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	14134
# of MS/MS scans	1253

**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	45
Peptide sequences	18
Protein groups	5
Proteins	35
Proteins (#Unique Peptides)	2 (>2); 0 (=2); 33 (=1);
FDR (Peptide-Spectrum Matches)	11.1%
FDR (Peptide Sequences)	27.8%
De Novo Only Spectra	66

**Table 4.** PTM profile.
Name	∆Mass	Position	#PSM	-10lgP	Area	AScore
Deamidation	.98	NQ	4	32.39	4.12E3	9.08

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+
Blank_SICAN	10	5	1	2	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: 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: Uni Taxon: All Searched Entry: 561911 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: Blank_Pa-MeOH1.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

Protein Accession Contains:

Protein Description Contains:

Peptide Sample Area >=

Protein Ptm Contains:

Protein Group	Protein ID	Accession	-10lgP	Coverage (%)	Coverage (%) Blank_SICAN	Area Blank_SICAN	#Peptides	#Unique	#Spec Blank_SICAN	PTM	Avg. Mass	Description
1	30	P0AEY0	95.05	15	15	2.96E5	5	5	26	Y	43388	Maltose/maltodextrin-binding periplasmic protein OS=Escherichia coli O157:H7 OX=83334 GN=malE PE=1 SV=1
1	29	P0AEX9	95.05	15	15	2.96E5	5	5	26	Y	43388	Maltose/maltodextrin-binding periplasmic protein OS=Escherichia coli (strain K12) OX=83333 GN=malE PE=1 SV=1
2	406641	B4SUT7	44.92	4	4	1.72E4	1	1	7	N	29820	50S ribosomal protein L2 OS=Salmonella newport (strain SL254) OX=423368 GN=rplB PE=3 SV=1
2	406642	A6TEW9	44.92	4	4	1.72E4	1	1	7	N	29834	50S ribosomal protein L2 OS=Klebsiella pneumoniae subsp. pneumoniae (strain ATCC 700721 / MGH 78578) OX=272620 GN=rplB PE=3 SV=1
2	406643	B4TXD9	44.92	4	4	1.72E4	1	1	7	N	29820	50S ribosomal protein L2 OS=Salmonella schwarzengrund (strain CVM19633) OX=439843 GN=rplB PE=3 SV=1
2	406644	Q5PIV5	44.92	4	4	1.72E4	1	1	7	N	29820	50S ribosomal protein L2 OS=Salmonella paratyphi A (strain ATCC 9150 / SARB42) OX=295319 GN=rplB PE=3 SV=1
2	406645	B5BGY3	44.92	4	4	1.72E4	1	1	7	N	29820	50S ribosomal protein L2 OS=Salmonella paratyphi A (strain AKU_12601) OX=554290 GN=rplB PE=3 SV=1
2	406646	B1LHD1	44.92	4	4	1.72E4	1	1	7	N	29860	50S ribosomal protein L2 OS=Escherichia coli (strain SMS-3-5 / SECEC) OX=439855 GN=rplB PE=3 SV=1
2	406647	B2VK61	44.92	4	4	1.72E4	1	1	7	N	29898	50S ribosomal protein L2 OS=Erwinia tasmaniensis (strain DSM 17950 / CIP 109463 / Et1/99) OX=465817 GN=rplB PE=3 SV=1
2	406648	C6DG71	44.92	4	4	1.72E4	1	1	7	N	29968	50S ribosomal protein L2 OS=Pectobacterium carotovorum subsp. carotovorum (strain PC1) OX=561230 GN=rplB PE=3 SV=1
2	406649	B5R288	44.92	4	4	1.72E4	1	1	7	N	29820	50S ribosomal protein L2 OS=Salmonella enteritidis PT4 (strain P125109) OX=550537 GN=rplB PE=3 SV=1
2	406650	P60427	44.92	4	4	1.72E4	1	1	7	N	29820	50S ribosomal protein L2 OS=Salmonella typhi OX=90370 GN=rplB PE=3 SV=1
2	406651	B5F8E9	44.92	4	4	1.72E4	1	1	7	N	29820	50S ribosomal protein L2 OS=Salmonella agona (strain SL483) OX=454166 GN=rplB PE=3 SV=1
2	406652	Q31VV9	44.92	4	4	1.72E4	1	1	7	N	29860	50S ribosomal protein L2 OS=Shigella boydii serotype 4 (strain Sb227) OX=300268 GN=rplB PE=3 SV=1
