1
Supporting Information
2
3
Protein substrates of the arginine methyltransferase Hmt1 identified by proteome arrays
4
5
Jason K.K. Low1,3, Hogune Im2, Melissa A. Erce1, Gene-Hart Smith1, Michael Snyder2, Marc R.
6
Wilkins1
7
8
1
9
South Wales, NSW 2052, Sydney, Australia
Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, The University of New
10
2
11
3
Department of Genetics, Stanford University School of Medicine, Palo Alto, California 94305, USA
Current address: School of Molecular Bioscience, The University of Sydney, NSW 2006, Australia
12
13
To whom correspondence should be addressed:
14
Marc R. Wilkins, School of Biotechnology and Biomolecular Sciences, The University of New South
15
Wales, NSW 2052, Sydney, Australia. Tel: (61) 2-93853633; Fax: (61) 2-93851483; E-mail:
16
marc.wilkins@unsw.edu.au
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18
Keywords:
19
Saccharomyces cerevisiae, proteome arrays
arginine
methylation,
Hmt1
methyltransferase,
post-translational
modification,
20
1
1
SUPPLEMENTARY METHODS
2
Immunoblots – For the immunodetection of arginine methylated proteins (e.g. Figure S1), the rabbit
3
anti-methylarginine (#ICP0801, Immunechem) antibody was used at a 1:1000 concentration (4 oC,
4
overnight) in the presence of 1% (w/v) BSA (#A3059, Sigma-Aldrich) and the secondary donkey anti-
5
rabbit horseradish peroxidise- (HRP) conjugated antibody (#sc-2313, Santa Cruz Biotechnology) was
6
used at a 1:5000 concentration in the presence of 0.8% (w/v) skim milk powder (room temperature,
7
1.5 h). To aid the visualisation of overexpressed proteins, immunoblots were probed with either the
8
mouse anti-penta-His-HRP conjugated antibody (#34460, Qiagen) (room temperature, 1.5 h) or the mouse
9
anti-HA antibody (#MMA-101P, Covance) (room temperature, 1.5 h) with the horse anti-mouse-HRP-
10
conjugated secondary antibody (#7076S, Cell Signalling Technology) (room temperature, 1 h).
11
Visualisation of the immunoblots was achieved using a LAS3000 imager (Fujifilm).
12
13
2
1
SUPPLEMENTARY TABLES
2
Table S1. Arginine-methylated proteins identified using proteome arrays
Gene
Name description
Biological processb
Cellular
locationc
name
ALO1
D-arabinono-1,4-lactone oxidase
Response to oxidative stress
C, Mt, M
BRR1
Pre-mRNA-splicing factor BRR1
mRNA processing, RNA splicing
N
CDC33
Eukaryotic translation initiation factor
RNA catabolic process, regulation
C, N
4E
of translation
CDC40
Pre-mRNA-processing factor 17
mRNA processing, RNA splicing
C, N
CTA1
Peroxisomal catalase A
Response to oxidative stress
C, Mt, P
DEG1
tRNA pseudouridine(38/39) synthase
tRNA processing
C, N
DHH1
ATP-dependent RNA helicase DHH1
RNA catabolic process
C
DOM34
Protein DOM34
RNA catabolic process
C
ELC1
Elongin-C
DNA repair, ubiquitin-dependent
C, N
catabolic process
EST1
Telomere elongation protein EST1
DNA replication, telomere
N, Nu
organisation
FUB1
Uncharacterised protein YCR076C
Unknown
Unknown
GCD10
tRNA (adenine(58)-N(1))-
tRNA processing
C, N
methyltransferase non-catalytic subunit
TRM6
3
GND1
6-phosphogluconate dehydrogenase,
Response to oxidative stress,
C, Mt
decarboxylating 1
carbohydrate metabolic process
GPM2
Phosphoglycerate mutase 2
Glycolysis
C, N
HSP60
Heat shock protein 60, mitochondriald
Protein folding, protein targeting
C, Mt
