Characterization of Disulfide Linkages in Proteins by 193 nm Ultraviolet Photodissociation (UVPD) Mass Spectrometry. Supporting Information

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1 Characterization of Disulfide Linkages in Proteins by 193 nm Ultraviolet Photodissociation (UVPD) Mass Spectrometry M. Montana Quick, Christopher M. Crittenden, Jake A. Rosenberg, and Jennifer S. Brodbelt * Department of Chemistry, University of Texas, Austin, TX *Correspondence to: Dr. Jennifer S. Brodbelt Department of Chemistry University of Texas at Austin Austin, TX, USA Phone: (512) jbrodbelt@cm.utexas.edu Supporting Information Supporting Information: The following items are included: a more detailed experimental section, tables summarizing the theoretical disulfide-containing tryptic peptides from lysozyme and serotransferrin, ion distributions observed upon MS/MS of insulin, the sequences of lysozyme and serotransferrin, a chromatogram of serotransferrin tryptic digest, a workflow scheme, a reaction pathway for TCEP reduction and NEM alkylation, and numerous EThcD, HCD, and UVPD mass spectra for tryptic peptides from lysozyme and serotransferrin. S 1

2 Experimental Details: Materials and reagents. Bovine insulin and human serotransferrin was purchased from Sigma- Aldrich (St. Louis, MO). Lysozyme was purchased from MP Biomedicals (Santa Ana, CA). Tris(2-carboxyethyl)phosphine (TCEP) and N-ethylmaleimide (NEM) were purchased from Thermo Fisher Scientific (Rockford, IL). Mass spectrometry-grade trypsin, endoproteinase Lys- C, and PNGase F were obtained from Promega (Madison, WI). LC-MS grade acetonitrile, methanol, and water were purchased from EMD Millipore (Darmstadt, Germany). Pierce C18 spin columns used for peptide desalting and LC-MS grade formic acid were obtained from Fisher Scientific (Fair Lawn, NJ). Sample preparation. For lysozyme digestion, 200 µg of lysozyme was obtained from 400 µm stock solution (~35 µl) and was added to 300µL of 2 M urea/50 mm TrisHCl (ph 7). To this solution 20 µg of trypsin was added to achieve a final lysozyme:trypsin ratio of 10:1. The sample was diluted to 500 µl to a final concentration of 400 ng/µl. Digestion proceeded overnight at 37 C and was terminated by the addition of 0.5 µl of formic acid. Serotransferrin digestion was prepared as described above but in addition to trypsin, Lys-C, and PNGase F were added to the solution to increase the fidelity of C-terminal lysine cleavage and to cleave glycosylations to simplify the complexity of samples. Partial reduction and alkylation was performed by obtaining 20 µl of digest and adding TCEP in a 1:1 molar ratio with the number of moles of disulfide bonds in the sample. Partial reduction and alkylation was performed in acid conditions (ph ~3) to prevent disulfide scrambling that can occur at alkaline ph. Samples were incubated at 55 C for 10, 30, or 60 minutes before being removed from heat. Alkylation was subsequently performed by the addition of excess NEM followed by incubation in the dark at room temperature for 1 hour. All samples were diluted with 0.1% formic acid to a final concentration of 100 ng/µl for 1 µl injections onto the HPLC system. S 2

