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