Summary

1. Notes

2. Result Statistics

Figure 1. False discovery rate (FDR) curve. X axis is the number of peptide-spectrum matches (PSM) being kept. Y axis is the corresponding FDR.

Figure 2. PSM score distribution. (a) Distribution of PEAKS peptide score; (b) Scatterplot of PEAKS peptide score versus precursor mass error.

**(a)**

**(b)**

Figure 3. De novo result validation. Distribution of residue local confidence: (a) Residues in de novo sequences validated by confident database peptide assignment; (b) Residues in "de novo only" sequences.

**(a)**

**(b)**

**Table 1.** Statistics of data.
# of MS Scans	1013
# of MS/MS Scans	1013

**Table 2.** Result filtration parameters.
Peptide -10lgP	≥15
Protein -10lgP	≥20
Proteins unique peptides	≥0
De novo ALC Score	≥50%

**Table 3.** Statistics of filtered result.
Peptide-Spectrum Matches	110
Peptide Sequences	60
Protein Groups	12
Proteins	28
Proteins (#Unique Peptides)	3 (>2); 0 (=2); 22 (=1);
FDR (Peptide-Spectrum Matches)	14.5%
FDR (Peptide Sequences)	26.7%
FDR (Protein)	67.9%
De Novo Only Spectra	23

**Table 4.** PTM profile.
Name	∆Mass	#PSM	Position
Carbamidomethyl	57.02	72	C
Deamidation	.98	4	N
Oxidation	15.99	2	M
Carbamidomethyl	57.02	2	N-term
Methyl ester	14.02	2	C-term
Sulfation	79.96	2	Y
NDA	175.04	1	N-term
Carbofuran	58.03	1	S
Dehydration	-18.01	1	N-term
DTT_ST	136.00	1	S
O-Diethylphosphate	136.03	1	Y

3. Experiment Control

Figure 4. Precursor mass error of peptide-spectrum matches (PSM) in filtered result. (a) Distribution of precursor mass error in ppm; (b) Scatterplot of precursor m/z versus precursor mass error in ppm.

**(a)**

**(b)**

Table 5. Number of identified peptides in each sample by the number of missed cleavages

Missed Cleavages	0	1	2	3	4+
Sample 28	36	14	8	2	0

4. Other Information

**Table 6.** Search parameters.
Query Type: Homology Match Fixed Modifications: Carbamidomethylation: 57.02 Variable Modifications: Oxidation (M): 15.99 Fragment ion tolerance: 0.5 L equals I: true Q equals K: true Report number: 1 Maximum # of PTMs: 3 De novo score (ALC%) threshold: 15 Peptide hit threshold (-10logP): 30.0 Peaks run ID: 219 Merge Options: 0.1 min. 0.5 Da Precursor Options: corrected Charge Options: no correction Filter Options: no filter Process: true

**Table 7.** Instrument parameters.
Fractions: 28_Seg1Ev1.mzXML Ion Source: ESI(nano-spray) Fragmentation Mode: CID, CAD(y and b ions) MS Scan Mode: 3D Ion Trap MS/MS Scan Mode: 3D Ion Trap