HTS1
Histidine--tRNA ligase, mitochondriald
tRNA aminoacylation for protein
C, Mt
translation, mitochondrial
translation, cellular amino acid
metabolic process
IMP4
U3 snoRNP protein IMP4
rRNA processing
N, Nu
LSM7
U6 snRNA-associated Sm-like protein
mRNA processing, rRNA
C, N, Nu
LSm7
processing, RNA splicing
MAK21
Ribosome biogenesis protein MAK21
Ribosome assembly
C, N, Nu
MAL32
Alpha-glucosidase MAL32
Carbohydrate metabolic process
M
MEU1
5'-methylthioadenosine phosphorylase
Cellular amino acid metabolic
C, N
process
MIP6
RNA-binding protein MIP6
Nuclear transport
C, N
MPP10
U3 snoRNA-associated protein MPP10
rRNA processing
N, Nu
MRD1
Multiple RNA-binding domain-
rRNA processing, ribosome
N, Nu
containing protein 1
assembly
54S ribosomal protein MRP49,
Mitochondrial translation
MRP49
C, Mt
mitochondriald
4
MRPL11
54S ribosomal protein L11,
Mitochondrial translation
C, Mt
Mitochondrial translation
C, Mt
Mitochondrial translation
C, Mt
54S ribosomal protein L9,
Translation (probably
C, Mt
mitochondriald
mitochondrial)
37S ribosomal protein S12,
Mitochondrial translation
C, Mt
Mitochondrial translation
C, Mt
mitochondriald
MRPL28
54S ribosomal protein L28,
mitochondriald
MRPL33
54S ribosomal protein L33,
mitochondriald
MRPL9
MRPS12
mitochondriald
MRPS18
37S ribosomal protein S18,
mitochondriald
MTR2
mRNA transport regulator MTR2
Nuclear transport
N, M
NAM2
Leucine--tRNA ligase, mitochondriald
tRNA aminoacylation for protein
C, Mt
translation, mitochondrial
translation, cellular amino acid
metabolic process
NCL1
Multisite-specific tRNA:(cytosine-
tRNA processing
N
RNA catabolic process
C
rRNA processing
C, N, Nu
C(5))-methyltransferase
NMD2
Nonsense-mediated mRNA decay
protein 2
NOC4
Nucleolar complex protein 4
5
NOP7
Nucleolar protein 7
rRNA processing
N, Nu
NPL3a
Nucleolar protein 3
mRNA processing, RNA splicing,
C, N
nuclear transport, regulation of
translation
NUG1
Nuclear GTP-binding protein NUG1
rRNA processing, nuclear transport
N, Nu
PAN3
PAB-dependent poly(A)-specific
mRNA processing, DNA repair
C
Carbohydrate metabolic process
C
ribonuclease subunit PAN3
PCK1
Phosphoenolpyruvate carboxykinase
[ATP]
PES4
DNA polymerase epsilon suppressor 4
Unknown
Unknown
POP6
Ribonucleases P/MRP protein subunit
mRNA processing, rRNA
N, Nu
POP6
processing, tRNA processing
Ribonucleases P/MRP protein subunit
mRNA processing, rRNA
POP8
processing, tRNA processing
Pre-mRNA-splicing factor ATP-
mRNA processing, rRNA
C, N, Nu,
dependent RNA helicase PRP43
processing, RNA splicing
Mt
Pre-mRNA-processing ATP-dependent
mRNA processing, RNA splicing
N
Carbohydrate metabolic process
C, N
rRNA processing, RNA catabolic
N
POP8
PRP43
PRP5
N, Nu
RNA helicase PRP5
QRI1
UDP-N-acetylglucosamine
pyrophosphorylase
RAI1
RAT1-interacting protein
process
6
RIO2
Serine/threonine-protein kinase RIO2
rRNA processing
C, N
RIT1
tRNA A64-2'-O-ribosylphosphate
tRNA processing
C
transferase
RMD1
Sporulation protein RMD1
Meiosis, sporulation
C
RNP1
Ribonucleoprotein 1
ribosome biogenesis
C
RNR4
Ribonucleoside-diphosphate reductase
DNA replication
C, N
Transcription
N, Nu
small chain 2
RPA43
DNA-directed RNA polymerase I
subunit RPA43
RPL11A
60 S ribosomal protein L11-A
Ribosome assembly
C
RRP12
Ribosomal RNA-processing protein 12
rRNA processing
C, N
RRP43
