Comparative Differential Cuproproteomes of Rhodobacter … · 2020-02-28 · S - 1 Comparative Differential Cuproproteomes of Rhodobacter capsulatus Reveal Novel Copper Homeostasis

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Comparative Differential Cuproproteomes of Rhodobacter capsulatus Reveal Novel Copper Homeostasis related Proteins

Nur Selamoglua, Özlem Öndera,b, Yavuz Öztürka,e, Bahia Khalfaoui-Hassania,#, Crysten E. Blaby-

Haasc, Benjamin A. Garciad, Hans-Georg Koche and Fevzi Daldala*

aDepartment of Biology, University of Pennsylvania, Philadelphia, PA, 19104 USA; bDepartment

of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania,

Philadelphia, PA 19104, USA; cBiology Department, Brookhaven National Laboratory, Upton,

NY 11973, USA; dDepartment of Biochemistry and Biophysics, Perelman School of Medicine,

University of Pennsylvania, Philadelphia, PA 19104, USA, eInstitute of Biochemistry and

Molecular Biology, ZBMZ, Faculty of Medicine, Albert-Ludwigs University of Freiburg, 79104

Freiburg, Germany.

Running Title: Rhodobacter capsulatus Copper-Responsive Proteome

Key words: Rhodobacter capsulatus proteome, differential cuproproteomics, copper

homeostasis, copper-responsive proteins, uncharacterized proteins.

#Present address: IPREM, UMR CNRS 5254, and Université de Pau et des Pays de l’Adour,

BP1155 Pau, France

*Corresponding author: Fevzi Daldal

[email protected]; +1 215-898-4394

Files in this Data Supplement:

Supplemental Information and Table S3 (Excel file)

Electronic Supplementary Material (ESI) for Metallomics.This journal is © The Royal Society of Chemistry 2020

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SUPPLEMENTAL INFORMATION

Experimental Procedures

ProteaseMAXTM Extraction/Trypsin Digestion (protocol B). Each 100 mg aliquot from the

resulting wet pellets was washed with ultrapure water, centrifuged and the supernatant discarded.

Solubilization and extraction of proteins was performed using ProteaseMAXTM Surfactant Trypsin

Enhancer (Promega Inc.), followed by trypsin digestion as follows (1). The pellet was resuspended

in 0.5 ml of ProteaseMAXTM solution (1 mg/ml in 10% acetonitrile and 90% 50mM NH4HCO3),

and 10 min of rigorous vortexing was followed by incubation in a 95 °C water bath for 10 min.

Following 15 min of sonication at 4 °C (BioruptorR, Diagenode), the sample was vortexed at room

temperature for 30 min. Estimates from separate BCA analysis of similar samples indicated that ~

400-500 µg protein was solubilized at this stage. Further reduction/alkylation and digestions steps

were completed without separating the insolubilized solid fraction. Proteins were reduced by

adding dithiothreitol (DTT) to a final concentration of 5mM and incubating for 30 min at 37 °C,

and alkylated by adding iodoacetamide to a final concentration of 15 mM and incubating for 30

min at room temperature in the dark. Trypsin (Promega, Sequencing Grade Modified Trypsin) was

added to the mixture at a 1/100 w/w ratio of enzyme/protein, and the extract was digested overnight

at 37 °C, pH 7. Digested mixtures were vortexed for 10 min and acidified with 10% formic acid

to pH 3-4 to halt digestion, vortexed and placed in a 45 °C shaker for 30 min to degrade

ProteaseMAXTM. Following a 15 min centrifugation at 15,000 x g, the supernatants were removed

and dried to completeness in a vacuum centrifuge at 45 °C. The digested peptide samples were

resuspended in 50 µL 5% acetonitrile/0.1% formic acid solution, and desalted using ZipTips

(Milipore U-C18 P10), eluted, lyophilized and stored at -80 °C. Samples were resuspended in 10

µL (5% acetonitrile/0.1% formic acid) prior to MS analysis.

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The efficacy of the extraction/digestion protocols A (thiourea/urea/trypsin/LysC) and B

(ProteaseMax/trypsin) was determined using the nano-LC-MS/MS Thermo Orbitrap Velos Pro

system described below. Protocol A yielded ~40% more identified proteins than protocol B (1784

versus 1244 in triplicate samples), indicating that the former with multiple digestion steps was

more effective than the latter. In addition, a further 15-20% increase in the number of identified

proteins was observed when the samples prepared by protocol A were analyzed using the higher

sensitivity Q-Exactive MS, rendering it the method of choice for the quantitative aspects of this

study. The qualitative data for the protein identification included all datasets, including those

treated by the procedure A or B, analyzed using Orbitrap Velos (c.f. below) as well as the Q-

Exactive MS runs (c.f. main text).

Data acquisition using nano-LC-MS/MS Thermo Orbitrap Velos Pro. Prepared digests were

loaded without pre-fractionation onto a 20 cm column (packed in-house, 75 µm ID, 3µm C18,

RepoSil-Pur Q18-AQ (Dr.Maisch GmbH)) with buffer A (0.1% formic acid). Peptides were

separated and eluted by nano-HPLC (EASY-nLC 1000, Thermo Inc.) using a 85 min linear

gradient from 4% to 40% buffer B (0.1% formic acid in acetonitrile), followed by a 20 min linear

gradient from 40% to 90% B, followed by 15 min washing with 90% B (flow rate 300 nL/min),

and re-equilibration with buffer A. The nLC system was coupled to an Orbitrap Velos Pro mass

spectrometer (Thermo Scientific), programmed to acquire spectra in data-dependent mode, with

high resolution full survey scans followed by the top 15 most abundant ions fragmented for MS2

(Thermo Xcalibur 2.2 software). The resolution for full MS scans (300-2000 m/z) was 60,000 (at

400 m/z) and dual lock mass was enabled with 371.101233 m/z and 445.120024 m/z polysiloxane

background ions. The 15 most intense ions were selected for fragmentation by collision induced

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dissociation (CID) with collision energy set at 35%, and with dynamic exclusion enabled to

minimize repeat fragmentation (repeat count: 3, repeat duration: 30 sec, exclusion duration: 90

sec; exclusion list: 500). Unassigned charge states and singly charged species were rejected. The

raw data were analyzed as described in the main text.

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REFERENCES

1. Pirmoradian, M., Budamgunta, H., Chingin, K., Zhang, B., Astorga-Wells, J., and Zubarev, R. A. (2013) Rapid and deep human proteome analysis by single-dimension shotgun proteomics. Mol Cell Proteomics 12, 3330-3338

2. Sambrook, J., and Russell, D. W. (2001) Molecular Cloning: a laboratory manual, 3rd ed. ed., Cold Spring harbor Laboratory Press, Cold Spring harbor

3. Simon, R., Priefer, U., and Puhler, A. (1983) A Broad Host Range Mobilization System for Invivo Genetic-Engineering - Transposon Mutagenesis in Gram-Negative Bacteria. Bio-Technology 1, 784-791

4. Zannoni, D., Prince, R. C., Dutton, P. L., and Marrs, B. L. (1980) Isolation and Characterization of a Cytochrome c2-Deficient Mutant of Rhodopseudomonas-capsulata. Febs Letters 113, 289-293

5. Yen, H. C., Hu, N. T., and Marrs, B. L. (1979) Characterization of the gene transfer agent made by an overproducer mutant of Rhodopseudomonas capsulata. J Mol Biol 131, 157-168

6. Ditta, G., Schmidhauser, T., Yakobson, E., Lu, P., Liang, X. W., Finlay, D. R., Guiney, D., and Helinski, D. R. (1985) Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid 13, 149-153

7. Brimacombe, C. A., Stevens, A., Jun, D., Mercer, R., Lang, A. S., and Beatty, J. T. (2013) Quorum-sensing regulation of a capsular polysaccharide receptor for the Rhodobacter capsulatus gene transfer agent (RcGTA). Mol Microbiol 87, 802-817

8. S. Ekici, S. Turkarslan, G. Pawlik, A. Dancis, N. S. Baliga, H. G. Koch and F. Daldal, Intracytoplasmic copper homeostasis controls cytochrome c oxidase production, MBio, 2014, 5, e01055-01013.

9. Y. Zhang, C. E. Blaby-Haas, S. Steimle, A. F. Verissimo, V. A. Garcia-Angulo, H. G. Koch, F. Daldal and B. Khalfaoui-Hassani, Cu Transport by the Extended Family of CcoA-like Transporters (CalT) in Proteobacteria, Sci Rep, 2019, 9, 1208.

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Table S1. Strains and plasmids used in this work. Strain or plasmid Description Phenotype Reference

Strains E. coli HB101

F- Δ(gpt-proA)62 leuB6 supE44 ara-14 galK2 lacY1 Δ(mcrC-mrr) rpsL20 (StrR) xyl-5 mtl-1recA13

Strr

(2)

S17-1 recA pro hsdR, RP4(Tc::Mu, Km::Tn7) (3)

R. capsulatus

aMT1131

crtD121

wild type (Rifr)

(4)

Y262 GTA overproducer (5)

YO-Δ00098 Δ(rcc00098) In-frame, markerless This work

YO-Δ00738 Δ(rcc00738::Gm) Gmr This work

YO- Δ00885 Δ(rcc00891-rcc00885::Gm) Gmr This work

YO-Δ01027 Δ(rcc01027-rcc01031::Gm) Gmr, This work

YO-Δ01423Su Δ(rcc01423) In-frame, markerless This work




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YO-Δ02111 Δ ( rcc02111) In-frame, markerless This work

YO-Δ03065 Δ(rcc03065-rcc03067::Gm) Gmr This work

Plasmids

pRK2013 Conjugation helper

Kanr

(6)

pRK415 Broad host-range vector

Tetr

(6)

pZDJ Puc promoter replaced with tetA promoter on the suicide plasmid pZJD29a

sacB, GmR (7)

pYO98 Δ(rcc01423::Gm) on pRK415 Gmr This work

pYO-Δ00738 Δ(rcc00738::Gm) on pRK415 Tetr This work

pYO-Δ00885 Δ(rcc00891-rcc00885::Gm) on pRK415 Tetr, Gmr This work


pYO-Δ01445 Δ(rcc01445::Gm) on pRK415 Tetr, Gmr This work


pYO-Δ02109 Δ(rcc02109::Gm) on pRK415 Tetr, Gmr This work

pYO97 Δ(rcc01423) on pZDJ; in-frame deletion of rcc01423 without its first 4 and last 4 codons.

