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)

Table 1. Statistics of data. #Scans #Features Identified #Peptides #Sequences #Proteins* MS1 MS2 #PSMs #Scans #Features Groups All Top Total 185381 832887 1232000 15714 15117 121172 1004 920 179 1221 690 F1 17747 25804 43172 151 145 1217 58 57 23 198 132 F10 14450 66767 101661 1025 1000 10570 180 159 78 393 235 F11 14901 60059 85015 702 688 8131 172 157 77 296 195 F12 13572 65943 90270 2771 2719 7830 372 350 106 680 328 F13 17804 25578 43974 851 840 2227 248 232 59 318 130 F2 13957 63001 81914 746 739 6004 151 147 38 198 114 F3 14375 66936 102262 835 824 10552 85 84 32 144 87 F4 13555 75415 120379 1243 1194 14161 105 104 43 132 96 F5 13188 79299 116370 1832 1731 14495 197 192 63 417 199 F6 13394 73671 113396 1838 1749 11085 257 245 73 455 234 F7 12386 78855 111633 1448 1370 10879 161 155 54 258 133 F8 13868 73656 103980 910 834 12269 150 141 56 309 171 F9 12184 77903 117974 1362 1284 11752 177 163 65 327 169 * proteins with significant peptides are used in counts.

Figure 3. Sample overlap for Proteins and Peptides (up to 8 samples). (a) All Proteins; (b) Top Proteins; (c) Peptides;

(a) Do not support more than 8 samples

(b) Do not support more than 8 samples

(c) Do not support more than 8 samples

Figure 4. Distribution of peptide feature detection. (a) Feature m/z distribution; (b) Feature RT distribution.

(a)

(b)

Figure 5. Distribution of identified peptide features. (a) Feature abundance distribution; (b) De novo sequencing validation.

(a)

(b)

Table 2. Result filtration parameters. Peptide -10lgP ≥41.8 PTM Ascore ≥0 Protein -10lgP ≥20 Proteins unique peptides ≥1 De novo score(%) ≥50% Table 3. Statistics of filtered result. FDR (Peptide-Spectrum Matches) 0.1% FDR (Peptide Sequences) 1.3% FDR (Protein Group) 7.6% De Novo Only Spectra 248152

Table 4. PTM profile. Name ∆Mass Position #PSM -10lgP Abundance AScore Carbamidomethyl 57.02 C 9818 103.21 6.19E7 1000.00 Oxidation 15.99 M 1088 91.90 6.56E8 1000.00

3. Experiment Control

Figure 6. 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+ F1 57 1 0 0 0 F10 138 32 10 0 0 F11 132 34 6 0 0 F12 297 71 4 0 0 F13 216 31 1 0 0 F2 123 27 1 0 0 F3 69 14 2 0 0 F4 77 26 2 0 0 F5 150 44 3 0 0 F6 201 53 3 0 0 ...

4. Other Information

Table 6. Search parameters. Search Engine Name: PEAKS Parent Mass Error Tolerance: 10.0 ppm Fragment Mass Error Tolerance: 0.6 Da Precursor Mass Search Type: monoisotopic Enzyme: Trypsin Max Missed Cleavages: 2 Digest Mode: Semispecific Fixed Modifications:   Carbamidomethylation: 57.02 Variable Modifications:   Oxidation (M): 15.99 Max Variable PTM Per Peptide: 3 Database: NCBI_Serpentes_NR Taxon: All Contaminant Database: cRAP_contaminants Searched Entry: 410049 FDR Estimation: Enabled Merge Options: no merge Precursor Options: corrected Charge Options: no correction Filter Charge: 2 - 8 Process: true Associate chimera: yes

Table 7. Instrument parameters. Fractions: NaNaKA16_F1.raw, NaNaKA16_F10.raw, NaNaKA16_F11.raw, NaNaKA16_F12.raw, NaNaKA16_F13.raw, NaNaKA16_F2.raw, NaNaKA1 6_F3.raw, NaNaKA16_F4.raw, NaNaKA16_F5.raw, NaNaKA16_F6.raw, NaNaKA16_F7.raw, NaNaKA16_F8.raw, NaNaKA16_F9.raw Ion Source: ESI(nano-spray) Fragmentation Mode: CID, CAD(y and b ions) MS Scan Mode: FT-ICR/Orbitrap MS/MS Scan Mode: FT-ICR/Orbitrap