Exosome complex component RRP43
rRNA processing, RNA catabolic
C, N, Nu
process
RRP6
Exosome complex exonuclease RRP6
rRNA processing, RNA catabolic
N, Nu
process
RUB1
NEDD8-like protein RUB1
Protein neddylation
C
SAD1
Pre-mRNA-splicing factor SAD1
mRNA processing, RNA splicing
C, N
SEC65
Signal recognition particle subunit
Protein targeting
C, ER
tRNA processing, RNA splicing
C, N, Mt,
SEC65
SEN34
tRNA-splicing endonuclease subunit
SEN34
M
7
SGN1
RNA-binding protein SGN1
mRNA metabolic process
C
SHE4
SWI5-dependent HO expression protein
Cytoskeleton organisation, mRNA
C
4
localisation
High osmolarity signalling protein
Response to osmotic stress,
SHO1
signalling
Suppressor of kinetochore protein 1
Cytoskeleton organisation, protein
SHO1
SKP1
C, M
C, N
neddylation
SNU114
114 kDa U5 small nuclear
mRNA processing, RNA splicing
N
13 kDa ribonucleoprotein-associated
mRNA processing, rRNA
N, Nu
protein
processing, RNA splicing
SOF1
U3 snoRNA-associated protein SOF1
rRNA processing
N, Nu
SPB4
ATP-dependent rRNA helicase SPB4
rRNA processing, ribosome
N, Nu
ribonucleoprotein component
SNU13
assembly
SPP381
Pre-mRNA-splicing factor SPP381
mRNA processing, RNA splicing
N
SRP21
Signal recognition particle subunit
Protein targeting
C, N, ER
SRP21
STI1
Heat shock protein STI1
Protein folding
C
TSA1
Peroxiredoxin TSA1
Protein folding, response to
C, PC
oxidative stress, regulation of
translation
8
TVP15
Golgi apparatus membrane protein
Unknown
C, M, G
TVP15
UTP21
U3 snoRNA-associated protein 21
rRNA processing
N, Nu
UTP4
U3 small nucleolar RNA-associated
rRNA processing, transcription
N, Nu
protein 4
UTP8
U3 snoRNA-associated protein 8
rRNA processing, nuclear transport, N, Nu
transcription
VAS1
Valine--tRNA ligase, mitochondriald
tRNA aminoacylation for protein
C, Mt
translation, cellular amino acid
metabolic process
YDR341C
Arginine--tRNA ligase, cytoplasmic
tRNA aminoacylation for protein
C, Mt
translation, cellular amino acid
metabolic process
YER187W
Uncharacterised protein YER187W
Unknown
Unknown
YGR250C
Uncharacterised RNA-binding protein
Unknown
C
Uncharacterised trans-sulfuration
Cellular amino acid metabolic
C, N
enzyme YHR112C
process
YJL218W
Putative acetyltransferase YJL218W
Unknown
Unknown
YKE2
Prefoldin subunit 6
Protein folding
C
YKU70
ATP-dependent DNA helicase II subunit
DNA repair, telomere organisation
C, N
YGR250C
YHR112C
1
9
YKU80
ATP-dependent DNA helicase II subunit
DNA repair, telomere organisation
N
Exocytosis, Golgi vesicle transport
C, Mt, M,
2
YPT31
GTP-binding protein YPT31/YPT8
G
1
a
Known substrates. bWhere available, biological process data were sourced from the Gene Ontology
2
Consortium [1] through the Saccharomyces Genome Database [2] and from the Uniprot Consortium [3].
3
Biological process GO data have been summarised for this table. cWhere available, localisation data
4
were sourced from the Gene Ontology Consortium [1] and from Huh et al. (2003) [4] through the
5
Saccharomyces Genome Database [2]. Legend: C – Cytoplasm, N – Nucleus, Nu – Nucleolus, M –
6
Membrane, Mt – Mitochondrion, V – Vacuole, G – Golgi apparatus, P – Peroxisome, PC – Punctate
7
composite, ER – Endoplasmic reticulum. dThese proteins are encoded in the nuclear genomic DNA.
8
While they have mitochondrial functions, some have cytoplasmic functions as well.