Protein List

Protein Group	Protein ID	Accession	-10lgP	Coverage (%)	#Peptides	#Unique	PTM	Avg. Mass	Description
2	10567	tr\|A0A1E1WVY8\|A0A1E1WVY8_TITOB	222.33	63	20	6	Y	10060	Putative sodium channel toxin OS=Tityus obscurus OX=1221240 PE=4 SV=1
1	44589	sp\|H1ZZI3\|SCX14_TITOB	200.46	46	15	0	Y	10106	Toxin To14 OS=Tityus obscurus OX=1221240 PE=1 SV=1
3	45508	sp\|C0HLM1\|SCX3_TITMA	170.46	39	12	0	Y	7836	Toxin Tma3 OS=Tityus macrochirus OX=2599738 PE=1 SV=1
4	45510	sp\|C0HLM0\|SCX2_TITMA	102.13	29	5	0	Y	7830	Toxin Tma2 OS=Tityus macrochirus OX=2599738 PE=1 SV=1
5	46545	tr\|A0A1E1WW05\|A0A1E1WW05_TITOB	99.63	58	4	4	Y	8972	Putative sodium channel toxin OS=Tityus obscurus OX=1221240 PE=4 SV=1
6	27165	tr\|A0A1E1WVV5\|A0A1E1WVV5_TITOB	71.46	38	4	4	Y	7194	Putative potassium channel toxin OS=Tityus obscurus OX=1221240 PE=4 SV=1
7	47494	tr\|A0A2L2YWB1\|A0A2L2YWB1_PARTP	38.97	8	1	1	N	6816	Knottin scorpion toxin-like domain-containing protein (Fragment) OS=Parasteatoda tepidariorum OX=114398 PE=2 SV=1
7	47496	tr\|A0A2S3ICR9\|A0A2S3ICR9_9POAL	38.97	7	1	1	N	8110	Knottin scorpion toxin-like domain-containing protein OS=Panicum hallii OX=206008 GN=PAHAL_8G046500 PE=4 SV=1
7	47497	tr\|A0A2T7CKN5\|A0A2T7CKN5_9POAL	38.97	7	1	1	N	8110	Knottin scorpion toxin-like domain-containing protein OS=Panicum hallii var. hallii OX=1504633 GN=GQ55_8G044400 PE=4 SV=1
7	46657	tr\|A0A453JPA3\|A0A453JPA3_AEGTS	32.47	5	1	1	N	11978	Knottin scorpion toxin-like domain-containing protein OS=Aegilops tauschii subsp. strangulata OX=200361 PE=4 SV=1
7	46658	tr\|A0A453JPC9\|A0A453JPC9_AEGTS	32.47	4	1	1	N	12513	Knottin scorpion toxin-like domain-containing protein OS=Aegilops tauschii subsp. strangulata OX=200361 PE=4 SV=1
7	17674	tr\|A0A1W7R9K5\|A0A1W7R9K5_9SCOR	38.97	1	1	1	N	48916	Putative serine incorporator OS=Hadrurus spadix OX=141984 PE=3 SV=1
7	4994	tr\|A0A1W7RAZ4\|A0A1W7RAZ4_9SCOR	32.47	1	1	1	N	106453	Anoctamin OS=Hadrurus spadix OX=141984 PE=3 SV=1