Gmr This work

pYO-Δ00098 Δ(rcc00098) on pZDJ; in-frame deletion of rcc02111 without its first 4 and last 4 codons.

Gmr This work

pYO-Δ02111Su Δ(rcc02111) on pZDJ; in-frame deletion of rcc02111 without its first 4 and last 4 codons.

Gmr This work

aR.capsulatus strain MT1131 is derived from SB1003 in multiple steps, as described in Zannoni et al. (1980): first a Ps-deficient mutant (TL1) was obtained using tetracycline suicide, then its crtD derivative was constructed by GTA cross to yield MT113, and then its Ps-proficient derivative was obtained via a second GTA cross.

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Table S2. Primers used in this work.

Gm-F 5-AAGCTTGTCGACCCGATCTGAGC-5

Gm-R 5- TCTAGAACTAGTGGATCCCCCG-3

01423-F1 5-CGGCCAGTGAATTCGAGCTCGGTACATGCGCAGGGTATAGGTGACGCC-3

01423-R1 5-TCATTTTTCGAACTGCGGGTGGCTCCAGGCGTTTTCTTCTTCGAAGTGCC-3

01423-F2 5-GAGCCACCCGCAGTTCGAAAAATGATCCGACTTCGCAAGCCAGGTCC-3

01423-R2 5-AAGCTTGCATGCCTGCAGGTCGACTGCGATCGTCTTTGCCGATCTGGC-3

01423Gm-R1 5-GAAAATGGCTCAGATCGGGTCGACAAGCTTAAGTGGCAGACATGACATGTCTCC-3

01423Gm-F3 5-TTCCCGGCCGGGGGATCCACTAGTTCTAGAGAAGAAAACGCCTGATCCGACTTCG -3

01423Su-F1 5-TACGCCAAGCTTGCATGCCTGCAGGGTCTGTTGCGCCAGCACGGCTTTGC-3

01423Su-R1 5-AGTGGCAGACATGACATGTCTCCTC-3

01423Su-F2 5-GAGGAGACATGTCATGTCTGCCACTGAAGAAAACGCCTGATCCGACTTCG-3

01423Su-R2 5-AAACGACGGCCAGTGAATTCGAGCTGGCGACGGACAATTGCCGGGGCTG-3

00738-F1 5-CGGCCAGTGAATTCGAGCTCGGTACACTGCGCCAAGACCTGCCCCAAGG-3

00738-R1 5-AAAAATGGCTCAGATCGGGTCGACAAGCTTAGACGATGCGGAAGAAGCAGCCCG -3

00738-F2 5-TTCCCGGCCGGGGGATCCACTAGTTCTAGAGATCTGGGCGGCGCGCTCTCGGC-3 00738-R2 5-AAGCTTGCATGCCTGCAGGTCGACTTGACCATGCTGCTGGAAGACGAC-3 00891-F1 5-CGGCCAGTGAATTCGAGCTCGGTACAGATGGTGATCCGCGCCATCGCG-3

00891-R1 5-AAAAATGGCTCAGATCGGGTCGACAAGCTTCGCCGAGGCCGAGAGATGTTCGG -3

00885-F2 5-TTCCCGGCCGGGGGATCCACTAGTTCTAGAACCCGAACCATCACTGCCCCTTCTG-3

00885-R2 5-AAGCTTGCATGCCTGCAGGTCGACTACGCCGCCGGATGGCATTCAAAAGG-3

01027-F1 5-CGGCCAGTGAATTCGAGCTCGGTACCCGCCCGAAAGCGCCAGAAGCGC-3

01027-R1 5-AAAAATGGCTCAGATCGGGTCGACAAGCTTGCTGCCCGTAGGCGTCGGCAACC-3

01031-F2 5-TTCCCGGCCGGGGGATCCACTAGTTCTAGAGCCGACCGGGTTCTGGTGCTGGC-3

01031-R2 5-AAGCTTGCATGCCTGCAGGTCGACTGCGATCGCGCAATTCGGCCTTGGC-3 02111Su-F1 5-TACGCCAAGCTTGCATGCCTGCAGGATCGAGCTGTGGCAGGTGATGCGC-3 02111Su-R1 5-CAGAATCCGCATGGGAGGAGCATGGC-3

02111Su-F2 5-CCATGCTCCTCCCATGCGGATTCTGCCGCCCCGCCTCTGATCTTCGCC-3

02111Su-R2 5-AAACGACGGCCAGTGAATTCGAGCTGGCAGCGGCACCACCGGGTTGCC-3

02109-R1 5-AAAAATGGCTCAGATCGGGTCGACAAGCTTAAGGCACCCGGCGACAAGGGCC-3

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02109-F2 5-TTCCCGGCCGGGGGATCCACTAGTTCTAGACTGATCACGGCGGACGGGCAAACC-3

02109-R2 5-AAGCTTGCATGCCTGCAGGTCGACTGTTCTAACCTTAGGCCGCCAGAC-3

00098Su-F1 5-TACGCCAAGCTTGCATGCCTGCAGGCGACGCTGGGCCTTGACCCGGAG-3

00098Su-R1 5-AAGGCGGCGCATGGGTGTCTCCAG-3

00098Su-F2 5-ACTGGAGACACCCATGCGCCGCCTTATCCGTTTCTGAGATCCGTTTCC-3

00098Su-R2 5-AAACGACGGCCAGTGAATTCGAGCTTCGTCCATGCGCAGATGCAGATC-3

01445-F1 5-CGGCCAGTGAATTCGAGCTCGGTACCGCTGGTGGCAGAACCGATCAGC-3

01445-R1 5-AAAAATGGCTCAGATCGGGTCGACAAGCTTGGTGATGACGATCGTGCCCAGCG-3

01445-F2 5-TTCCCGGCCGGGGGATCCACTAGTTCTAGAGACGGCGCTTTTGATCGACGCGC-3

01445-R2 5-AAGCTTGCATGCCTGCAGGTCGACTTGGCATGGATGCGATGCTGATCG-3

03065-F1 5-CGGCCAGTGAATTCGAGCTCGGTACTCCAGCGGCAGATGCGTGGCGGC-5

03065-R1 5-AAAAATGGCTCAGATCGGGTCGACAAGCTTCGGCGGCGCGCATCCAGAACACC-3

03067-F2 5-TTCCCGGCCGGGGGATCCACTAGTTCTAGATTCGGCCCGATGGTCGAGGACCGC-3

03067-R2 5-AAGCTTGCATGCCTGCAGGTCGACtCCAGCCGATGCGCGATGACATTCG-3

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Table S3. Excel spreadsheet compiled datasets. Data related to the identification, annotations,

quantitation and statistics of R. capsulatus proteins are cumulated in Table S3. Sheet 1 (R.

capsulatus Proteins): Full genome listing of R. capsulatus (3632 entries, UniprotKB). Various

annotations are included: PsortB localizations, TIGRFAM and TIGR roles, GO annotations,

Interpro, and KEGG classifications, as well as protein identifiers. Those proteins experimentally

identified in the current study are marked with asterisks (first column), and those unidentified are

designated in gray colored rows. Sheet 2 (Identified Proteins): Proteins experimentally identified

in the current study (2430 entries), with the number of unique peptides, MS/MS counts and sequence

coverage listed along with other information. Sheet 3 (Unidentified Proteins): Proteins unidentified

in the present study are listed along with functional annotations where available. Sheet 4 (Single

Peptide-ID): List of proteins identified with only one unique peptide (59 entries). Those proteins

identified with single MS/MS spectra are highlighted in yellow (13 entries). Annotated spectra for

all single peptides are provided in Figure S4. Sheet 5 (Quantified-Statistics): The 1926 proteins

quantified in this study are listed, along with T-test p-values and Fold-changes for +Cu vs control

and for +BCS vs control (see Experimental Procedures). The 75 proteins that were significantly

affected by changes in Cu levels (i.e., surpassed the criteria of p<0.05 and FC>2.0) are highlighted

as noted in the table headings.

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Supplemental FIGURES

Figure S1. Nadi staining of R. capsulatus colonies grown under different Cu concentrations. Cu-sufficient (control), Cu-excess

(+Cu) and Cu-depleted (+BCS) refer to MedA medium containing 0.3 µM Cu, 5 µM Cu and 5mM BCS, respectively.

Figure S1. Selamoglu et al.

Cu-sufficient(control)

Cu-excess(+Cu)

Cu-depleted(+BCS)

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Figure S2. Pearson correlation for individual experimental runs. Color-coded Pearson r-values are shown for the 31 individual runs

using lysis/digestion protocol A and Q-Exactive mass spectrometer (Experimental Procedures). The samples are identified by Cu

condition (Ctrl, +Cu and +BCS), biological repeat (B1, B2, B3) and technical repeat (R1, R2, R3, R4). All r values were consistently

high, within 0.92 to 0.99, except the sample +Cu(B2R1) that had r-values between 0.75-0.83 and was subsequently discarded.

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Figure S3. The NEBuilder HiFi DNA Assembly Method. (a) the version used for GTA mediated chromosomal inactivation, and (b)

that used for construction of chromosomal in-frame seamless deletions of R. capsulatus chromosomal genes. The dotted lines indicate

the overlapping regions between the vector arms and the DNA fragments being inserted.

a)Vector: pRK415 (KpnI/XbaI), 10.5 kb

GmR cassette20-30 nt

overlapping sequence

N-terminal part of gene

C-terminal part of gene

-Kpn

I

-Xba

I

20-30 ntoverlapping sequence

5’

-3’

3’3’

5’

5’ 5’

3’

3’

5’

5’

5’3’

3’3’ 5’

TetR

b)Vector: pZDJ (SalI/SacI), 4.8 kb

25 ntoverlapping sequence

Upstream of gene-(5’ flanking region)

-Sal

I

-Sac

I

ATG NNN NNN NNN

ATG NNN NNN NNN NNN NNN NNN TGA

TAC NNN NNN NNN

TAC NNN NNN NNN NNN NNN NNN ACT

Downstream of gene(3’ flanking region)

Start and first 3 codons

Last 3 codons and Stop 25 ntoverlapping sequence

-3’

3’

3’ 5’

5’

5’3’ 5’

-5’

5’

3’

3’

GmRsacB


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Figure S4. Annotated spectra corresponding to proteins identified by a single unique peptide (see Sheet 4 (Single Peptide-ID) in Table S3).