9
10
1
Table S2. Functional enrichment analysis reveals statistically significant GO terms for
2
methylarginine containing proteins
Category/Term
Count
P-value
ribonucleoprotein complex biogenesis
32
1.9E-12
RNA processing
36
4.4E-12
rRNA metabolic process
23
4.5E-10
ribosome biogenesis
26
2.4E-09
mRNA metabolic process
21
2.6E-07
tRNA metabolic process
14
3.7E-05
RNA splicing
12
1.6E-04
translation
21
3.2E-02
RNA binding
32
1.5E-10
RNA splicing factor activity, transesterification mechanism
9
2.9E-04
ribonuclease activity
7
2.7E-03
telomeric DNA binding
4
5.6E-03
ATP-dependent helicase activity
6
1.6E-02
tRNA-specific ribonuclease activity
3
4.7E-02
tRNA binding
3
4.7E-02
Biological processa
Molecular functionb
11
Cellular componentc
1
a
2
b
3
c
ribonucleoprotein complex
40
2.4E-14
nucleolus
24
8.5E-12
90S preribosome
12
1.5E-07
nuclear lumen
26
2.5E-07
spliceosome
8
1.1E-03
heterochromatin
3
2.2E-02
mitochondrial ribosome
6
2.5E-02
mitochondrial lumen
9
4.0E-02
22 out of the input of 88 genes were not in the output.
39 out of the input of 88 genes were not in the output.
36 out of the input of 88 genes were not in the output.
4
12
1
Table S3. Putative Hmt1 substrates identified using the antibody-based fluorescently-labelled
2
proteome arrays
Gene
Name description
Biological processb
Cellular
locationc
name
New Hmt1 substrates found
ARA2
D-arabinose 1-dehydrogenase
Unknown (Note: possibly response to
Unknown
oxidative stress)
DIA4
Serine--tRNA ligase,
tRNA aminoacylation for protein
mitochondriald
translation, mitochondrial translation,
C, Mt, PC
cellular amino acid metabolic process
MER1
Meiotic recombination 1 protein
mRNA processing, RNA splicing,
N
DNA recombination
MTR3
Exosome complex component
rRNA processing, RNA catabolic
C, N, Nu
MTR3
process
NAM8
Protein NAM8
mRNA processing, RNA splicing
C, N
NOP56
Nucleolar protein 56
rRNA processing
N, Nu
STM1a
Suppressor protein STM1
Regulation of translation, signalling
C
WHI3
Protein WHI3
Pseudohyphal growth, invasive growth
C
in response to glucose limitation
YNL284C-A
Transposon Ty1-NL1 Gag
Transposition (Note: RNA binding
polyprotein
function)
N
Substrates also seen in Table S1 (non-Hmt1-treated arrays)
13
BRR1
Pre-mRNA-splicing factor BRR1
mRNA processing, RNA splicing
N
DEG1
tRNA pseudouridine (38/39)
tRNA processing
C, N
synthase
DOM34
Protein DOM34
RNA catabolic process
C
GPM2
Phosphoglycerate mutase 2
Glycolysis
C, N
HTS1
Histidine--tRNA ligase,
tRNA aminoacylation for protein
C, Mt
mitochondriald
translation, mitochondrial translation,
cellular amino acid metabolic process
MAL32
Alpha-glucosidase MAL32
Carbohydrate metabolic process
M
MEU1
5'-methylthioadenosine
Cellular amino acid metabolic process
C, N
rRNA processing
N, Nu
rRNA processing
N, Nu
Mitochondrial translation
C, Mt
Mitochondrial translation
C, Mt
Mitochondrial translation
C, Mt
rRNA processing
C, N, Nu
phosphorylase
MPP10
U3 snoRNA-associated protein
MPP10
MRD1
Multiple RNA-binding domaincontaining protein 1
MRPL11
54S ribosomal protein L11,
mitochondriald
MRPL33
54S ribosomal protein L33,
mitochondriald
MRPS12
37S ribosomal protein S12,
mitochondriald
NOC4
Nucleolar complex protein 4
14
NOP7
Nucleolar protein 7
rRNA processing
N, Nu
NPL3a
Nucleolar protein 3
mRNA processing, RNA splicing,
C, N
nuclear transport, regulation of
translation
NUG1
Nuclear GTP-binding protein NUG1
rRNA processing, nuclear transport
N, Nu
POP8
Ribonucleases P/MRP protein
rRNA processing, mRNA processing,
N, Nu
subunit POP8
tRNA processing
UDP-N-acetylglucosamine