11	6162	tr\|A0A510A9R9\|A0A510A9R9_MESMA	23.54	1	1	1	N	209391	LDscam3 OS=Mesobuthus martensii OX=34649 PE=2 SV=1
16	47507	tr\|A0A507ZTD3\|A0A507ZTD3_9ACTO	22.48	8	1	1	Y	8454	Arthropod defensin OS=Actinomyces johnsonii OX=544581 GN=FK256_14200 PE=4 SV=1
16	16596	tr\|U1PXD7\|U1PXD7_9ACTO	22.48	8	1	1	Y	8454	Arthropod defensin OS=Actinomyces johnsonii F0510 OX=1227262 GN=HMPREF1549_01193 PE=4 SV=1
16	47017	tr\|A0A397ZYR2\|A0A397ZYR2_BRACM	22.48	7	1	1	Y	9423	Knottin scorpion toxin-like domain-containing protein OS=Brassica campestris OX=3711 GN=BRARA_C02325 PE=4 SV=1
16	47020	tr\|A0A508B976\|A0A508B976_9ACTO	22.48	6	1	1	Y	11189	Arthropod defensin OS=Actinomyces oris OX=544580 GN=FK267_13255 PE=4 SV=1
8	34601	tr\|M7YWV1\|M7YWV1_TRIUA	22.07	20	1	1	Y	9168	Defensin Tk-AMP-D1 OS=Triticum urartu OX=4572 GN=TRIUR3_01070 PE=3 SV=1
12	47511	tr\|A0A1V1WBR0\|A0A1V1WBR0_9SCOR	21.79	9	1	1	N	6339	Putative NDBP (Fragment) OS=Superstitionia donensis OX=311983 PE=4 SV=1
12	47514	tr\|A0A1V1WC73\|A0A1V1WC73_9SCOR	21.79	7	1	1	N	8271	Putative NDBP OS=Superstitionia donensis OX=311983 PE=4 SV=1
12	47522	sp\|P0DMF2\|NDB4T_CHATY	21.79	6	1	1	N	9235	Peptide Ctry2346 OS=Chaerilus tryznai OX=1464547 PE=3 SV=1
12	47534	tr\|D0UMT6\|D0UMT6_HADAR	21.79	3	1	1	N	20076	SH2 domain binding protein (Fragment) OS=Hadrurus arizonensis OX=88316 PE=2 SV=1
12	21658	tr\|A0A1W7RA09\|A0A1W7RA09_9SCOR	21.79	1	1	1	N	45956	Poly(RC)-binding protein 3 OS=Hadrurus spadix OX=141984 PE=4 SV=1
12	10485	tr\|A0A1W7RAL8\|A0A1W7RAL8_9SCOR	21.79	1	1	1	N	61091	EH domain-containing protein 3 OS=Hadrurus spadix OX=141984 PE=4 SV=1
12	13586	tr\|A0A1S5QMZ5\|A0A1S5QMZ5_TITSE	21.79	1	1	1	N	71868	Angiotensin-converting enzyme OS=Tityus serrulatus OX=6887 PE=2 SV=1
14	47537	tr\|A0A0U1SCI0\|A0A0U1SCI0_ISOMC	20.56	2	1	1	N	24078	SCP domain-containing protein (Fragment) OS=Isometrus maculatus OX=497827 PE=2 SV=1
14	4987	tr\|A0A077S8H7\|A0A077S8H7_WHEAT	20.56	0	1	1	N	207714	Uncharacterized protein OS=Triticum aestivum OX=4565 GN=TRAES_3BF139100070CFD_c1 PE=3 SV=1
total 28 proteins

