479.2900]2+

H[485.3232]2+

L

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m/z480.0 481.0 482.0 483.0 484.0 485.0 486.0

0

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6.033Th


C


675.6 676.4 677.2 678.0 678.8m/z

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H3F3AEIAQDFK(me1)TDLR (73-83)

L[675.3566]2+

H[677.3677]2+

2.011Th

D

Kme1

Kme1


L[682.3649]2+

H[686.3865]2+

L

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Kme2

Kme2

683.0 684.0 685.0 686.0 687.0m/z

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H3F3AEIAQDFK(me2)TDLR (73-83)

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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

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