Electronic Supplementary Material for the manuscript entitled:
Effective selenium detoxification in the seed proteins of a hyperaccumulator plant: the analysis of selenium containing proteins of monkeypot nut (Lecythis minor) seeds
by A. Németh and M. Dernovics, published in Journal of Biological Inorganic Chemistry
Table S 2 Instrumental parameters of the HPLC-ESI-QTOF-MS set-up.
6530 Accurate Mass QTOF LC-MS (Agilent)
ESI source Dual ESI (Agilent)
Operational mode Positive
Precursor ion isolation in MS/MS mode medium (4 m/z)
Mass accuracy < 2 ppm
Mass resolution > 10000
Detection frequency 4 GHz
Fragmentor voltage 150 V/-150 V
Curtain voltage 65 V/ -65 V
Drying gas 13 L/min
Capillary voltage 800 V
Nebulizer pressure 40 psig
Gas temperature 325 ºC
Data analysis software Mass Hunter Acquisition B.02.01 with SP3
Mass Hunter Qualitative Analysis B.03.01 with SP3
Table S 1 Chromatographic parameters applied.
Column XDB C18, 4.6 mm x 100 mm x 5 µm
Injection volume (µL) 10
Flow rate (mL min-1) 0.35
Buffer A 0.1% formic acid in water
Buffer B acetonitrile with 0.1% formic acid
Gradient program 0-2 10% B
4 up to 20% B
10 up to 45% B
15 up to 100% B
20 100% B
22 down to 10% B
Table S 4. Protein database search identification results. The coverage was calculated by the sotware based on the sequence of the precursor protein. In the case of 2S albumin and 2S sulfur-rich proteins, the signal and propeptides are removed during the maturation of the proteines. The revised coverages were calculated based on the sequence of the mature proteins. Methionine coverage is the coverage of methionine containing peptides. Se-containing peptides are highlighted.
Accession number
Description OrganismSoftware coverage
Calculated coverage
Methionine coverage
Peptides
Sequence m/z z Mass Mass error Start End
Q84ND2 11S globulin B. excelsa 31 32 25
VIRPP 291.1921 2 581.3768 0.03 260 264VQIVDHR 289.499 3 866.4822 2.24 360 366HFFLAGNIQR 401.5506 3 1202.637 4.78 185 194CAGVAALR 409.2206 2 817.4338 1.15 64 71LYYVTGR 500.2654 2 999.5234 2.27 90 97NGETVFDDNLR 583.2778 2 1165.5482 0 367 376VQHTASDLNQLDQNPR 612.6377 3 1835.8983 0.47 169 184NTIRPQGLLLPVYTNAPK 665.7175 3 1995.1377 0.27 72 89KGDIIAIPAGVALW 712.4186 2 1423.8298 0.62 142 155GILGVLMPGCPETF 745.8737 2 1490.74 1.32 98 111GILGVLBPGCPETF 769.8457 2 1538.684 0.97 98 111LTTVNSLKVPILTF 773.4643 2 1545.9212 2.46 310 323FIQNIDNPAEDFYNPR 1012.4823 2 2023.9572 3.18 290 306VPILTFLQL 1043.6495 2 2086.2916 0.39 318 326
B6EU54 2S albumin B. excelsa 18 29 29
GEQMR 310.6446 2 620.2818 4.78 112 116CEGLR 317.6519 2 634.2964 1.83 93 97EMQPR 330.6594 2 660.3114 2.77 107 111GEQBR 334.615 2 668.2226 5.61 112 116EBQPR 354.6315 2 708.2556 2.96 107 111QQMLSH 372.1772 2 743.347 4.53 46 51pyro-QQBLSH 396.1521 2 791.2968 2.43 46 51CNLSPQR 437.7131 2 874.4188 1.18 129 135MAENLPSR 459.229 2 917.4506 0.15 121 128BAENLPSR 483.2005 2 965.3936 1.73 121 128
P044032S sulfur-rich
storage protein 1
B. excelsa 15 32 32
GEQMR 310.6446 2 620.2818 0.26 113 117CEGLR 317.6519 2 634.2964 1.83 94 98EMQPR 330.6594 2 660.3114 2.77 108 112GEQBR 334.615 2 668.2226 5.61 113 117EBQPR 354.6315 2 708.2556 2.96 108 112QQMLSH 372.1772 2 743.347 4.53 47 52AENLPSR 393.7075 2 786.4076 3.04 123 129pyro-QQBLSH 396.1521 2 791.2968 2.43 47 52MQQEEMQPR 588.7606 2 1176.5138 0.3 104 112BQQEEMQPR 612.7319 2 1224.4564 1.29 104 112BQQEEBQPR 636.7047 2 1272.402 0.38 104 112QQBLSH 774.2685 1 774.2685 0.14 47 52
P0C8Y82S sulfur-rich
storage protein 2
B. excelsa 5 15 19
MYLR 291.6574 2 582.3074 3.11 62 65BYLR 315.6289 2 630.2504 1.03 62 65CEGLR 317.6519 2 634.2964 4.75 103 107CNLSPQR 437.7121 2 874.4168 1.18 138 144
Table S 3. Database search and protein validation settings.