2	406653	B7LRT3	44.92	4	4	1.72E4	1	1	7	N	29860	50S ribosomal protein L2 OS=Escherichia fergusonii (strain ATCC 35469 / DSM 13698 / CDC 0568-73) OX=585054 GN=rplB PE=3 SV=1
2	406654	B5FJL1	44.92	4	4	1.72E4	1	1	7	N	29820	50S ribosomal protein L2 OS=Salmonella dublin (strain CT_02021853) OX=439851 GN=rplB PE=3 SV=1
2	406655	Q3YWU2	44.92	4	4	1.72E4	1	1	7	N	29860	50S ribosomal protein L2 OS=Shigella sonnei (strain Ss046) OX=300269 GN=rplB PE=3 SV=1
2	43538	A9MSZ5	44.92	4	4	1.72E4	1	1	7	N	29820	50S ribosomal protein L2 OS=Salmonella paratyphi B (strain ATCC BAA-1250 / SPB7) OX=1016998 GN=rplB PE=3 SV=1
2	406656	Q32B34	44.92	4	4	1.72E4	1	1	7	N	29832	50S ribosomal protein L2 OS=Shigella dysenteriae serotype 1 (strain Sd197) OX=300267 GN=rplB PE=3 SV=1
2	406657	A9MN51	44.92	4	4	1.72E4	1	1	7	N	29820	50S ribosomal protein L2 OS=Salmonella arizonae (strain ATCC BAA-731 / CDC346-86 / RSK2980) OX=41514 GN=rplB PE=3 SV=1
2	406658	Q57J35	44.92	4	4	1.72E4	1	1	7	N	29820	50S ribosomal protein L2 OS=Salmonella choleraesuis (strain SC-B67) OX=321314 GN=rplB PE=3 SV=1
2	406659	Q0SZY6	44.92	4	4	1.72E4	1	1	7	N	29860	50S ribosomal protein L2 OS=Shigella flexneri serotype 5b (strain 8401) OX=373384 GN=rplB PE=3 SV=1
2	406660	B5XN97	44.92	4	4	1.72E4	1	1	7	N	29834	50S ribosomal protein L2 OS=Klebsiella pneumoniae (strain 342) OX=507522 GN=rplB PE=3 SV=1
2	406661	B4TKL2	44.92	4	4	1.72E4	1	1	7	N	29820	50S ribosomal protein L2 OS=Salmonella heidelberg (strain SL476) OX=454169 GN=rplB PE=3 SV=1
2	406662	A4WFC5	44.92	4	4	1.72E4	1	1	7	N	29836	50S ribosomal protein L2 OS=Enterobacter sp. (strain 638) OX=399742 GN=rplB PE=3 SV=1
2	406663	P60428	44.92	4	4	1.72E4	1	1	7	N	29820	50S ribosomal protein L2 OS=Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) OX=99287 GN=rplB PE=3 SV=1
2	406664	B2U2T5	44.92	4	4	1.72E4	1	1	7	N	29860	50S ribosomal protein L2 OS=Shigella boydii serotype 18 (strain CDC 3083-94 / BS512) OX=344609 GN=rplB PE=3 SV=1
2	406665	B5RH18	44.92	4	4	1.72E4	1	1	7	N	29820	50S ribosomal protein L2 OS=Salmonella gallinarum (strain 287/91 / NCTC 13346) OX=550538 GN=rplB PE=3 SV=1
2	406666	Q6CZX3	44.92	4	4	1.72E4	1	1	7	N	29894	50S ribosomal protein L2 OS=Pectobacterium atrosepticum (strain SCRI 1043 / ATCC BAA-672) OX=218491 GN=rplB PE=3 SV=1
2	406667	Q1R607	44.92	4	4	1.72E4	1	1	7	N	29860	50S ribosomal protein L2 OS=Escherichia coli (strain UTI89 / UPEC) OX=364106 GN=rplB PE=3 SV=1
2	406668	P60429	44.92	4	4	1.72E4	1	1	7	N	29860	50S ribosomal protein L2 OS=Shigella flexneri OX=623 GN=rplB PE=3 SV=2
2	406669	B4F1I7	44.92	4	4	1.72E4	1	1	7	N	30057	50S ribosomal protein L2 OS=Proteus mirabilis (strain HI4320) OX=529507 GN=rplB PE=3 SV=1
5	74807	P93422	22.62	1	1	0E0	1	1	1	N	85548	Histidine--tRNA ligase, cytoplasmic OS=Oryza sativa subsp. japonica OX=39947 GN=Os05g0150900 PE=2 SV=2
6	406672	Q13Z16	22.17	5	5	0E0	1	1	1	Y	49879	Trigger factor OS=Paraburkholderia xenovorans (strain LB400) OX=266265 GN=tig PE=3 SV=1
8	406674	O58814	20.02	9	9	0E0	1	1	1	N	31485	Homoserine kinase OS=Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3) OX=70601 GN=thrB PE=3 SV=2
total 35 proteins