tr|A0A1E1WVY8|A0A1E1WVY8_TITOB

Peptide	Uniq	-10lgP	Mass	ppm	m/z	z	RT	Scan	#Spec	Start	End	PTM
R.NC(+57.02)MLDC(+57.02)NILDDK.G	Y	78.52	1509.6262	-0.5	755.8200	2	28.31	912	3	32	43	Carbamidomethylation
K.AAC(+57.02)YC(+57.02)YGLPDDSPTK.T	N	77.51	1716.7123	-15.7	859.3500	2	23.65	736	7	65	79	Carbamidomethylation
C.KKDDYPVDTAKR.N	N	70.16	1434.7466	-0.8	718.3800	2	11.05	250	4	20	31
C.KKDDYPVDTAK.R	N	69.53	1278.6455	0.0	640.3300	2	12.39	306	3	20	30
R.KADSGYC(+57.02)YK.L	N	66.40	1090.4753	247.7	546.3800	2	7.93	124	14	54	62	Carbamidomethylation
Y.C(+57.02)YGLPDDSPTK.T	N	57.19	1251.5441	1.2	626.7800	2	21.46	654	4	69	79	Carbamidomethylation
K.KDDYPVDTAK.R	N	52.31	1150.5505	-4.4	576.2800	2	14.04	370	2	21	30
R.N(+.98)C(+57.02)MLDC(+57.02)NILDDK.G	Y	51.27	1510.6102	605.9	756.7700	2	28.36	914	1	32	43	Deamidation (NQ); Carbamidomethylation
R.NC(+57.02)MLDC(+57.02)NILDDKGYC(+57.02)DK.F	Y	48.34	2132.8635	-16.6	711.9500	3	27.77	892	1	32	48	Carbamidomethylation
R.N(+.98)C(+57.02)MLDC(+57.02)NILDDKGYC(+57.02)DKF.C	Y	44.14	2280.9160	496.4	761.6900	3	32.53	1076	1	32	49	Deamidation (NQ); Carbamidomethylation
K.DDYPVDTAKR.N	N	44.10	1178.5568	7.3	590.2900	2	15.69	434	2	22	31
D.C(-18.01)NILDDKGYC(+57.02)DK.F	Y	42.40	1424.6064	-84.9	713.2500	2	27.99	900	1	37	48	Dehydration (C@N-term); Carbamidomethylation
K.KDDYPVDTAKR.N	N	38.90	1306.6517	-4.8	654.3300	2	13.42	346	1	21	31
D.SGYC(+57.02)YK.L	N	35.46	776.3163	-4.6	777.3200	1	6.38	58	4	57	62	Carbamidomethylation
R.K(+26.02)ADSGYC(+57.02)YK.L	N	32.06	1116.4910	-4.9	559.2500	2	14.34	382	1	54	62	Acetaldehyde +26; Carbamidomethylation
D.YPVDTAK.R	N	27.24	792.4017	-11.3	793.4000	1	13.52	350	1	24	30
R.N(+.98)C(+57.02)M(+15.99)LDC(+57.02)NILDDK.G	Y	25.74	1526.6051	-654.8	763.8100	2	24.84	782	1	32	43	Deamidation (NQ); Carbamidomethylation; Oxidation (M)
L.PDDSPTK.T	N	25.61	758.3446	-15.6	759.3400	1	23.16	716	2	73	79
Y.PVDTAK.R	N	25.03	629.3384	1564.0	631.3300	1	12.29	300	1	25	30
I.LDDKGY(+79.96)C(+57.02)DKFC(+57.02)K.G	Y	17.88	1627.6316	-605.9	814.3300	2	34.24	1138	1	40	51	Sulfation; Carbamidomethylation
D.KGY(+79.96)C(+57.02)DKFC(+57.02)K.G	Y	16.51	1284.4937	24.8	643.2700	2	28.72	928	1	43	51	Sulfation; Carbamidomethylation
Y.PVDTAKR.N	N	15.75	785.4395	-43.4	393.7100	2	11.61	272	1	25	31
R.KADSGYC(+57.02)YE(sub K).L	N	15.66	1091.4230	625.3	547.0600	2	6.09	46	1	54	62	Carbamidomethylation; Mutation
R.NC(+57.02)MLDC(+57.02)NILDDKGYC(+57.02)DKF.C	Y	15.12	2279.9319	441.4	761.3200	3	32.10	1060	1	32	49	Carbamidomethylation
I.L(+175.04)DDKGYC(+57.02)DKFC(+57.02)K.G	Y	15.05	1722.7170	-88.0	862.2900	2	26.74	852	1	40	51	Naphthalene-2,3-dicarboxaldehyde; Carbamidomethylation
total 25 peptides