Database search settings
Name and version of the program PeptideShaker1-0-23-0
Enzyme Trypsin
Fixed modification Carbamidomethyl cysteineVariable modifications Phosphorylation of serine or tyrosine
Oxidation of methionine
Selenomethionine
Selenocysteine
Precursor mass tolerance 10 ppm
Fragment ion types b and y
Maximum missed cleavages 2
Fragment mass tolerance 0.05 mDa
Precursor charge 2-3
Validation settings
Validated hits 8
Confidence 95%
Lowest confidence of validated results 100%
FDR 1
FNR 1
x102
0
1.2
2.4
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400
y2
y1
y4
y5
y7
y8
m/z
Inte
nsity
, cp
s
y12
G V L M P938.36311051.25981150.52721207.5487
683.4041552.3636439.2795340.2111
I L1320.63281433.7168
283.18961170.1055
C* P E T F267.1298493.2352653.2491
1324.65201223.60431094.5617997.5089
G710.2815807.3180
837.4783780.4568
396.1766
G166.0863
57.0215
x102
0
1
2
200 300 400 500 600 700 800 900 1000m/z
Inte
nsity
. cp
s
y2y1
y4
y5
y7
y8
y9y5’
y7’y6’
G V L B P986.31921099.42281198.47171255.4932
731.3486552.3636439.2795340.2111
I L1368.57731481.6613
283.1896170.1055
C P E T F267.1298493.2352653.2491
1372.59651271.54881142.50621045.4535
G710.2815807.3180
885.4228828.4013
396.1766
G166.0863
57.0215
G V L M P986.31921099.42281198.47171255.4932
683.4041552.3636439.2795340.2111
I L1368.57731481.6613
283.1896170.1055
SeC P E T F267.1298493.2352701.1545
1372.59651271.54881142.50621045.4535
G758.1760855.2287
837.4783780.4568
396.1766
G166.0863
57.0215
y7
y5’y7’y6’
m/z
Inte
nsity
, cp
s
x102
0
0.8
1.6807.31804
733.05634
700 725 750 775 800 825 850
(‘)
Figure S 1. The MS-MS spectra and the identification details of the peptide GILGVLMPGCPETF and its selenium analogue. While the selenopeptide was identified by the search engines as a Sec containing peptide, the manual checking of the MS-MS spectra revealed the absence of Sec containing fragments (y5’, y6’, y7’). In the peptide sequence C* corresponds to carbamydomethyl-cysteine, B denotes to selenomethionine and Sec represents selenocysteine.
m/z
Inte
nsity
, cp
s
x103
0
4
8
100 200 300 400 500 600 700
B S K147.1130234.1449
576.0425489.0105
M B413.1325592.1152
310.0255131.0405
B S K147.1130234.1449
576.0425489.0105
B M413.1307544.1713
310.0255178.9852
M S K147.1131234.1451
576.0425489.0105
B B365.1855544.1716
357.9704178.9857
y1
y2
y3 y4
y2’
y3’ y4’ y4’’
y2’’
0
0,5
1
100 200 300 400 500 600
x104
m/z
Inte
nsity
, cp
s
M S K147.1130234.1451
528.0981441.0665
B M365.1829496.2265
310.0255178.9857
M S K147.1130234.1451
528.0981441.0665
M B365.1829544.1716
310.0255131.0405
B S K147.1131234.1451
528.0981441.0665
M M413.1301544.1716
262.0812131.0405
y1
y3
y4
y2’’
y3’ y3’’
y4’ y4’’b2
y2 y2’ y2’’
Figure S 2. MS-MS spectra and details of de novo sequencing of selenopeptides.