Peptide	Uniq	-10lgP	Mass	Length	ppm	m/z	z	RT	Fraction	Scan	Source File	Area Blank_SICAN	#Spec	#Spec Blank_SICAN	Start	End	PTM
R.TAVINAASGR.Q	Y	48.32	958.5196	10	-0.1	480.2670	2	26.48	140	1622	Blank_Pa-MeOH1.raw	2.81E5	22	22	371	380
K.AGLTFLVDLIK.N	Y	45.11	1188.7118	11	-0.2	595.3630	2	27.60	140	1753	Blank_Pa-MeOH1.raw	1.08E4	1	1	216	226
M.PNIPQMSAFWYAVR.T	Y	37.42	1678.8290	14	-0.8	840.4211	2	27.62	140	1755	Blank_Pa-MeOH1.raw	0	1	1	357	370
R.IAATMENAQKGEIMPNIPQ(+.98)MSAFWYAVR.T	Y	32.39	3167.5242	28	9.0	1056.8582	3	27.61	140	1754	Blank_Pa-MeOH1.raw	4.12E3	1	1	343	370	Deamidation (NQ)
K.DVGVDNAGAK.A	Y	17.99	944.4563	10	-0.7	473.2351	2	26.88	140	1666	Blank_Pa-MeOH1.raw	0	1	1	206	215
total 5 peptides

Peptide	Uniq	-10lgP	Mass	Length	ppm	m/z	z	RT	Fraction	Scan	Source File	Area Blank_SICAN	#Spec	#Spec Blank_SICAN	Start	End	PTM
R.TAVINAASGR.Q	Y	48.32	958.5196	10	-0.1	480.2670	2	26.48	140	1622	Blank_Pa-MeOH1.raw	2.81E5	22	22	371	380
K.AGLTFLVDLIK.N	Y	45.11	1188.7118	11	-0.2	595.3630	2	27.60	140	1753	Blank_Pa-MeOH1.raw	1.08E4	1	1	216	226
M.PNIPQMSAFWYAVR.T	Y	37.42	1678.8290	14	-0.8	840.4211	2	27.62	140	1755	Blank_Pa-MeOH1.raw	0	1	1	357	370
R.IAATMENAQKGEIMPNIPQ(+.98)MSAFWYAVR.T	Y	32.39	3167.5242	28	9.0	1056.8582	3	27.61	140	1754	Blank_Pa-MeOH1.raw	4.12E3	1	1	343	370	Deamidation (NQ)
K.DVGVDNAGAK.A	Y	17.99	944.4563	10	-0.7	473.2351	2	26.88	140	1666	Blank_Pa-MeOH1.raw	0	1	1	206	215
total 5 peptides

Peptide	Uniq	-10lgP	Mass	Length	ppm	m/z	z	RT	Fraction	Scan	Source File	Area Blank_SICAN	#Spec	#Spec Blank_SICAN	Start	End	PTM
R.SAGTYVQIVAR.D	Y	44.92	1163.6299	11	0.2	582.8223	2	25.86	140	1560	Blank_Pa-MeOH1.raw	1.72E4	7	7	157	167
total 1 peptides

Peptide	Uniq	-10lgP	Mass	Length	ppm	m/z	z	RT	Fraction	Scan	Source File	Area Blank_SICAN	#Spec	#Spec Blank_SICAN	Start	End	PTM
R.SAGTYVQIVAR.E	Y	44.92	1163.6299	11	0.2	582.8223	2	25.86	140	1560	Blank_Pa-MeOH1.raw	1.72E4	7	7	157	167
total 1 peptides

Peptide	Uniq	-10lgP	Mass	Length	ppm	m/z	z	RT	Fraction	Scan	Source File	Area Blank_SICAN	#Spec	#Spec Blank_SICAN	Start	End	PTM
T.TQVGSIAAGGR.Y	Y	22.62	1015.5410	11	-0.7	508.7774	2	27.66	140	1761	Blank_Pa-MeOH1.raw	0	1	1	641	651
total 1 peptides

Peptide	Uniq	-10lgP	Mass	Length	ppm	m/z	z	RT	Fraction	Scan	Source File	Area Blank_SICAN	#Spec	#Spec Blank_SICAN	Start	End	PTM
R.QDLEQRGVPNAKDAPIPAAM(+15.99)FK.E	Y	22.17	2411.2266	22	-11.1	804.7405	3	49.44	140	4509	Blank_Pa-MeOH1.raw	0	1	1	331	352	Oxidation (M)
total 1 peptides

Peptide	Uniq	-10lgP	Mass	Length	ppm	m/z	z	RT	Fraction	Scan	Source File	Area Blank_SICAN	#Spec	#Spec Blank_SICAN	Start	End	PTM
K.LKKGIRPKSGLGSSGASAVAGALGAAK.L	Y	20.02	2451.4285	27	8.9	818.1574	3	44.24	140	3848	Blank_Pa-MeOH1.raw	0	1	1	78	104
total 1 peptides