Carbohydrate metabolic process
C, N
QRI1
pyrophosphorylase
RMD1
Sporulation protein RMD1
Meiosis, sporulation
C
RNP1
Ribonucleoprotein 1
Ribosome biogenesis
C
RNR4
Ribonucleoside-diphosphate
DNA replication
C, N
Pre-mRNA-splicing factor ATP-
mRNA processing, rRNA processing,
C, N, Nu,
dependent RNA helicase PRP43
RNA splicing
Mt
Exosome complex component
rRNA processing, RNA catabolic
C, N, Nu
RRP43
process
RUB1
NEDD8-like protein RUB1
Protein neddylation
C
SAD1
Pre-mRNA-splicing factor SAD1
mRNA processing, RNA splicing
C, N
SEN34
tRNA-splicing endonuclease subunit
tRNA processing, RNA splicing
C, N, Mt,
reductase small chain 2
PRP43
RRP43
SEN34
M
15
SHO1
High osmolarity signalling protein
Response to osmotic stress, signalling
C, M
13 kDa ribonucleoprotein-associated
rRNA processing, mRNA processing,
N, Nu
protein
RNA splicing
SPP381
Pre-mRNA-splicing factor SPP381
mRNA processing, RNA splicing
N
TSA1
Peroxiredoxin TSA1
Regulation of translation, response to
C, PC
SHO1
SNU13
oxidative stress, protein folding
UTP4
U3 small nucleolar RNA-associated
rRNA processing, transcription
N, Nu
Unknown
C
Cellular amino acid metabolic process
C, N
protein 4
YGR250C
Uncharacterised RNA-binding
protein YGR250C
YHR112C
Uncharacterised trans-sulfuration
enzyme YHR112C
1
a
Known substrates. bWhere available, biological process data were sourced from the Gene Ontology
2
Consortium [1] through the Saccharomyces Genome Database [2] and from the Uniprot Consortium [3].
3
Biological process GO data have been summarised for this table. cWhere available, localisation data
4
were sourced from the Gene Ontology Consortium [1] and from Huh et al. (2003) [4] through the
5
Saccharomyces Genome Database [2]. Legend: C – Cytoplasm, N – Nucleus, Nu – Nucleolus, M –
6
Membrane, Mt – Mitochondrion, PC – Punctate composite. dThese proteins are encoded in the nuclear
7
genomic DNA. While they have mitochondrial functions, some have cytoplasmic functions as well.
8
16
1
SUPPLEMENTARY FIGURES
2
3
Figure S1. Signal from the anti-methylarginine antibody increases as the amount of methylated
4
Npl3 increases. Methylated Npl3 (10 ng, 100 ng, and 500 ng per spot) were spotted onto PVDF
5
membrane and then probed with the anti-methylarginine antibody. As the amount of available methylated
6
Npl3 increases, the detected signal from the antibody also increases.
17
1
2
3
18
1
2
19
1
2
Figure S2. Electron-transfer dissociation (ETD) tandem MS spectra of the Brr1 derived tryptic
3
peptides A. Annotated ETD-MS/MS spectrum obtained for the doubly-charged tryptic Brr1 peptide
4
QEALR(methyl)TNAISIK observed at 679.3947 m/z , and identified with Mascot ion scores and expect
5
values of 46 and 3.1x10-3, respectively. In this peptide, one monomethylarginine site was identified and
6
diagnostic neutral losses for methylarginines were observed. B. Annotated ETD-MS/MS spectrum
7
obtained for the triply-charged tryptic Brr1 peptide R(methyl)GESQAPDAIFGQSR observed at
8
544.9412 m/z , and identified with Mascot ion scores and expect values of 78 and 1.8x10-6, respectively.
9
In this peptide, one monomethylarginine site was identified and diagnostic neutral losses for
10
methylarginines were observed. C. Annotated ETD-MS/MS spectrum obtained for the doubly-charged
11
tryptic Brr1 peptide R(methyl)GESQAPDAIFGQSR observed at 816.9112 m/z , and identified with
12
Mascot ion scores and expect values of 55 and 1.1x10-4, respectively. In this peptide, one
13
monomethylarginine site was identified and a diagnostic neutral loss for methylarginines was observed.
