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 1124 Peptide sequences 793 Protein groups 241 Proteins 475 Proteins (#Unique Peptides) 81 (>2); 88 (=2); 117 (=1); FDR (Peptide-Spectrum Matches) 4.9% FDR (Peptide Sequences) 6.8% De Novo Only Spectra 2956

Table 4. PTM profile. Name ∆Mass Position #PSM -10lgP Area AScore Carbamidomethyl 57.02 C 134 50.03 5.39E5 1000.00 Deamidation .98 NQ 67 53.44 4.21E5 30.46 Oxidation 15.99 M 59 54.06 1.44E5 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 715 72 5 1 0

4. Other Information

Table 6. Search parameters. Search Engine Name: PEAKS Parent Mass Error Tolerance: 20.0 ppm Fragment Mass Error Tolerance: 0.8 Da Precursor Mass Search Type: monoisotopic Enzyme: LysCTryp Max Missed Cleavages: 3 Non-specific Cleavage: both Fixed Modifications:   Carbamidomethylation: 57.02 Variable Modifications:   Oxidation (M): 15.99   Deamidation (NQ): 0.98 Max Variable PTM Per Peptide: 3 Database: Uniprot BLOOD Taxon: All Searched Entry: 1159819 FDR Estimation: Enabled 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