y₁-NH₃130.0863

y₁147.1128

b₂-H₂O199.0713

y₂-NH₃258.1448

y₂275.1714

y₃346.2085

b₄-H₂O357.1405

y₄417.2456

b₅-H₂O486.1831

y₅504.2776

b₆-H₂O557.2202

y₆575.3148

y₇646.3519

y₈717.389

y₉846.4316

y₁₀917.4687

020

4060

80100

120

00.2

0.40.6

0.81

1.21.4

1.61.8

2

[1e4]

200 400 600 800

Raw File Scan Method Score m/zMT1131-minusCu-Set3-02 10728 FTMS; HCD 56.23 710.83

- E S G T E A

y₁₀

E

y₉

A

y₈

A

y₇

A

y₆

S

y₅

A

y₄

A

y₃

Q

y₂

K

y₁

-

Nur Selamoglu

RCAP_rcc00013 D5AKD6 Inner membrane transport permease

a₂143.1179

b₂171.1128

y₁175.119

b₃299.1714

b₄430.2119

b₅501.249

b₆-NH₃583.2908

b₆600.3174

y₆798.4621

y₇-NH₃878.4883

y₇895.5148

y₈1008.599

y₉1107.667

y₁₀1178.704

y₁₁1309.745

020

4060

80100

120

00.2

0.40.6

0.81

1.21.4

1.6

[1e5]

200 400 600 800 1000 1200

Raw File Scan Method Score m/zMT1131-minusCu-plusBC-Set3-01 42715 FTMS; HCD 59.07 804.76

- I Gb₂

Qb₃

M

y₁₁

b₄A

y₁₀

b₅V

y₉

b₆L

y₈

P

y₇

P

y₆

W L N L R

y₁

-

Nur Selamoglu

RCAP_rcc00270 D5ALH9 Lipoprotein signal peptidase

y₁-NH₃158.0924

b₂-NH₃169.0608

y₁175.119

b₂186.0873

y₂-NH₃257.1608

b₃-NH₃266.1135

y₂274.1874

b₃283.1401

b₄-NH₃379.1976

y₃-NH₃385.2194

b₄396.2241

y₃402.2459

y₄-H₂O471.2674

y₄-NH₃472.2514

y₄489.278

b₅-NH₃507.2562

b₅524.2827

y₅-H₂O584.3515

y₅-NH₃585.3355

y₅602.362

b₆-NH₃654.3246

b₆671.3511

y₆-H₂O731.4199

y₆-NH₃732.4039

y₆749.4304

y₇-H₂O859.4785

y₇-NH₃860.4625

y₇877.489

y₈-NH₃973.5465

y₈990.5731

y₉-NH₃1070.599

y₉1087.626

020

4060

80100

120

00.5

11.5

22.5

33.5

[1e6]

200 400 600 800 1000 1200


- A Nb₂

P

y₉

b₃I

y₈

b₄Q

y₇

b₅F

y₆

b₆L

y₅

S

y₄

Q

y₃

V

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc00285 D5ALY0 Protein translocase subunit SecE

y₁-NH₃158.0924

a₂161.0743

y₁175.119

b₂189.0692

b₃288.1376

y₂289.1619

b₄-NH₃385.154

b₄402.1806

y₃402.2459

b₅-NH₃484.2224

b₅501.249

y₄533.2864

b₆-H₂O584.2861

b₆-NH₃585.2701

b₆602.2967

y₅-NH₃644.3185

y₅661.345

b₁₃²⁺685.3156

y₆-NH₃715.3556

y₆732.3821

b₈-NH₃785.3498

y₇-NH₃862.424

y₇879.4505

b₉-H₂O915.4063

b₁₈²⁺949.4428

y₈-NH₃977.4509

y₈994.4775

y₉-NH₃1048.488

y₉1065.515

b₁₀1070.476

y₁₉²⁺1088.02

y₁₀-H₂O1175.563

y₁₀-NH₃1176.547

y₁₀1193.573

b₁₁1199.518

b₁₂1256.54

y₁₁1294.621

b₁₃1369.624

y₁₂1407.705

y₁₄1577.81

y₁₆1843.912

y₁₈2045.99

y₂₀-NH₃2259.053

y₂₀2276.08

y₂₁2375.148

y₂₂2489.191

020

4060

80100

120

00.2

0.40.6

0.81

1.21.4

1.61.8

[1e5]

500 1000 1500 2000 2500

Raw File Scan Method Score m/zMT1131-plusCu-Set2-01 41976 FTMS; HCD 148.95 926.45

- M Gb₂

Vb₃

N

y₂₂

b₄V

y₂₁

b₅T

y₂₀

b₆E

y₁₉²⁺

A

y₁₈

M H

y₁₆

b₁₀Eb₁₁

G

y₁₄

b₁₂L

b₁₃L

y₁₂

T

y₁₁

Q

y₁₀

A

y₉

D

y₈

b₁₈²⁺

F

y₇

A

y₆

Q

y₅

M

y₄

I

y₃

N

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc00423 D5AMP9 Uncharacterized protein

y₄-NH₃411.24

y₄428.26

b₆525.27

y₅-NH₃597.31

y₅614.34

b₇624.34

y₆727.42

b₈752.39

y₇798.46

b₉809.42

y₈869.5

b₁₀922.5

y₉982.58

y₁₀1111.63

b₁₂1122.58

y₁₁-H₂O1164.65

y₁₁1182.66

b₁₃1235.66

b₁₄1306.7

y₁₃1352.77

b₁₅1377.74

y₁₄1480.83

b₁₆1490.82

y₁₅1579.9

b₁₇-H₂O1658.89

b₁₇-NH₃1659.87

b₁₇1676.9

y₁₆1678.96

b₁₈-H₂O1755.94

y₁₇1793.99

b₁₉-NH₃1813.95

b₁₉1830.97

b₂₀1930.04

y₁₉-NH₃1945.05

020

4060

80100

120

0200

400600

8001000

12001400

400 600 800 1000 1200 1400 1600 1800 2000

Raw File Scan Method Score m/zCTRL-minusCu-Biorep1-Run2 23785 ITMS; CID 154.46 1053.09

- A A P A D

y₁₇

V

y₁₆

b₆V

y₁₅

b₇Q

y₁₄

b₈G

y₁₃

b₉I

b₁₀A

y₁₁

E

y₁₀

b₁₂I

y₉

b₁₃A

y₈

b₁₄A

y₇

b₁₅L

y₆

b₁₆W

y₅

b₁₇P

y₄

Gb₁₉

Vb₂₀

R -

Nur Selamoglu

RCAP_rcc00522 D5AMZ8 Ferrous-iron efflux pump FieF

y₁-NH₃158.09

y₁175.12

a₂187.11

b₂-H₂O197.09

y₂-NH₃215.11

y₂232.14

b₃286.14

y₄-NH₃385.22

y₄402.25

b₄417.18

y₅-NH₃498.3

y₅515.33

b₅530.26

y₆628.41

b₆643.35

y₇-NH₃742.43

y₈-NH₃813.47

y₈830.49

b₉870.48

020

4060

80100

120

01

23

45

6

[1e5]

200 400 600 800 1000

Raw File Scan Method Score m/zPlusBC-Biorep1-Run1 10767 ITMS; CID 114.5 523.3

- D Va₂

A

y₈

b₃Mb₄

L

y₆

b₅L

y₅

b₆G

y₄

L G

y₂

b₉R

y₁

-

Nur Selamoglu

RCAP_rcc00633 D5ANP4 ABC transporter, permease protein

y₁132.1019

a₂200.1394

b₂-NH₃211.1077

y₂-H₂O215.139

b₂228.1343

y₂233.1496

b₃-NH₃308.1605

b₄-NH₃365.1819

y₄-H₂O425.2758

y₄443.2864

y₅-H₂O553.3708

y₅571.3814

y₆-H₂O682.4134

y₆700.424

b₇-NH₃719.3723

y₇-H₂O739.4349

y₇757.4454

y₈-H₂O836.4876

y₈854.4982

020

4060

80100

120

00.5

11.5

22.5

[1e5]

100 200 300 400 500 600 700 800 900


- L Nb₂

P

y₈

G

y₇

E

y₆

K

y₅

P

y₄

I T

y₂

L

y₁

-

Nur Selamoglu

RCAP_rcc00681 P17057 Protein CrtK

y₁-NH₃158.0924

a₂169.1335

y₁175.119

b₂197.1285

y₂246.1561

b₃268.1656

b₄325.187

y₃333.1881

y₄-NH₃430.2045

y₄447.231

b₅456.2275

y₅-NH₃501.2416

y₅518.2681

b₆-H₂O539.2646

b₆557.2752

y₆631.3522

y₇702.3893

b₈-H₂O737.3651

y₈865.4526

b₁₀883.4342

y₉922.4741

y₁₀-NH₃1006.495

y₁₀1023.522 y₁₁

1094.559

y₁₂1151.58

b₁₃1204.567

y₁₃-H₂O1234.617

y₁₃1252.628

y₁₄-H₂O1331.67

y₁₄1349.681

y₁₅-H₂O1432.718

y₁₅-NH₃1433.702

y₁₅1450.728

y₁₆1581.769

y₁₇1638.79

y₁₈1709.828

y₁₉-H₂O1788.87

y₁₉1806.88

020

4060

80100

120

00.2

0.40.6

0.81

1.21.4

1.61.8

[1e6]

200 400 600 800 1000 1200 1400 1600 1800

Raw File Scan Method Score m/zMT1131-minusCu-plusBC-01 26292 FTMS; HCD 150.12 953.98

- V P

y₁₉

b₂A

y₁₈

b₃G

y₁₇

b₄M

y₁₆

b₅T

y₁₅

b₆P

y₁₄

T

y₁₃

G

y₁₂

A

y₁₁

b₁₀T

y₁₀

G

y₉

Y

y₈

b₁₃A

y₇

L

y₆

A

y₅

N

y₄

S

y₃

A

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc00722 D5APB5 Lipoprotein, putative

y₁-NH₃130.0863

y₁147.1128

y₂-NH₃187.1077

a₂203.1026

y₂204.1343

b₂-H₂O213.087

b₂231.0975

y₃305.1819

b₃-H₂O310.1397

b₃328.1503

b₄-H₂O409.2082

y₄-H₂O415.23

y₄-NH₃416.214

b₄427.2187

y₄433.2405

b₅-H₂O480.2453

b₅498.2558

y₅532.3089

y₆-H₂O611.3511

y₆629.3617

b₇694.377

y₇-H₂O740.3937

y₇758.4043

b₈795.4247

y₈857.4727

y₉-H₂O940.5098

y₉-NH₃941.4938

y₉958.5204

y₁₀1057.589

y₁₁-H₂O1136.631

y₁₁-NH₃1137.615

y₁₁1154.642

y₁₂-H₂O1207.668

y₁₂1225.679

y₁₃1324.747

y₁₄1421.8

020

4060

80100

120

00.2

0.40.6

0.81

1.21.4

[1e6]