Peptide	Uniq	-10lgP	Mass	ppm	m/z	z	RT	Scan	#Spec	Start	End	PTM
KKDDYPVDTAKR.N	N	70.16	1434.7466	-0.8	718.3800	2	11.05	250	4	1	12
KKDDYPVDTAK.R	N	69.53	1278.6455	0.0	640.3300	2	12.39	306	3	1	11
Y.C(+57.02)YGLPDDSPTK.K	N	57.19	1251.5441	1.2	626.7800	2	21.46	654	4	50	60	Carbamidomethylation
K.KDDYPVDTAK.R	N	52.31	1150.5505	-4.4	576.2800	2	14.04	370	2	2	11
K.DDYPVDTAKR.N	N	44.10	1178.5568	7.3	590.2900	2	15.69	434	2	3	12
K.KDDYPVDTAKR.N	N	38.90	1306.6517	-4.8	654.3300	2	13.42	346	1	2	12
N.AS(+136.00)C(+57.02)YC(+57.02)YGLPDDSPTK.K	N	37.48	1868.7089	-87.4	935.2800	2	24.79	780	1	46	60	Dithiothreitol (DTT); Carbamidomethylation
N.A(+57.02)S(+79.97)C(+57.02)YC(+57.02)YGLPDDSPTK.K	N	35.16	1869.6951	112.5	935.9600	2	25.11	792	1	46	60	Carbamidomethylation (DHKE, X@N-term); Phosphorylation (STY); Carbamidomethylation
N.ASC(+57.02)Y(+136.03)C(+57.02)YGLPDDSPTK.K	N	34.95	1868.7362	-112.8	935.2700	2	24.90	784	1	46	60	Carbamidomethylation; O-Diethylphosphorylation
A.S(+58.03)C(+57.02)YC(+57.02)YGLPDDSPTK.K	N	28.41	1719.6995	-194.2	860.6900	2	23.60	734	1	47	60	2,3-dihydro-2,2-dimethyl-7-benzofuranol N-methyl carbamate; Carbamidomethylation
D.YPVDTAK.R	N	27.24	792.4017	-11.3	793.4000	1	13.52	350	1	5	11
L.PDDSPTK.K	N	25.61	758.3446	-15.6	759.3400	1	23.16	716	2	54	60
Y.PVDTAK.R	N	25.03	629.3384	1564.0	631.3300	1	12.29	300	1	6	11
Y.PVDTAKR.N	N	15.75	785.4395	-43.4	393.7100	2	11.61	272	1	6	12
total 14 peptides

Peptide	Uniq	-10lgP	Mass	ppm	m/z	z	RT	Scan	#Spec	Start	End	PTM
K.GC(+57.02)YC(+57.02)YDVAPSTK.M	Y	52.34	1419.5798	-10.2	710.7900	2	19.49	578	2	63	74	Carbamidomethylation
R.AGATN(+.98)GKGDC(+57.02)INK.G	Y	50.60	1305.5983	-9.8	653.8000	2	10.33	220	1	50	62	Deamidation (NQ); Carbamidomethylation
K.IYC(+57.02)PDDAVC(+57.02)KDTC(+57.02)K.N	Y	41.92	1743.7266	-7.7	582.2450	3	18.31	534	2	34	47	Carbamidomethylation
K.MYPGRLPC(+57.02)N	Y	32.06	1106.5001	4.8	554.2600	2	19.82	590	2	75	83	Carbamidomethylation
total 4 peptides

Peptide	Uniq	-10lgP	Mass	ppm	m/z	z	RT	Scan	#Spec	Start	End	PTM
R.GKGTGYC(+57.02)DKDVC(+57.02)K.C	Y	43.10	1486.6544	-6.0	744.3300	2	10.63	232	1	46	58	Carbamidomethylation
A.SPFGK.C	Y	32.06	534.2802	-13.8	535.2800	1	9.09	170	2	28	32
K.G(+57.02)TGYCDKDVC(+57.02)K.C	Y	24.78	1301.5380	5.8	651.7800	2	12.73	318	1	48	58	Carbamidomethylation (DHKE, X@N-term); Carbamidomethylation
K.PLNC(+57.02)AK.E	Y	16.25	701.3530	-0.5	702.3600	1	13.99	368	1	35	40	Carbamidomethylation
total 4 peptides

Peptide	Uniq	-10lgP	Mass	ppm	m/z	z	RT	Scan	#Spec	Start	End	PTM
H.ALFGK.C	Y	38.97	534.3165	-81.9	535.2800	1	9.82	200	3	40	44
total 1 peptides

Peptide	Uniq	-10lgP	Mass	ppm	m/z	z	RT	Scan	#Spec	Start	End	PTM
V.ALFGK.G	Y	38.97	534.3165	-81.9	535.2800	1	9.82	200	3	53	57
total 1 peptides

Peptide	Uniq	-10lgP	Mass	ppm	m/z	z	RT	Scan	#Spec	Start	End	PTM
M.AIFGK.S	Y	32.47	534.3165	-81.9	535.2800	1	9.82	200	3	22	26
total 1 peptides

Peptide	Uniq	-10lgP	Mass	ppm	m/z	z	RT	Scan	#Spec	Start	End	PTM
Q.ALFGK.V	Y	38.97	534.3165	-81.9	535.2800	1	9.82	200	3	149	153
total 1 peptides