14
D. Annotated ETD-MS/MS spectrum obtained for the triply-charged tryptic Brr1 peptide
15
R(dimethyl)GESQAPDAIFGQSR observed at 549.6142 m/z , and identified with Mascot ion scores and
16
expect values of 72 and 2.7x10-5, respectively. In this peptide, one dimethylarginine site was identified
17
and diagnostic neutral losses for methylarginines were observed. E. Annotated ETD-MS/MS spectrum
20
1
obtained for the doubly-charged tryptic Brr1 peptide R(dimethyl)GESQAPDAIFGQSR observed at
2
823.9169 m/z , and identified with Mascot ion scores and expect values of 57 and 1.4x10-4, respectively.
3
In this peptide, one dimethylarginine site was identified and diagnostic neutral losses for methylarginines
4
were observed. Included above is the summarized ion-fragment coverage where c- and z-ions and their
5
derivatives are shown. Precursor and charge-reduced precursor ions, c- and z-ions and prominent ions
6
resulting from –NH3 and methylarginine-associated losses are labelled in the spectra. Methylarginine-
7
associated neutral losses are abbreviated as follows: monomethylamine (MMA), monomethylguanidine
8
(MMG), dimethylamine (DMA) and dimethylguanidine (DMG). Losses from NH3 are shown as ‘*’.
21
1
2
Figure S3. Electron-transfer dissociation (ETD) tandem MS spectrum of the Hts1 derived tryptic
3
peptide. Annotated ETD-MS/MS spectrum obtained for the doubly-charged tryptic Hts1 peptide
4
LIYNLEDQGGELC(carbamidomethyl)SLR(methyl) observed at 947.4738 m/z , and identified with
5
Mascot ion scores and expect values of 61 and 3.5x10-4, respectively. In this peptide, one
6
monomethylarginine site was identified and a diagnostic neutral loss for methylarginines was observed.
7
Included above is the summarized ion-fragment coverage where c- and z-ions and their derivatives are
8
shown. Precursor and charge-reduced precursor ions, c- and z-ions and prominent ions resulting from –
9
NH3 and methylarginine-associated losses are labelled in the spectrum. The methylarginine-associated
10
neutral loss is abbreviated as monomethylguanidine (MMG). Losses from NH3 are shown as ‘*’.
22
1
2
Figure S4. Electron-transfer dissociation (ETD) tandem MS spectrum of the Mpp10 derived tryptic
3
peptide. Annotated ETD-MS/MS spectrum obtained for the doubly-charged tryptic Mpp10 peptide
4
SR(methyl)SGPDSTNIKL observed at 644.8466 m/z, and identified with Mascot ion scores and expect
5
values of 43 and 3.4x10-3, respectively. In this peptide, one monomethylarginine site was identified and a
6
diagnostic neutral loss for methylarginines was observed. Included above is the summarized ion-fragment
7
coverage where c- and z-ions and their derivatives are shown. Precursor and charge-reduced precursor
8
ions, c- and z-ions and prominent ions resulting from –NH3 and methylarginine-associated losses are
9
labelled in the spectrum. The methylarginine-associated neutral loss is abbreviated as
10
monomethylguanidine (MMG). Losses from NH3 are shown as ‘*’.
23
1
2
Figure S5. Electron-transfer dissociation (ETD) tandem MS spectra of the Mrd1 derived tryptic
3
peptides. A. Annotated ETD-MS/MS spectrum obtained for the doubly-charged tryptic Mrd1 peptide
4
R(dimethyl)FKDGIIYLER observed at 719.4142 m/z, and identified with Mascot ion scores and expect
5
values of 18 and 2.8x10-2, respectively. In this peptide, one dimethylarginine site was identified and a
6
diagnostic neutral loss for methylarginines was observed. B. Annotated ETD-MS/MS spectrum obtained
24
1
for the triply-charged tryptic Mrd1 peptide LVMQYAEEDAVDAEEEIAR(methyl) observed at
2
732.3434 m/z, and identified with Mascot ion scores and expect values of 43 and 2.4x10-4, respectively. In
3
this peptide, one monomethylarginine site was identified and a diagnostic neutral loss for methylarginines
4
was observed. Included above is the summarized ion-fragment coverage where c- and z-ions and their
5
derivatives are shown. Precursor and charge-reduced precursor ions, c- and z-ions and prominent ions
6
resulting from –NH3 and methylarginine-associated losses are labelled in the spectra. Methylarginine-
7
associated neutral losses are abbreviated as follows: monomethylguanidine (MMG) and dimethylamine
8
(DMA). Losses from NH3 are shown as ‘*’ and interfering co-eluting ions are shown as ‘I’.