200 400 600 800 1000 1200 1400 1600


- T Eb₂

P

y₁₄

b₃V

y₁₃

b₄A

y₁₂

b₅P

y₁₁

V

y₁₀

b₇T

y₉

b₈V

y₈

E

y₇

P

y₆

V

y₅

Q

y₄

T

y₃

G

y₂

K

y₁

-

Nur Selamoglu

RCAP_rcc00908 D5AQ86 Lipoprotein, putative

y₃439.16

b₆441.25

y₄496.19

b₇512.28

b₁₃²⁺526.79

y₁₀²⁺534.72

y₅553.21

b₉640.34

y₆666.29

y₁₄²⁺677.78

y₇723.31

b₁₀753.43

b₁₈²⁺776.39

b₁₉²⁺849.91

b₁₁866.51 b₂₁²⁺

934.96

b₁₂937.55

y₁₀-NH₃1051.4

y₁₀1068.42

b₂₅²⁺1091.05

b₂₇²⁺1155.08

y₁₁1169.47

b₁₅-H₂O1176.64

b₁₅1194.65

b₂₉²⁺1247.14

y₁₃1297.53

b₁₆1309.67

y₁₄-NH₃1337.52

b₃₂²⁺1339.68

y₁₄1354.55

b₁₇1437.73

y₃₁²⁺1455.64

y₁₆1538.67

b₃₆²⁺1562.76

y₁₇-H₂O1591.7

y₃₄²⁺1604.25

y₁₈-NH₃1649.7

y₁₈1666.73

y₄₀²⁺1796.33

b₂₀1797.88

y₂₀1850.85

b₂₁-NH₃1851.89

b₂₁1868.92

b₂₃1996.98

020

4060

80100

120

050

100150

200250

300

400 600 800 1000 1200 1400 1600 1800 2000

Raw File Scan Method Score m/zPlusBC-Biorep2-Run1 34218 ITMS; CID 74.87 962.96

- A L A G

y₄₀²⁺

G Ab₆

Ab₇

G Ab₉

I

y₃₄²⁺

b₁₀I

b₁₁Ab₁₂

D

y₃₁²⁺

b₁₃²⁺A A

b₁₅Db₁₆

Qb₁₇

Nb₁₈²⁺

Mox

b₁₉²⁺Vb₂₀

Ab₂₁

G Ab₂₃

A

y₂₀

Lb₂₅²⁺

G

y₁₈

Ab₂₇²⁺

L

y₁₆

Ab₂₉²⁺

G

y₁₄

G

y₁₃

Ab₃₂²⁺

T

y₁₁

C

y₁₀

N Ab₃₆²⁺

G

y₇

I

y₆

G

y₅

G

y₄

P

y₃

C Y -

Nur Selamoglu

RCAP_rcc00910 D5AQ88 Uncharacterized protein

a₂145.0972

b₂-H₂O155.0815

y₁-NH₃158.0924

b₂173.0921

y₁175.119

b₃-H₂O226.1186

b₃244.1292

b₄-H₂O297.1557

y₃417.2456

y₆676.326

y₇773.3788

y₈-NH₃853.405

y₈870.4316

y₉941.4687

y₁₀1012.506

020

4060

80100

120

00.2

0.40.6

0.81

1.21.4

[1e5]

200 400 600 800 1000 1200 1400


- A Tb₂

A

y₁₀

b₃A

y₉

P

y₈

P

y₇

S

y₆

G D I

y₃

E R

y₁

-

Nur Selamoglu

RCAP_rcc01014 D5AQX1 Conserved domain protein

y₁-NH₃158.0924

y₁175.119

a₂201.1234

b₂229.1183

y₂-NH₃286.151

b₃300.1554

y₂303.1775

b₄-H₂O369.1769

y₄417.2205

y₅504.2525

y₆617.3365

y₇704.3686

y₈-H₂O757.3951

y₈775.4057

020

4060

80100

120

01

23

45

6

[1e4]

100 200 300 400 500 600 700 800


- D Ib₂

A

y₈

b₃S

y₇

L

y₆

S

y₅

G

y₄

G Q

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc01045 D5AQZ9 Iron siderophore/cobalamin ABC transporter, ATP-binding protein

y₁-NH₃158.0924

y₁175.119

b₂-NH₃253.1659

b₂270.1925

y₂288.203

y₃359.2401

b₄424.2667

y₄430.2772

y₅531.3249

b₅553.3093

y₁₁²⁺609.304

b₆650.362

y₆660.3675

b₇721.3991

y₇761.4152

y₈-H₂O872.4472

y₈890.4578

b₉-H₂O929.4839

b₉947.4945

y₉-H₂O973.4949

y₉991.5055

b₁₀-NH₃1031.516

y₁₀-H₂O1102.537

y₁₀-NH₃1103.522

y₁₀1120.548

b₁₁1177.585

y₁₁-H₂O1199.59

y₁₁1217.601

020

4060

80100

120

00.2

0.40.6

0.81

[1e5]

200 400 600 800 1000 1200 1400 1600 1800

Raw File Scan Method Score m/zMT1131-minusCu-Set2-01 18462 FTMS; HCD 96.14 647

- L Rb₂

P Gb₄

Eb₅

Pb₆

Ab₇

P

y₁₁

E

y₁₀

b₉T

y₉

E

y₈

b₁₁T

y₇

E

y₆

T

y₅

A

y₄

A

y₃

L

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc01054 D5AR08 Gas vesicle structural protein

b₂115.0502

y₁-NH₃158.0924

y₁175.119

b₃229.0931

y₂288.203

b₄342.1772

y₃389.2507

b₅-NH₃396.1878

b₅413.2143

b₆-NH₃524.2463

b₆541.2729

y₄552.314

b₇-NH₃637.3304

y₅653.3617

b₈-H₂O751.3733

y₇-H₂O820.4312

y₇838.4417

y₈953.4687

y₉1066.553

y₁₀1181.58

y₁₁1294.664

y₁₂-NH₃1405.696

y₁₂1422.722

y₁₃1493.759

020

4060

80100

120

01

23

45

67

89

[1e4]

200 400 600 800 1000 1200 1400 1600 1800


- G Gb₂

Nb₃

Lb₄

A

y₁₃

b₅Q

y₁₂

b₆I

y₁₁

D

y₁₀

L

y₉

D

y₈

G

y₇

Q T

y₅

Y

y₄

T

y₃

L

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc01112 D5ARJ9 Uncharacterized protein

y₁-NH₃158.0924

y₁175.119

a₂201.1234

b₂-H₂O211.1077

b₂229.1183

b₃-H₂O339.1663

b₃357.1769

y₄-NH₃373.183

y₄390.2096

b₄-H₂O452.2504

b₄470.2609

y₅503.2936

y₆-H₂O582.3358

y₆-NH₃583.3198

y₆600.3464

y₇-H₂O681.4042

y₇699.4148

y₈812.4989

y₉-H₂O922.5469

y₉940.5574

020

4060

80100

120

00.2

0.40.6

0.81

1.21.4

1.61.8

[1e5]

200 400 600 800 1000 1200


- E Vb₂

Q

y₉

b₃L

y₈

b₄V

y₇

P

y₆

L

y₅

S

y₄

G A R

y₁

-

Nur Selamoglu

RCAP_rcc01219 D5AS85 Urease subunit beta

y₁175.119

b₂187.1077

b₃-H₂O270.1448

y₂303.1775

y₃440.2364

y₄527.2685

y₅640.3525

y₆-H₂O723.3896

y₆741.4002

y₇-NH₃852.4322

y₇869.4588

y₈984.4857

y₉1055.523

020

4060

80100

120

00.5

11.5

22.5

[1e4]

200 400 600 800 1000 1200


- V Sb₂

T A

y₉

D

y₈

Q

y₇

T

y₆

L

y₅

S

y₄

H

y₃

Q

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc01262 D5ASC8 Recombinase

y₁-NH₃158.0924

a₂159.0764

y₁175.119

b₂187.0713

y₂312.1779

b₃316.1139

b₄387.151 y₃

425.2619

y₄554.3045

y₅668.3474

y₆-H₂O778.3955

y₆-NH₃779.3795

y₆796.406

y₇-NH₃907.4381

y₇924.4646

y₈995.5017

y₉1066.539

020

4060

80100

120

00.5

11.5

22.5

3

[1e4]

200 400 600 800 1000


- D Ab₂

Eb₃

A

y₉

b₄A

y₈

Q

y₇

Q

y₆

N

y₅

E

y₄

L

y₃

H

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc01320 D5ASI0 Uncharacterized protein

a₂157.1335

y₁-NH₃158.0924

y₁175.119

b₂185.1285

b₃282.1812

y₆²⁺379.6826

b₄395.2653

y₄534.2671

y₉²⁺549.7882

y₅-H₂O603.2885

y₁₀²⁺606.3302

y₅621.2991

y₆-H₂O740.3474

y₆758.358

y₁₅²⁺867.4885

y₇-H₂O868.406

y₇-NH₃869.39

y₇886.4166

y₈999.5007

y₉-NH₃1081.543

y₉1098.569

y₁₀-NH₃1194.627

y₁₀1211.653

y₁₁1339.712

y₁₂-NH₃1419.738

y₁₂1436.764

y₁₃-H₂O1489.791

y₁₃1507.802

y₁₄1620.886

020

4060

80100

120

00.2

0.40.6

0.81

1.2

[1e6]

200 400 600 800 1000 1200 1400 1600

Raw File Scan Method Score m/zMT1131-plusCu-01_150512102711 44969 FTMS; HCD 73.17 672.72

- I Ab₂

Pb₃

L

y₁₅²⁺

b₄L

y₁₄

A

y₁₃

P

y₁₂

Q

y₁₁

I

y₁₀

V

y₉

I

y₈

Q

y₇

H

y₆

S

y₅

P

y₄

F D R

y₁

-

Nur Selamoglu

RCAP_rcc01348 D5ASK3 Exonuclease family protein

y₁-NH₃158.0924

y₁175.119

a₂187.1441

b₂-H₂O197.1285

b₂215.139

y₂-NH₃229.1295

y₂246.1561 y₃-NH₃

392.1928

y₃409.2194

y₄480.2565

y₆608.3151

y₇-NH₃648.31

y₇665.3365

y₈-NH₃776.3686

y₈793.3951

y₉894.4428

020

4060

80100

120

01

23

45

[1e4]