Peptide	Uniq	-10lgP	Mass	ppm	m/z	z	RT	Scan	#Spec	Start	End	PTM
N.AIFGK.R	Y	32.47	534.3165	-81.9	535.2800	1	9.82	200	3	266	270
total 1 peptides

Peptide	Uniq	-10lgP	Mass	ppm	m/z	z	RT	Scan	#Spec	Start	End	PTM
R.IQPSDASVLVGR.S	Y	23.54	1240.6775	-138.7	621.2600	2	18.44	538	1	707	718
total 1 peptides

Peptide	Uniq	-10lgP	Mass	ppm	m/z	z	RT	Scan	#Spec	Start	End	PTM
H.C(+57.02)LSTGY.R	Y	22.48	699.2898	-10.1	700.2900	1	16.16	452	1	53	58	Carbamidomethylation
total 1 peptides

Peptide	Uniq	-10lgP	Mass	ppm	m/z	z	RT	Scan	#Spec	Start	End	PTM
R.C(+57.02)ISTGY.K	Y	22.48	699.2898	-10.1	700.2900	1	16.16	452	1	46	51	Carbamidomethylation
total 1 peptides

Peptide	Uniq	-10lgP	Mass	ppm	m/z	z	RT	Scan	#Spec	Start	End	PTM
R.M(+15.99)AAAPAAALLIVLLLVA.T	Y	22.07	1635.9996	480.4	819.4000	2	24.68	776	4	7	23	Oxidation (M)
total 1 peptides

Peptide	Uniq	-10lgP	Mass	ppm	m/z	z	RT	Scan	#Spec	Start	End	PTM
K.SLLGK.R	Y	21.79	516.3271	-105.3	517.2800	1	9.62	192	1	36	40
total 1 peptides

Peptide	Uniq	-10lgP	Mass	ppm	m/z	z	RT	Scan	#Spec	Start	End	PTM
S.SILGK.R	Y	21.79	516.3271	-105.3	517.2800	1	9.62	192	1	40	44
total 1 peptides

Peptide	Uniq	-10lgP	Mass	ppm	m/z	z	RT	Scan	#Spec	Start	End	PTM
Q.SLLGK.R	Y	21.79	516.3271	-105.3	517.2800	1	9.62	192	1	31	35
total 1 peptides

Peptide	Uniq	-10lgP	Mass	ppm	m/z	z	RT	Scan	#Spec	Start	End	PTM
P.SLLGK.A	Y	21.79	516.3271	-105.3	517.2800	1	9.62	192	1	18	22
total 1 peptides

Peptide	Uniq	-10lgP	Mass	ppm	m/z	z	RT	Scan	#Spec	Start	End	PTM
G.SIIGK.K	Y	21.79	516.3271	-105.3	517.2800	1	9.62	192	1	31	35
total 1 peptides

Peptide	Uniq	-10lgP	Mass	ppm	m/z	z	RT	Scan	#Spec	Start	End	PTM
N.SILGK.I	Y	21.79	516.3271	-105.3	517.2800	1	9.62	192	1	481	485
total 1 peptides

Peptide	Uniq	-10lgP	Mass	ppm	m/z	z	RT	Scan	#Spec	Start	End	PTM
L.SILGK.A	Y	21.79	516.3271	-105.3	517.2800	1	9.62	192	1	110	114
total 1 peptides

Peptide	Uniq	-10lgP	Mass	ppm	m/z	z	RT	Scan	#Spec	Start	End	PTM
K.DPTGK.L	Y	20.56	516.2543	16.3	517.2700	1	9.14	172	1	82	86
total 1 peptides

Peptide	Uniq	-10lgP	Mass	ppm	m/z	z	RT	Scan	#Spec	Start	End	PTM
I.DPTGK.K	Y	20.56	516.2543	16.3	517.2700	1	9.14	172	1	983	987
total 1 peptides