9
25
1
26
1
2
27
1
2
Figure S6. Electron-transfer dissociation (ETD) tandem MS spectra of the Nug1 derived tryptic
3
peptides, confirming the methylation of Nug1. A. Annotated ETD-MS/MS spectrum obtained for the
4
doubly-charged tryptic Nug1 peptide R(methyl)TSTKLK observed at 424.2717 m/z, and identified with
5
Mascot ion scores and expect values of 39 and 9.8x10-3, respectively.. In this peptide, one
6
monomethylarginine site was identified and diagnostic neutral losses for methylarginines were observed.
7
B. Annotated ETD-MS/MS spectrum obtained for the doubly-charged tryptic Nug1 peptide
8
ASAHR(methyl)KK observed at 406.2485 m/z, and identified with Mascot ion scores and expect values
9
of 37 and 2.0x10-2, respectively. In this peptide, one monomethylarginine site was identified and
10
diagnostic neutral losses for methylarginines were observed. C. Annotated ETD-MS/MS spectrum
11
obtained for the triply-charged tryptic Nug1 peptide KDVTWR(dimethyl)SR(methyl) observed at
12
363.8770 m/z, and identified with Mascot ion scores and expect values of 42 and 1.3x10-2, respectively. In
13
this peptide, one monomethylarginine and one dimethylarginine site were identified and diagnostic
14
neutral losses for methylarginines were observed. D. Annotated ETD-MS/MS spectrum obtained for the
15
doubly-charged tryptic Nug1 peptide KDVTWR(methyl)SR(dimethyl) observed at 545.3121 m/z, and
16
identified with Mascot ion scores and expect values of 42 and 8.9x10-3, respectively. In this peptide, one
17
monomethylarginine and one dimethylarginine site were identified and diagnostic neutral losses for
28
1
methylarginines were observed. E. Annotated ETD-MS/MS spectrum obtained for the triply-charged
2
tryptic Nug1 peptide KDVTWR(dimethyl)SR(dimethyl)SK observed at 440.2585 m/z, and identified with
3
Mascot ion scores and expect values of 51 and 3.0x10-4, respectively. In this peptide, twp
4
dimethylarginine sites were identified and diagnostic neutral losses for methylarginines were observed.
5
Included above is the summarized ion-fragment coverage where c- and z-ions and their derivatives are
6
shown. Precursor and charge-reduced precursor ions, c- and z-ions and prominent ions resulting from –
7
NH3 and methylarginine-associated losses are labelled in the spectra. Methylarginine-associated neutral
8
losses are abbreviated as follows: monomethylamine (MMA), monomethylguanidine (MMG),
9
dimethylamine (DMA) and dimethylguanidine (DMG). Losses from NH3 are shown as ‘*’.
10
29
1
2
Figure S7. Electron-transfer dissociation (ETD) tandem MS spectrum of the Rpa43 derived
3
chymotryptic peptide. Annotated ETD-MS/MS spectrum obtained for the triply-charged chymotryptic
4
Rpa43 peptide SQVKR(methyl)ANENRETARF observed at 607.3217 m/z, and identified with Mascot
5
ion scores and expect values of 53 and 1.1x10-5, respectively. In this peptide, one monomethylarginine
6
site was identified and a diagnostic neutral loss for methylarginines was observed. Included above is the
7
summarized ion-fragment coverage where c- and z-ions and their derivatives are shown. Precursor and
8
charge-reduced precursor ions, c- and z-ions and prominent ions resulting from –NH3 and methylarginine-
9
associated losses are labelled in the spectrum. The methylarginine-associated neutral loss is abbreviated
10
as monomethylguanidine (MMG). Losses from NH3 are shown as ‘*’.