200 400 600 800

Raw File Scan Method Score m/zMT1131-minusCu-01 6607 FTMS; HCD 84.57 504.27

- L T

y₉

b₂Q

y₈

G

y₇

G

y₆

A A

y₄

Y

y₃

A

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc01467 D5ATA5 Glycine cleavage system transcriptional activator

y₂246.16

b₃258.14

y₃361.18

y₄-H₂O458.2

y₄476.21

b₅-H₂O532.24

b₅550.25

y₅623.28

y₆-H₂O676.3

y₆-NH₃677.29

b₆-H₂O692.27

y₆694.32

b₆710.28 y₇-NH₃

791.33

y₇808.36

b₈-H₂O896.36

b₈914.37

y₈955.43

b₉-NH₃1011.39

y₉1012.45

b₉1028.41

b₁₀-H₂O1081.44

b₁₀-NH₃1082.42

b₁₀1099.45

y₁₀-NH₃1155.45

y₁₀1172.48

b₁₁-NH₃1229.49

b₁₁1246.52

y₁₁-H₂O1283.51

y₁₁-NH₃1284.49

y₁₁1301.52

b₁₂1361.55

y₁₂1464.58

b₁₃1476.57

b₁₄-NH₃1530.58

y₁₃1535.62

b₁₄1547.61

y₁₄-NH₃1605.63

y₁₄1622.65

020

4060

80100

120

01

23

45

[1e4]

200 400 600 800 1000 1200 1400 1600

Raw File Scan Method Score m/zPlusCu-Biorep2-Run1 13938 ITMS; CID 220.37 861.87

- V S

y₁₄

A

y₁₃

b₃Y

y₁₂

E

y₁₁

b₅C

y₁₀

b₆G

y₉

F

y₈

b₈N

y₇

b₉A

y₆

b₁₀F

y₅

b₁₁D

y₄

b₁₂D

y₃

b₁₃A

y₂

b₁₄R -

Nur Selamoglu

RCAP_rcc01517 D5ATH3 NADH-quinone oxidoreductase subunit A

y₁175.119

b₄-H₂O271.1037

y₂272.1717

y₅553.3457

y₆640.3777

y₇753.4618

y₉1029.609

y₁₁1199.715

y₁₂1314.742

y₁₃1385.779

020

4060

80100

120

00.2

0.40.6

0.81

1.21.4

1.6

[1e4]

200 400 600 800 1000 1200 1400


- S G G S L Q A

y₁₃

D

y₁₂

A

y₁₁

V Y

y₉

L I

y₇

S

y₆

P

y₅

A L P

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc01528 D5ATI4 Uncharacterized protein

y₁-NH₃158.0924

a₂173.1285

y₁175.119

b₂-H₂O183.1128

b₂201.1234

y₂288.203

b₃348.1918

y₃389.2507

b₄-H₂O445.2082

b₄463.2187

y₄552.314

b₅560.2715

y₅665.3981

b₆-H₂O671.3035

b₆689.3141

y₆778.4822

b₇-H₂O784.3876

b₇802.3981

y₇835.5036

b₈899.4509

y₈948.5877

y₉1063.615

b₁₀1112.562

b₁₁-NH₃1208.62

y₁₀1210.683

y₁₁1323.767

y₁₂1437.81

y₁₃1536.878

y₁₄-NH₃1616.905

y₁₄1633.931

y₁₅1747.015

y₁₇1973.111

020

4060

80100

120

00.5

11.5

22.5

[1e6]

500 1000 1500 2000 2500


- T Vb₂

Fb₃

Db₄

P

y₁₇

b₅Eb₆

I

y₁₅

b₇P

y₁₄

b₈V

y₁₃

N

y₁₂

b₁₀I

y₁₁

F

y₁₀

D

y₉

L

y₈

G

y₇

L

y₆

I

y₅

Y

y₄

T

y₃

I

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc01601 D5ATQ7 FeS assembly SUF system protein

y₁-NH₃158.0924

a₂173.0921

y₁175.119

b₂-H₂O183.0764

b₂201.087

y₂-NH₃257.1608

b₃-H₂O298.1034

y₃-NH₃314.1823

b₃316.1139

y₃331.2088

b₄-H₂O369.1405

y₄-NH₃443.2249

y₄460.2514

y₅531.2885

y₆-H₂O600.31

y₆618.3206

y₇-H₂O731.3505

y₇749.361

y₈820.3982

y₉935.4251

020

4060

80100

120

00.2

0.40.6

0.81

1.21.4

[1e5]

200 400 600 800


- E Ab₂

D

y₉

b₃A

y₈

M

y₇

S

y₆

A

y₅

E

y₄

G

y₃

V R

y₁

-

Nur Selamoglu

RCAP_rcc01627 D5ATT3 Isoprenyl transferase

y₁-NH₃158.0924

y₁175.119

a₂189.087

b₂217.0819

b₃-H₂O296.1241

y₂303.2139

b₃314.1347

b₄-H₂O367.1612

b₄385.1718

y₃416.298

y₄529.382

y₅616.4141

b₆-H₂O627.2773

b₆645.2879

b₇-H₂O728.325

y₆729.4981

b₇746.3355

y₇844.5251

y₈915.5622

y₉1002.594

y₁₀-H₂O1085.631

y₁₀-NH₃1086.615

y₁₀1103.642

y₁₁-H₂O1248.695

y₁₁-NH₃1249.679

y₁₁1266.705

y₁₂-H₂O1345.747

y₁₂1363.758

y₁₃1434.795

y₁₄-H₂O1513.837

y₁₄1531.848

y₁₅-H₂O1642.88

020

4060

80100

120

01

23

45

67

8

[1e6]

200 400 600 800 1000 1200 1400 1600

Raw File Scan Method Score m/zMT1131-plusCu-03 26579 FTMS; HCD 137.6 874.47

- S Eb₂

P

y₁₄

b₃A

y₁₃

b₄P

y₁₂

Y

y₁₁

b₆T

y₁₀

b₇S

y₉

A

y₈

D

y₇

L

y₆

S

y₅

L

y₄

I

y₃

K

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc01634 D5ATU0 3-hydroxyacyl-[acyl-carrier-protein] dehydratase FabZ

y₁-NH₃130.0863

y₁147.1128

a₂173.1285

b₂-H₂O183.1128

b₂201.1234

y₂-H₂O230.1499

y₂248.1605

b₃-H₂O312.1554

b₃330.166

y₃-H₂O359.1925

y₃377.2031

y₄-H₂O416.214

y₄434.2245

b₄-H₂O441.198

b₄459.2086

b₅-H₂O512.2351

y₅-H₂O529.298

b₅530.2457

y₅547.3086

y₆-H₂O644.325

y₆-NH₃645.309

y₆662.3355

y₇-H₂O807.3883

y₇825.3989

y₈-H₂O878.4254

y₈896.436

y₉967.4731

y₁₀-H₂O1078.505

y₁₀1096.516

y₁₁-H₂O1207.548

y₁₁1225.558

020

4060

80100

120

00.5

11.5

22.5

33.5

4

[1e5]

200 400 600 800 1000 1200 1400 1600


- S Lb₂

E

y₁₁

b₃E

y₁₀

b₄A

y₉

b₅A

y₈

Y

y₇

D

y₆

L

y₅

G

y₄

E

y₃

T

y₂

K

y₁

-

Nur Selamoglu

RCAP_rcc01894 D5AUK0 Polyamine ABC transporter, permease protein PotI-2

a₂175.09

b₂203.08

b₃274.12

b₄345.16

b₅416.2

y₃417.25

y₄516.31

y₅-NH₃600.34

b₇601.28

b₈-NH₃685.3

y₆745.42

y₇-H₂O784.43

y₇-NH₃785.42

y₇802.44

y₈-H₂O855.47

y₈-NH₃856.45

y₈873.48

b₁₁-H₂O1025.51

b₁₁-NH₃1026.49

b₁₁1043.52

020

4060

80100

120

01000

20003000

40005000

60007000

80009000

200 400 600 800 1000 1200

Raw File Scan Method Score m/zCTRL-minusCu-Biorep1-Run3 4792 ITMS; CID 83.65 609.82

- A Mb₂

Ab₃

Ab₄

A

y₈

b₅G

y₇

Q

y₆

b₇T V

y₄

E

y₃

Ib₁₁

R -

Nur Selamoglu

RCAP_rcc01933 D5AUN8 Uncharacterized protein

y₁-NH₃158.0924

a₂173.1285

y₁175.119

b₂-H₂O183.1128

b₂201.1234

y₂-NH₃287.135

y₂304.1615

y₃-H₂O415.1936

y₃433.2041

y₄504.2413

y₅591.2733

y₆-H₂O701.3213

y₆-NH₃702.3053

y₆719.3319 y₇

820.3795

y₈-NH₃890.385

y₈907.4116

020

4060

80100

120

01

23

45

[1e4]

200 400 600 800 1000


- L S

y₈

b₂T

y₇

Q

y₆

S

y₅

A

y₄

E

y₃

E

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc01938 D5AUP3 Membrane protein, putative

y₁-NH₃158.0924

a₂173.0921

y₁175.119

b₂201.087

b₃330.1296

y₂338.1823

b₄443.2136

y₄-NH₃547.2511

y₄564.2776

b₆585.2879

y₅677.3617

y₆748.3988

y₇819.4359

y₈-NH₃873.4465

y₈890.473

y₉1003.557

y₁₀1132.6

y₁₁-NH₃1244.616

020

4060

80100

120

00.2

0.40.6

0.81

[1e5]

200 400 600 800 1000 1200 1400 1600


- A Eb₂

E

y₁₀

b₃L

y₉

b₄A

y₈

A

y₇

b₆A

y₆

L

y₅

P

y₄

E Y

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc02041 D5AV03 Cobalamin biosynthesis protein CbiG

y₁-NH₃158.0924

y₁175.119

a₂187.1441

b₂-H₂O197.1285

b₂215.139

y₂-NH₃286.151

b₃-H₂O298.1761

y₂303.1775

b₃316.1867

b₄-H₂O399.2238

y₃-H₂O414.2096

y₃-NH₃415.1936

y₃432.2201

y₄-NH₃514.262

b₅-H₂O528.2664

y₄531.2885

b₅546.277

y₅-NH₃627.3461

y₅644.3726

b₆-H₂O656.325

b₆674.3355

y₆-NH₃758.3865

b₇-H₂O769.409

y₆775.4131

b₇787.4196

y₇-NH₃871.4706

y₇888.4971

b₈-H₂O900.4495

y₈-H₂O998.5452

y₈-NH₃999.5292

y₈1016.556

y₉-H₂O1127.588

y₉-NH₃1128.572

y₉1145.598 y₁₀

1246.646

y₁₁-H₂O1329.683

y₁₁-NH₃1330.667

y₁₁1347.694

020

4060

80100

120

00.5

11.5

22.5

33.5

44.5

[1e5]

