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 5931 # of MS/MS scans 9771 Table 2. Result filtration parameters. Peptide -10lgP ≥15 Peptide Ascore ≥0 Protein -10lgP ≥20 Proteins unique peptides ≥0 De novo ALC Score ≥50% Table 3. Statistics of filtered result. Peptide-Spectrum Matches 413 Peptide sequences 373 Protein groups 19 Proteins 20 Proteins (#Unique Peptides) 11 (>2); 7 (=2); 2 (=1); FDR (Peptide-Spectrum Matches) 91.5% FDR (Peptide Sequences) 93.8% De Novo Only Spectra 3660

Table 4. PTM profile. Name ∆Mass Position #PSM -10lgP Area AScore Deamidation .98 NQ 118 33.07 2.51E4 1000.00 Carbamidomethyl 57.02 C 71 33.07 2.51E4 1000.00 Phosphorylation 79.97 ST 35 34.74 1.55E4 28.36 Oxidation 15.99 M 34 24.09 7.25E4 1000.00 Dehydration -18.01 STY 19 34.89 3.61E4 1000.00 Methylation(KR) 14.02 KR 16 27.89   9.32 Acetylation 42.01 N-term 12 23.61   1000.00 Sodium 21.98 D,C-term 9 24.54 6.87E4 1000.00 Dihydroxy 31.99 KPRY 9 22.14 1.3E5 17.01 Acetylation 42.01 Protein N-term 8 25.79 2.33E4 1000.00 Dimethylation(KR) 28.03 KR 7 25.26   21.33 Acetylation 42.01 K 7 21.30   10.94 Carbamylation 43.01 K,N-term 6 45.73 1.84E5 145.30 Formylation 27.99 N-term 6 43.73 4.48E4 90.85 Arg->Npo 80.99 R 6 23.15 2.71E4 18.34 AEC-MAEC 59.02 S 5 28.64   1000.00 Amidation -.98 C-term 4 28.08 2.42E4 1000.00

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+ SIMCA 165 141 46 21 0

4. Other Information

Table 6. Search parameters. Search Engine Name: PEAKS Parent Mass Error Tolerance: 15.0 ppm Fragment Mass Error Tolerance: 0.8 Da Precursor Mass Search Type: monoisotopic Enzyme: Trypsin Max Missed Cleavages: 3 Non-specific Cleavage: both Fixed Modifications:   Carbamidomethylation: 57.02 Variable Modifications:   Oxidation (M): 15.99   Deamidation (NQ): 0.98   Acetylation (K): 42.01   Acetylation (Protein N-term): 42.01   Acetylation (N-term): 42.01   Amidation: -0.98   Beta-methylthiolation: 45.99   Biotinylation: 226.08   and 304 more... Max Variable PTM Per Peptide: 3 Database: egg Taxon: All Searched Entry: 63 FDR Estimation: Enabled De novo score (ALC%) threshold: 15 Peptide hit threshold (-10logP): 30.0 Peaks run ID: 259 Merge Options: no merge Precursor Options: corrected Charge Options: no correction Filter Options: no filter Process: true

Table 7. Instrument parameters. Fractions: Sican.raw Ion Source: ESI(nano-spray) Fragmentation Mode: CID, CAD(y and b ions) MS Scan Mode: FT-ICR/Orbitrap MS/MS Scan Mode: Linear Ion Trap