11
30
1
2
Figure S8. Electron-transfer dissociation (ETD) tandem MS spectrum of the Rrp43 derived tryptic
3
peptide. Annotated ETD-MS/MS spectrum obtained for the doubly-charged tryptic Rrp43 peptide
4
GR(methyl)VGACTDEEMTISQK observed at 906.4155 m/z, and identified with Mascot ion scores and
5
expect values of 44 and 5.7x10-4, respectively. In this peptide, one monomethylarginine site was
6
identified and a diagnostic neutral loss for methylarginines was observed. Included above is the
7
summarized ion-fragment coverage where c- and z-ions and their derivatives are shown. Precursor and
8
charge-reduced precursor ions, c- and z-ions and prominent ions resulting from –NH3 and methylarginine-
9
associated losses are labelled in the spectrum. The methylarginine-associated neutral loss is abbreviated
10
as monomethylguanidine (MMG). Losses from NH3 are shown as ‘*’.
31
1
2
Figure S9. Electron-transfer dissociation (ETD) tandem MS spectrum of the Spp381 derived tryptic
3
peptide. Annotated ETD-MS/MS spectrum obtained for the triply-charged tryptic Spp381 peptide
4
R(dimethyl)LDTSSADESSSADEEHPDQNVSLTEK observed at 992.4454 m/z, and identified with
5
Mascot ion scores and expect values of 23 and 2.6x10-2, respectively. In this peptide, one
6
dimethylarginine site was identified and diagnostic neutral losses for methylarginines were observed.
7
Included above is the summarized ion-fragment coverage where c- and z-ions and their derivatives are
8
shown. Precursor and charge-reduced precursor ions, c- and z-ions and prominent ions resulting from –
9
NH3 and methylarginine-associated losses are labelled in the spectrum. Methylarginine-associated neutral
10
losses are abbreviated as follows: dimethylamine (DMA) and dimethylguanidine (DMG). Losses from
11
NH3 are shown as ‘*’.
32
1
33
1
2
Figure S10. Electron-transfer dissociation (ETD) tandem MS spectra of the Utp4 derived tryptic
3
peptides, confirming the methylation of Utp4. A. Annotated ETD-MS/MS spectrum obtained for the
4
triply-charged tryptic Utp4 peptide NNR(methyl)WVNSSNR observed at 420.8782 m/z, and identified
5
with Mascot ion scores and expect values of 40 and 5.7x10-3, respectively. In this peptide, one
6
monomethylarginine site was identified and a diagnostic neutral loss for methylarginines was observed.
7
B. Annotated ETD-MS/MS spectrum obtained for the triply-charged tryptic Utp4 peptide
8
NNR(dimethyl)WVNSSNR observed at 425.5499 m/z, and identified with Mascot ion scores and expect
9
values of 29 and 2.5x10-2, respectively. In this peptide, one dimethylarginine site was identified and a
10
diagnostic neutral loss for methylarginines was observed. C. Annotated ETD-MS/MS spectrum obtained
11
for the doubly-charged tryptic Utp4 peptide DDFVIGGCSDGR(methyl) observed at 656.2852 m/z, and
12
identified with Mascot ion scores and expect values of 21 and 3.1x10-2, respectively. In this peptide, one
13
monomethylarginine site was identified and a diagnostic neutral loss for methylarginines was observed.
14
Included above is the summarized ion-fragment coverage where c- and z-ions and their derivatives are
15
shown. Precursor and charge-reduced precursor ions, c- and z-ions and prominent ions resulting from –
16
NH3 and methylarginine-associated losses are labelled in the spectra. Methylarginine-associated neutral
17
losses are abbreviated as follows: monomethylamine (MMA), monomethylguanidine (MMG) and
34
1
dimethylamine (DMA). Losses from NH3 are shown as ‘*’ and interfering co-eluting ions are shown as
2
‘I’.
3
35
1
REFERENCES
2
[1] Ashburner, M., Ball, C. A., Blake, J. A., Botstein, D., et al., Gene Ontology: tool for the unification of
3
biology. Nature Genetics 2000, 25, 25-29.
4
[2] Cherry, J. M., Hong, E. L., Amundsen, C., Balakrishnan, R., et al., Saccharomyces Genome Database:
5
the genomics resource of budding yeast. Nucleic Acids Research 2012, 40, D700-D705.
6
[3] Consortium, T. U., Reorganizing the protein space at the Universal Protein Resource (UniProt).
7
Nucleic Acids Research 2012, 40, D71-D75.
8
[4] Huh, W.-K., Falvo, J. V., Gerke, L. C., Carroll, A. S., et al., Global analysis of protein localization in
9
budding yeast. Nature 2003, 425, 686-691.
10
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