200 400 600 800 1000 1200


- T Lb₂

T

y₁₁

b₃T

y₁₀

E

y₉

b₅Q

y₈

b₆L

y₇

b₇M

y₆

L

y₅

V

y₄

E

y₃

Q

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc02152 D5AVA0 TM2 domain protein

y₁-NH₃158.0924

y₁175.119

a₂191.1179

b₂219.1128

y₂-NH₃287.135

y₂304.1615

y₃403.23

y₄-NH₃483.2562

y₄500.2827

y₅-NH₃540.2776

y₅557.3042

y₆-NH₃641.3253

y₆658.3519

y₇-H₂O769.3839

y₇787.3945

020

4060

80100

120

00.2

0.40.6

0.81

1.2

[1e5]

100 200 300 400 500 600 700 800 900


- A Fb₂

E

y₇

T

y₆

G

y₅

P

y₄

V

y₃

E

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc02158 D5AVA6 Flagellar motor switch protein FliG

a₂115.0866

y₁-NH₃130.0863

b₂143.0815

y₁147.1128

b₃-H₂O226.1186

b₃244.1292

y₂-H₂O258.1448

y₂276.1554

b₄-H₂O341.1456

y₃347.1925

b₄359.1561

y₄418.2296

y₅517.298

y₆588.3352

b₈-H₂O639.3097

y₇645.3566

y₈716.3937

y₉831.4207

y₁₀932.4684

020

4060

80100

120

00.5

11.5

22.5

3

[1e4]

200 400 600 800 1000


- A Ab₂

T

y₁₀

b₃D

y₉

b₄A

y₈

G

y₇

A

y₆

V

y₅

A

y₄

A

y₃

E

y₂

K

y₁

-

Nur Selamoglu

RCAP_rcc02209 D5AKU9 Propanediol utilization protein PduA-2

y₁175.119

a₂199.1805

b₂227.1754

b₃342.2023

b₅584.329

y₁₂²⁺653.8468

y₆673.3628

y₁₆²⁺845.9472

y₁₉²⁺1015.045

y₂₀²⁺1079.566

y₁₃1419.77

y₁₈1915.998

y₂₀2158.125

y₂₁2271.209

y₂₂2386.236

020

4060

80100

120

00.2

0.40.6

0.81

1.21.4

[1e5]

500 1000 1500 2000


- I Ib₂

D

y₂₂

b₃I

y₂₁

E

y₂₀

b₅L

y₁₉²⁺

P

y₁₈

Q G

y₁₆²⁺

V D L

y₁₃

P

y₁₂²⁺

R P A P D

A

y₆

Q A L D R

y₁

-

Nur Selamoglu

RCAP_rcc02238 D5AKX8 Sulfoacetaldehyde acetyltransferase

a₂157.1335

y₁-NH₃158.0924

y₁175.119

b₂185.1285

b₃313.187

b₄442.2296

y₅459.2674

b₅541.298

y₆558.3358

y₇-NH₃688.3777

y₇705.4042

b₇785.4192

y₈802.457 y₉

949.5254

y₁₀1048.594 y₁₁

1177.636

y₁₂-NH₃1288.668

y₁₂1305.695

020

4060

80100

120

01

23

45

6

[1e5]

200 400 600 800 1000 1200 1400 1600 1800

Raw File Scan Method Score m/zMT1131-plusCu-03 44457 FTMS; HCD 60 745.42

- A Lb₂

Q

y₁₂

b₃E

y₁₁

b₄V

y₁₀

b₅F

y₉

P

y₈

b₇F

y₇

V

y₆

A

y₅

A A A R

y₁

-

Nur Selamoglu

RCAP_rcc02270 D5AL10 Transcriptional regulator, LuxR family

y₁-NH₃158.0924

y₁175.119

a₂187.1077

b₂215.1026

b₃312.1554

y₃-NH₃371.2037

y₃388.2303

y₄501.3144

y₅-NH₃571.3198

y₅588.3464

y₆-NH₃628.3413

y₆645.3678

y₇-H₂O698.3944

y₇716.405

y₈787.4421

y₉-H₂O866.4843

y₉-NH₃867.4683

y₉884.4948

020

4060

80100

120

00.2

0.40.6

0.81

1.2

[1e6]

200 400 600 800 1000 1200


- V Db₂

P

y₉

b₃A

y₈

A

y₇

G

y₆

S

y₅

L

y₄

N

y₃

V R

y₁

-

Nur Selamoglu

RCAP_rcc02321 D5ALJ0 Uncharacterized protein

y₁-NH₃158.0924

y₁175.119

b₂-NH₃227.1139

b₂244.1404

y₄625.3093

b₆669.3427

b₇-NH₃767.3431

b₁₄²⁺784.3478

b₇784.3696

y₆854.3791 y₈

1054.459y₁₀-NH₃

1205.522

y₁₀1222.549

b₁₃1381.609

y₁₅1762.778

020

4060

80100

120

00.5

11.5

22.5

33.5

44.5

5

[1e5]

200 400 600 800 1000 1200 1400 1600 1800 2000


- S Rb₂

P

y₁₅

G R Db₆

Db₇

P

y₁₀

A A

y₈

E N

y₆

Db₁₃

W

y₄

b₁₄²⁺Y T R

y₁

-

Nur Selamoglu

RCAP_rcc02344 D5ALL3 Uncharacterized protein

a₂131.0815

b₂-H₂O141.0659

y₁-NH₃158.0924

b₂159.0764

y₁175.119

b₃-H₂O212.103

b₃230.1135

b₄-H₂O311.1714

y₂-NH₃321.1557

b₄329.1819

y₂338.1823

y₃-NH₃408.1878

b₅-H₂O412.2191

y₃425.2143 y₄-NH₃

479.2249

y₄496.2514

y₅-H₂O575.2936

y₅-NH₃576.2776

y₅593.3042

y₆-H₂O662.3257

y₆-NH₃663.3097

y₆680.3362

y₇-H₂O763.3733

y₇781.3839

y₈-NH₃863.4258

y₈880.4523

y₉951.4894

y₁₀1008.511

020

4060

80100

120

00.5

11.5

22.5

33.5

[1e5]

200 400 600 800 1000 1200


- T G

y₁₀

b₂A

y₉

b₃V

y₈

b₄T

y₇

S

y₆

P

y₅

A

y₄

S

y₃

Y

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc02403 D5ALS2 Peptidoglycan-binding domain 1 protein

a₂115.0866

y₁-NH₃130.0863

b₂143.0815

y₁147.1128

b₃214.1186

y₂218.1499

b₄285.1557

y₃305.1819

b₅382.2085

b₇578.3297

y₆-H₂O627.3097

y₆645.3202

y₇-H₂O756.3523

y₇774.3628

y₈845.3999

y₉942.4527

y₁₀1041.521

y₁₁1138.574

y₁₂1235.627

020

4060

80100

120

00.5

11.5

22.5

[1e5]

200 400 600 800 1000 1200


- A Ab₂

Ab₃

Ab₄

P

y₁₂

b₅P

y₁₁

V

y₁₀

b₇P

y₉

A

y₈

E

y₇

P

y₆

Q D S

y₃

A

y₂

K

y₁

-

Nur Selamoglu

RCAP_rcc02415 D5ALT4 Uncharacterized protein

y₁-NH₃158.0924

y₁175.119

y₂-NH₃215.1139

y₂232.1404

a₂233.1648

b₂261.1598

y₃345.2245 b₃

375.2027y₄

474.2671 y₆716.3937

y₇-H₂O769.4203

y₇787.4308

y₈-NH₃867.4571

y₈884.4836

y₁₆²⁺904.4705

y₁₇²⁺978.0047 y₉-H₂O

1003.532

y₉-NH₃1004.516

y₉1021.543

y₁₀1134.627

y₁₁1247.711

y₁₂1344.763

y₁₃-H₂O1455.795

y₁₃1473.806

y₁₄1530.827

y₁₅1693.891

y₁₆-NH₃1790.907

020

4060

80100

120

00.2

0.40.6

0.81

1.2

[1e6]

200 400 600 800 1000 1200 1400 1600 1800


- L F

y₁₇²⁺

b₂N

y₁₆²⁺

b₃Y

y₁₅

G

y₁₄

E

y₁₃

P

y₁₂

L

y₁₁

L

y₁₀

H

y₉

P

y₈

A

y₇

L

y₆

E E

y₄

L

y₃

G

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc02439 D5ALV8 Uncharacterized protein

y₁-NH₃158.0924

a₂159.1128

b₂-H₂O169.0972

y₁175.119

b₂187.1077

y₂274.1874

b₃302.1347

b₄-H₂O385.1718

y₃-NH₃385.2194

y₃402.2459

b₄403.1823

y₄-NH₃484.2878

b₅-H₂O499.2147

b₅-NH₃500.1987

y₄501.3144

y₅602.362

b₆-H₂O646.2831

b₆664.2937

y₆-H₂O698.3944

y₆-NH₃699.3784

y₆716.405

y₇-H₂O845.4628

y₇-NH₃846.4468

y₇863.4734

y₈-NH₃960.4898

y₈977.5163

y₉-NH₃1061.537

y₉1078.564

y₁₀-H₂O1175.58

y₁₀-NH₃1176.564

y₁₀1193.591

020

4060

80100

120

00.2

0.40.6

0.81

1.21.4

[1e6]

200 400 600 800 1000 1200


- S Vb₂

D

y₁₀

b₃T

y₉

b₄N

y₈

F

y₇

b₆N

y₆

T

y₅

V

y₄

Q

y₃

V

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc02462 D5AMB6 Membrane protein, putative

y₁-NH₃130.0863

y₁147.1128

a₂201.1234

b₂-H₂O211.1077

b₂229.1183

y₂248.1605

b₃-H₂O324.1918

b₃342.2023

y₃361.2445

b₄-H₂O395.2289

b₄413.2395 b₅-H₂O

466.266

y₄476.2715

y₅662.3508

y₆719.3723

y₇-H₂O798.4145

y₇816.425

y₈887.4621

y₉-H₂O940.4887

y₉958.4993

y₁₀1029.536

y₁₁1142.62

020

4060

80100

120

00.2

0.40.6

0.81

1.2

[1e5]