x104
0
0,7
1,4
100 200 300 400 500 600m/z
Inte
nsity
, cp
s
M S K147.1126234.1452
480.1535393.1215
M M365.1860496.2284
263.0893131.0405
y1
y2
y3 y4
m/z
Inte
nsity
, cp
s
x103
0
2.5
5
50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950
y1 y2
y3
y4
S F B585.1870732.2584
363.1431216.0746
E819.2912
129.0426
G S R175.1192262.1512
773.2136686.1815
S319.1725406.2034
629.1601542.1285
y5 y6
x104
0
0,5
1
50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950
m/z
Inte
nsity
, cp
s
y1
y2
y3
y4
y5y6
G K N750.3392878.4336
332.1849204.0899
F935.4249
147.0684
C A M306.1577377.1963
776.3642705.3269
V537.2294636.2913
545.2962446.2278
R175.1183
907.4045
y7
y8
x103
0
1,2
2,4
50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050m/z
Inte
nsity
, cp
s
y1
y2y3
y4
y5
y6
G K N798.2741926.3781
332.1849204.0899
F983.3703
147.0684
C A B354.1755425.1411
776.3642705.3269
V585.1718684.2458
545.2962446.2278
R175.1191
955.3490
y7y8
x104
0
0,5
1
100 200 300 400 500 600 700 800 900m/z
Inte
nsity
, cp
s
y1
y2
y3
y4
S F M537.2441684.3134
363.1431216.0746
E771.3455
129.0426
G S R175.1192262.1512
725.2691638.2374
S319.1725406.2027
581.2156494.1835
y5
y6
y7
M G M933.4095990.4787
367.0815310.0255
B1121.5171
178.9855
E N P488.2464602.2893
812.2101698.1246
A731.3882802.45233
569.1246498.0874
E391.1936
909.2629
S262.1510
1038.4432
R175.1191
1125.3371
B G M933.4095990.4787
367.0815310.0255
M1169.5054
131.0405
E N P488.2464602.2893
812.2101698.1246
A731.3882802.45233
569.1246498.0874
E391.1936
909.2629
S262.1510
1038.4432
R175.1191
1125.3371
M G B981.35421038.4432
319.1025262.0812
M1121.5171
131.0405
E N P488.2464602.2893
812.2101698.1246
A731.3882802.45233
569.1246498.0874
E391.1936
909.2629
S262.1510
1038.4432
R175.1191
1125.3371
x102
0
1.3
2.6
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300m/z
Inte
nsity
, cp
s
y1 y2
y3
y5y6
y7
y8 y10
y4 y8’ y10’’
y9’’
y9 y9’
y10’
x103
0
2
4
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100m/z
Inte
nsity
, cp
s
E N A M M605.2911676.3329790.3750919.4025
877.4117746.3712675.3341561.2911
I Y1082.47661195.5610
432.2485269.1823
P R175.1193270.1820
1176.5423
A343.2074474.2419
1079.48921008.4521
R
156.1011
y1
y2
y3 y4 y5 y6
y7y8
y9
y10
m/z
Inte
nsity
, cp
s
x102
0
2.5
5
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400
E N A B M653.2148724.3034837.3211967.3614
925.3562746.3712675.3341561.2911
I Y1130.42791243.5041
432.2485269.1823
P R175.1193270.1820
1224.4873
A343.1984474.2369
1127.43451056.3972
R
156.1011
y1
y2
y3
y4 y5y6
y7 y8y9
y10
x102
0
2.5
5
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250m/z
Inte
nsity
, cp
s
y1 y2
y4 y5
y6
y7
y8
M G M933.4817990.4955
319.1088262.0814
M1121.5171
131.0405
E N P488.2464602.3191
764.2656650.2227
A731.3963802.4511
521.1948450.1429
E391.1936
861.3184
S262.1512
990.3612
R175.1191
1077.3932
y9
y10