200 400 600 800 1000 1200 1400


- E Vb₂

L

y₁₁

b₃A

y₁₀

b₄A

y₉

A

y₈

P

y₇

G

y₆

W

y₅

D

y₄

L

y₃

T

y₂

K

y₁

-

Nur Selamoglu

RCAP_rcc02485 D5AMD9 Methyltransferase, type 12 family

b₂277.15

y₃-NH₃358.21

y₃375.24

y₄-H₂O454.28

y₄-NH₃455.26

y₄472.29

y₅571.36

b₅592.31

b₆-H₂O689.33

y₆700.4

b₇-H₂O788.4

b₇806.41

y₇829.44

b₈877.45

y₈916.47

b₉-H₂O946.47

b₉964.48

y₉987.51

b₁₀-H₂O1075.51

y₁₀1086.58

b₁₀1093.52

y₁₁1201.61

b₁₁1222.56

y₁₂1272.64

b₁₂-H₂O1303.62

b₁₂1321.63

y₁₃1419.71

y₁₄1516.76

b₁₄1531.77

b₁₅1618.80

2040

6080

100

120

00.5

11.5

22.5

3

[1e4]

400 600 800 1000 1200 1400 1600 1800

Raw File Scan Method Score m/zPlusCu-Biorep2-Run2 13179 ITMS; CID 164.48 897.46

- Y Lb₂

P

y₁₄

F

y₁₃

A

y₁₂

b₅D

y₁₁

V

y₁₀

b₇A

y₉

b₈S

y₈

b₉E

y₇

b₁₀E

y₆

b₁₁V

y₅

b₁₂P

y₄

L

y₃

b₁₄Sb₁₅

R -

Nur Selamoglu

RCAP_rcc02532 D5AMI6 Protein PucC-2

a₂127.0866

b₂155.0815

y₁-NH₃158.0924

y₁175.119

b₃-H₂O224.103

y₂-NH₃229.1295

b₃242.1135

y₂246.1561

b₄-H₂O281.1244

b₄299.135

y₃-NH₃342.2136

y₃359.2401

b₅-H₂O382.1721

b₅400.1827

y₄-NH₃489.282

y₄506.3085

y₅619.3926

y₆-NH₃699.4188

y₆716.4454

y₇817.4931

y₈-H₂O856.5039

y₈874.5145

y₉-H₂O943.536

y₉-NH₃944.52

y₉961.5465

y₁₀-H₂O1000.557

y₁₀1018.568

020

4060

80100

120

00.2

0.40.6

0.81

1.21.4

1.61.8

[1e5]

200 400 600 800 1000


- P G

y₁₀

b₂S

y₉

b₃G

y₈

b₄T

y₇

b₅P

y₆

L

y₅

F

y₄

L

y₃

A

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc02593 D5AN33 Uncharacterized protein

y₃-NH₃373.12

y₃390.14

y₄518.2

y₅617.27

y₆-NH₃701.29

y₆718.32

b₈-H₂O800.32

b₈818.33

y₈904.42

y₉991.45

b₁₁-H₂O1101.49

y₁₀1104.54

b₁₁1119.5

y₁₁-NH₃1188.56

y₁₁1205.58

b₁₂1206.53

y₁₂1292.62

b₁₃1293.56

b₁₄-H₂O1374.62

b₁₄1392.63

b₁₅1493.68

b₁₆-H₂O1574.74

b₁₆1592.75

y₁₅1607.76

b₁₇-H₂O1702.8

b₁₇-NH₃1703.78

b₁₇1720.81

b₁₈-H₂O1816.84

b₁₈-NH₃1817.82

b₁₈1834.85

020

4060

80100

120

0500

10001500

20002500

30003500

40004500

400 600 800 1000 1200 1400 1600 1800

Raw File Scan Method Score m/zPlusCu-Biorep2-Run3 11287 ITMS; CID 170 1055.98

- C E G G V S

y₁₅

D Lb₈

S

y₁₂

T

y₁₁

L

y₁₀

b₁₁S

y₉

b₁₂S

y₈

b₁₃Vb₁₄

T

y₆

b₁₅V

y₅

b₁₆Q

y₄

b₁₇N

y₃

b₁₈

P C -

Nur Selamoglu

RCAP_rcc02596 D5AN36 Lipoprotein, putative

b₂115.0502

y₁-NH₃158.0924

y₁175.119

b₃186.0873

y₂-NH₃321.1557

y₂338.1823

y₃-NH₃434.2398

y₃451.2663

y₄-NH₃571.2987

y₄588.3253

y₅-NH₃668.3515

y₅685.378

y₆-NH₃815.4199

y₆832.4464

020

4060

80100

120

01

23

45

67

[1e5]

200 400 600 800


- G Gb₂

Ab₃

L F

y₆

P

y₅

H

y₄

L

y₃

Y

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc02618 D5AN58 Uncharacterized protein

a₂127.0866

b₂155.0815

y₁-NH₃158.0924

y₁175.119

b₃252.1343

y₂-NH₃273.1193

y₄448.215

y₅519.2522

y₆-H₂O558.2631

y₆576.2736

y₇673.3264

y₈786.4104

y₉-H₂O865.4526

y₉883.4632

y₁₀-NH₃1022.538

y₁₀1039.564

y₁₁1168.607

y₁₂-NH₃1222.617

y₁₂1239.644

y₁₃1310.681

y₁₄1407.734

y₁₅-NH₃1447.729

y₁₅1464.755

020

4060

80100

120

00.2

0.40.6

0.81

1.21.4

[1e5]

200 400 600 800 1000 1200 1400


- P G

y₁₅

b₂P

y₁₄

b₃A

y₁₃

A

y₁₂

E

y₁₁

R

y₁₀

P

y₉

L

y₈

P

y₇

G

y₆

A

y₅

S

y₄

A D R

y₁

-

Nur Selamoglu

RCAP_rcc02748 D5ANX2 Uncharacterized protein

y₁-NH₃158.0924

y₁175.119

b₂-H₂O213.087

b₂231.0975

y₂232.1404

b₃-H₂O314.1347

b₃332.1452

y₄460.2514

b₅-H₂O484.2402

y₅588.31

y₆717.3526

y₇830.4367

y₈977.5051 y₉

1124.573

y₁₀1195.611

020

4060

80100

120

00.2

0.40.6

0.81

1.21.4

1.6

[1e4]

200 400 600 800 1000 1200 1400 1600


- T Eb₂

Tb₃

V A

y₁₀

F

y₉

F

y₈

L

y₇

E

y₆

Q

y₅

L

y₄

D G

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc02760 D5ANY4 TfoX N-terminal domain protein

y₁-NH₃158.0924

b₂-NH₃169.0608

y₁175.119

b₂186.0873

b₃-NH₃240.0979

b₃257.1244

y₂-NH₃286.151

y₂303.1775

b₄-H₂O340.1615

y₃-NH₃357.1881

y₃374.2146

y₄503.2572

y₅560.2787

y₆631.3158

y₇762.3563

y₈863.404

y₉-H₂O916.4305

y₉934.4411

020

4060

80100

120

00.5

11.5

22.5

33.5

4

[1e4]

200 400 600 800 1000


- G Qb₂

A

y₉

b₃T

y₈

M

y₇

A

y₆

G

y₅

E

y₄

A

y₃

Q

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc02775 D5ANZ9 Polar amino acid ABC transporter, permease protein HisQ

y₁-NH₃158.0924

a₂173.0921

y₁175.119

b₂-H₂O183.0764

b₂201.087

b₃272.1241

y₂289.1619

b₄-H₂O369.1405

b₄387.151

y₃388.2303

y₇²⁺435.7489

b₅458.1882

y₅-NH₃569.3042

y₅586.3307

b₁₂²⁺620.3018

y₆-NH₃725.4053

y₁₃²⁺728.8606

y₇-NH₃853.4639

y₈-NH₃924.501

y₈941.5275

y₉-NH₃1053.544

y₉1070.57

y₁₀1141.607

y₁₁1256.634

y₁₂-H₂O1309.661

y₁₂1327.671

020

4060

80100

120

01

23

45

67

89

[1e4]

200 400 600 800 1000 1200


- A E

y₁₃²⁺

b₂A

y₁₂

b₃D

y₁₁

b₄A

y₁₀

b₅E

y₉

A

y₈

Q

y₇²⁺

R P

y₅

T V

y₃

b₁₂²⁺N

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc02879 D5APN9 Monosacharide ABC transporter, permease protein

y₁-NH₃158.0924

y₁175.119

a₂201.1234

b₂229.1183

y₂-NH₃271.1765

y₂288.203

y₃-NH₃384.2605

y₃401.2871

y₄-NH₃512.3191

y₄529.3457

y₅642.4297

y₆729.4618

y₇-H₂O768.4726

y₇-NH₃769.4567

y₇786.4832

y₈-H₂O865.5254

y₈883.536

y₉998.56290

2040

6080

100

120

00.2

0.40.6

0.81

1.21.4

1.61.8

22.2

[1e5]

200 400 600 800 1000 1200


- L D

y₉

b₂P

y₈

G

y₇

S

y₆

L

y₅

Q

y₄

I

y₃

L

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc03010 D5AQE8 Uncharacterized protein

y₁-NH₃158.0924

y₁175.119

a₂193.0972 b₂

221.0921

y₂-NH₃229.1295

y₂246.1561

b₃335.135

y₃359.2401

b₄392.1565

y₄-NH₃470.2722

y₄487.2987

y₅-H₂O598.3307

y₅-NH₃599.3148

y₅616.3413

y₆-H₂O695.3835

y₆-NH₃696.3675

y₆713.3941

y₇-H₂O752.405

y₇-NH₃753.389

y₇770.4155

y₈-NH₃867.4319

y₈884.4585

y₉-NH₃924.4534

y₉941.4799

020

4060

80100

120

00.2

0.40.6

0.81

1.21.4

1.61.8

22.2

[1e5]

200 400 600 800 1000


- Y G

y₉

b₂N

y₈

b₃G

y₇

b₄P

y₆

E

y₅

Q

y₄

I

y₃

A

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc03112 D5AQP7 Uncharacterized protein

y₁116.0706

a₂145.0972

b₂-H₂O155.0815

b₂173.0921

y₂272.1717

y₃-H₂O383.2037

y₃-NH₃384.1878

y₆²⁺384.2298

y₃401.2143

y₄-H₂O480.2565

y₄-NH₃481.2405

y₄498.2671

y₅-NH₃637.3416

y₁₁²⁺664.8879

y₆-NH₃750.4257

y₇-NH₃863.5098

y₇880.5363

y₈-NH₃976.5938

y₈993.6204 y₉

1156.684

y₁₀1257.731

020

4060

80100

120

00.2

0.40.6

0.81

1.21.4

1.61.8

2

[1e5]

200 400 600 800 1000 1200


- T A

y₁₁²⁺

b₂T

y₁₀

Y

y₉

I

y₈

L

y₇

L

y₆²⁺

R P

y₄

E

y₃

R

y₂

P

y₁

-

Nur Selamoglu

RCAP_rcc03185 D5ARA4 Membrane protein, putative

y₁-NH₃130.0863

y₁147.1128

y₂-H₂O216.1343

a₂217.1335

y₂234.1448

b₂245.1285

b₃316.1656

y₃381.2132

b₄387.2027

y₄494.2973

b₆-H₂O607.2987

y₅607.3814

b₆625.3093

y₆694.4134

b₇-H₂O720.3828

b₇738.3933

y₇807.4975

b₈835.4461 y₈

864.5189

b₉-H₂O904.4676

b₉922.4781

y₉-H₂O943.5611

y₉961.5717

b₁₀-H₂O1051.536

b₁₀1069.547

y₁₀1074.656

b₁₁-H₂O1108.557

b₁₁1126.568

b₁₂-H₂O1237.6

b₁₂1255.611

b₁₃1368.695

y₁₅1591.8730

2040

6080

100

120

00.2

0.40.6

0.81

1.21.4

1.6

[1e6]

200 400 600 800 1000 1200 1400 1600


- F Pb₂

Ab₃

Ab₄

T Hb₆

Lb₇

P

y₁₅

b₈Sb₉

Fb₁₀

Gb₁₁

Eb₁₂

I

y₁₀

b₁₃P

y₉

G

y₈

L

y₇

S

y₆

L

y₅

I

y₄

F

y₃

S

y₂

K

y₁

-

Nur Selamoglu

RCAP_rcc03234 D5ARF3 Monosacharide ABC transporter, permease protein

y₁-NH₃158.0924

y₁175.119

a₂199.1805

b₂227.1754

y₂288.203

b₃298.2125

y₃359.2401

y₄-NH₃457.2405

y₄474.2671

y₉²⁺500.2375

y₅-NH₃528.2776

y₅545.3042

y₁₂²⁺671.8064

y₆676.3447

y₁₄²⁺771.8645

y₇-NH₃796.377

y₇813.4036

y₈928.4305

y₉999.4676

y₁₀-NH₃1097.468

y₁₀1114.495

y₁₁-H₂O1211.511

y₁₁1229.522

y₁₂-H₂O1324.595

y₁₂1342.606

y₁₃1455.69

y₁₄-H₂O1524.711

y₁₄1542.722

y₁₅-H₂O1671.78

y₁₅1689.79

y₁₆1790.838

y₁₇-NH₃1886.895

y₁₇1903.922

y₁₈-H₂O1982.964

y₁₈2000.975

y₁₉2072.012

020

4060

80100

120

00.2

0.40.6

0.81

1.21.4

1.61.8

22.2

[1e5]

200 400 600 800 1000 1200 1400 1600 1800 2000


- L Lb₂

A

y₁₉

b₃P

y₁₈

L

y₁₇

T

y₁₆

F

y₁₅

S

y₁₄

L

y₁₃

L

y₁₂

D

y₁₁

D

y₁₀

A

y₉

D

y₈

H

y₇

M

y₆

A

y₅

D

y₄

A

y₃

L

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc03250 D5ARG9 Energy-coupling factor transporter transmembrane protein BioN

y₁-NH₃158.0924

b₂-NH₃169.0608

y₁175.119

b₂186.0873

b₃-NH₃266.1135

b₃283.1401

y₂-NH₃321.1557

y₂338.1823

b₄-H₂O380.1565

b₄-NH₃381.1405

y₃-NH₃449.2143

y₃466.2409

b₅-H₂O479.2249

b₆-H₂O536.2463

y₄-NH₃546.2671

y₄563.2936

y₅-NH₃603.2885

y₅620.3151

y₆-NH₃702.357

y₆719.3835

y₇-NH₃817.3839

y₇834.4104

y₈931.4632

y₉988.4847

020

4060

80100

120

00.2

0.40.6

0.81

1.21.4

1.61.8

22.2

[1e5]

200 400 600 800 1000


- Q G

y₉

b₂P

y₈

b₃D

y₇

V

y₆

G

y₅

P

y₄

Q

y₃

Y

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc03257 D5ARV9 Peptide methionine sulfoxide reductase MsrA

a₂158.0924

b₂-NH₃169.0608

b₂186.0873

y₂-H₂O225.0982

y₂243.1088

b₃-NH₃282.1448

b₃299.1714

y₃-H₂O388.1615

b₄-H₂O396.1878

b₄-NH₃397.1718

y₃406.1721 b₄

414.1983

y₄-H₂O459.1987

b₅-NH₃510.2558

y₅564.2413

b₆-NH₃611.3035

y₇-H₂O732.2947

y₇750.3053

b₈-NH₃849.4101

y₈887.3642

b₉-NH₃906.4316

y₉-H₂O970.4013

y₉988.4119

b₁₀-H₂O1034.49

b₁₀-NH₃1035.474

y₁₀-H₂O1071.449

y₁₀1089.46

b₁₁-H₂O1121.522

b₁₂-NH₃1193.543

y₁₁1202.544

b₁₂1210.57

y₁₂1317.571

b₁₃-H₂O1355.623

b₁₃1373.633

020

4060

80100

120

00.2

0.40.6

0.81

[1e5]

200 400 600 800 1000 1200 1400 1600


- Q Gb₂

Lb₃

D

y₁₂

b₄L

y₁₁

T

y₁₀

T

y₉

H

y₈

G

y₇

E S

y₅

Ab₁₂

Y

y₃

b₁₃H

y₂

S -

Nur Selamoglu

RCAP_rcc03386 D5ASL9 Ammonium transporter

y₁133.0608

y₂-H₂O244.0928

a₂272.1757

b₂300.1707

y₃-NH₃359.1197

y₃376.1463 b₃

415.1976

b₄571.2987

b₅-H₂O668.3151

b₅-NH₃669.2991

b₅686.3257

y₆-NH₃743.3319

y₆760.3584

b₆799.4097

y₇-H₂O857.3748

y₇-NH₃858.3588

y₇875.3854

b₇-NH₃896.4261

b₇913.4526

b₈1042.495

y₈-H₂O1043.454

y₈-NH₃1044.438

y₈1061.465

020

4060

80100

120

00.2

0.40.6

0.81

[1e5]

200 400 600 800 1000


- I W

y₈

b₂D

y₇

b₃R

y₆

b₄Db₅

Ib₆

N

y₃

b₇Eb₈

N

y₁

-

Nur Selamoglu

RCAP_rcc03402 D5ASN5 Membrane protein, putative

b₂145.0608

y₁-NH₃158.0924

y₁175.119

b₃274.1034

y₂288.203

b₄403.146

y₃403.23 y₄

474.2671

b₅531.2045

y₅-H₂O585.2991

y₅603.3097

b₆630.2729

y₆716.3937

y₇-NH₃796.4199

y₇813.4465

b₈824.3533

y₈-H₂O923.4945

y₈-NH₃924.4785

y₈941.5051

b₉971.4217

y₉-NH₃981.5

y₉998.5265

y₁₀-NH₃1052.537

b₁₀-H₂O1054.459

y₁₀1069.564

b₁₀1072.469

b₁₁-H₂O1167.543

y₁₁1182.648

b₁₁1185.553

b₁₂1298.638

020

4060

80100

120

00.5

11.5

22.5

33.5

[1e5]

200 400 600 800 1000 1200 1400


- S Gb₂

Eb₃

Eb₄

Qb₅

Vb₆

G Hb₈

Fb₉

Tb₁₀

Lb₁₁

I

y₁₁

b₁₂A

y₁₀

G

y₉

Q

y₈

P

y₇

L

y₆

E

y₅

A

y₄

D

y₃

L

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcc03440 D5ASS3 Uncharacterized protein

a₂101.0709

b₂129.0659

y₁-NH₃158.0924

y₁175.119

b₃242.1499

y₂-NH₃286.151

y₂303.1775

y₃-NH₃343.1724

y₃360.199

y₄-NH₃414.2096

y₄431.2361

y₅-NH₃501.2416

y₅518.2681

y₆-NH₃600.31

y₆617.3365

y₇-NH₃687.342

y₇704.3686

y₈-H₂O787.4057

y₈805.4163

020

4060

80100

120

01

23

45

67

[1e4]

200 400 600 800 1000


- A Gb₂

Lb₃

T

y₈

S

y₇

V

y₆

S

y₅

A

y₄

G

y₃

Q

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcp00001 D5AVC4 Polar amino acid ABC transporter, permease protein

y₁175.119

a₂205.1005

b₂233.0954

y₂288.203

b₃346.1795

y₃389.2507

b₄-H₂O456.2275

b₄474.2381

y₄504.2776

b₁₀²⁺554.7582

b₁₁²⁺628.2924

b₁₂²⁺701.8266

y₆702.3781

y₇773.4152

y₈844.4523

b₈922.4451

y₉941.5051

y₁₀1012.542

b₉1037.472

b₁₀1108.509

y₁₁1127.569

y₁₂1240.653

b₁₁1255.578

b₁₂1402.646

b₁₃-NH₃1456.657

b₁₃1473.683

b₁₆1772.831

b₁₇1843.868

020

4060

80100

120

01

23

45

6

[1e5]

200 400 600 800 1000 1200 1400 1600 1800


- T Mb₂

Lb₃

Qb₄

P V D Hb₈

Db₉

Ab₁₀

Fb₁₁

Fb₁₂

Ab₁₃

A L

y₁₂

D

y₁₁

b₁₆A

y₁₀

b₁₇P

y₉

A

y₈

A

y₇

P

y₆

T D

y₄

T

y₃

L

y₂

R

y₁

-

Nur Selamoglu

RCAP_rcp00141 D5AVR1 Uncharacterized protein

S- 15

Figure S5. Total cellular metal contents of cells grown in Cu-sufficient control (-Cu), or + Cu (5

µM), or +BCS (5 mM) medium, determined by ICP-MS as described earlier (8, 9) (note the

logarithmic scale of y axis).


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