3 Mass spectrometry, liquid chromatography, and photodissociation. A 3 cm trap column (100 µm ID, New Objective Integrafrit, Woburn, MA) and a 15 cm analytical column (75 µm ID, New Objective Picofrit, Woburn, MA) were used for LC experiments. Both columns were packed inhouse. The trap column was packed with 3 µm UChrom C18 stationary phase (Waters, Milford, MA), and the analytical column was paced with 1.8 µm UChrom C18 stationary phase (Waters, Milford, MA). Each injection (approximately 100 ng) was pre-concentrated on the trap column with 2% acetonitrile and 0.1% formic acid. Mobile phases for separation on the analytical column consisted of 0.1% formic acid in water as eluent A and 0.1% formic acid in acetonitrile as eluent B. Unless otherwise stated, a linear gradient from 2% B to 41% B over 90 minutes was applied, then increased linearly to 50% during the following 10 minutes. The column was washed with 80% eluent B for 5 minutes before returning to 2% eluent B for 10 minutes to equilibrate prior to the next LC run. The flow rate was 300 nl/min. For the elucidation of disulfide bonds 6-8 of serotransferrin, a special linear gradient from 30% B to 80% B over 20 minutes was used to allow this complex peptide to be targeted. For nanospray, 1.8 kv was applied at a precolumn liquid voltage junction. The ion isolation width was 5 m/z, and all MS/MS spectra were acquired using a resolution of 30,000 (at m/z 200). UVPD was performed in the low pressure trap. For EThcD the ETD reaction time was set at 50 ms, an AGC target for the reagent anion was set to , and the HCD NCE was 32%. Typically each spectral acquisition consisted of 3 μscans. An AGC target of was used for MS1 and for MS 2. Data processing. Disulfide bonds were searched as a crosslink between cysteines with a change in mass of Da. Alkylations were searched as a dead-end crosslink with a change in mass of Da. Since Kojak does not use expected disulfide connectivities to process data, all possible disulfide bonds were searched (the connection of any two cysteines by a disulfide bond). S 3

4 Kojak does not consider peptides produced by homolytic cleavage of disulfide bonds. As a result, sequence ions from homolytic cleavage were confirmed manually. All analyzed spectra were deconvoluted with Xtract using a signal to noise threshold of 3. The deconvoluted spectra were processed by Prosight Lite to identify sequence ions of disulfide linked peptide pairs. All data analyzed by UV-POSIT, Kojak, and Prosight Lite were searched using a 10 ppm tolerance based off theoretical values for precursor and fragment ion assignments. S 4

5 Disulfide Bond Disulfide Linkage Residues Sequence Theoretical Mass [M+3H] 3+ [M+4H] 4+ 1 C6-C CELAAAMK GCR 2 C30-C GYSLGNWVCAAK CK 3 C64-C WWCNDGR 4 C76-C NLCNIPCSALLSSDITASVNCAK 3 C64-C WWCNDGR 4 (Red/Alk) ** NLC*NIPCSALLSSDITASVNC*AK Table S1. Theoretical disulfide-containing tryptic peptides from lysozyme. Peptides are arranged according to disulfide connectivity and contain zero missed cleavages. Cysteines are shown in bold. An asterisk denotes an NEM tag. The numbering of residues corresponds to lysozyme without the signal peptide. S 5

and the calculated m/z values assume zero missed cleavages.

6 Table S2. Theoretical disulfide-containing peptides of serotransferrin after in silico digestion with PNGase F, Lys- C, and trypsin. Peptides are arranged according to disulfide connectivity, and the calculated m/z values assume zero missed cleavages. Cysteines are shown in bold font. The numbering of residues corresponds to transferrin without the signal peptide. S 6

current, and categorized according to their origin from the A or B chain.

7 Figure S1. Ion distributions for UVPD, EThcD, and HCD activation of insulin (5+) (a). Fragment ion abundances were summed according to fragment ion type, normalized to the total ion current, and categorized according to their origin from the A or B chain. A chain and B chain sequence ion distributions of insulin from EThcD (b,c). A chain and B chain sequence ion distributions of insulin from UVPD (d,e). S 7

8 Figure S2. Sequence of lysozyme (M r 16 kda) with expected disulfide bonds represented by blue lines and numbered. The signal peptide is not shown. S 8

9 Figure S3. Deconvoluted EThcD (a) and HCD (b) spectra of peptide pair containing disulfide bond 1 of lysozyme (Cys6-Cys127) (Table S1) (3+). Fragment ions denoted with a blue dot contain the intact mass of the B-chain. Fragment ions denoted with a red dot contain the intact mass of the A-chain. S 9

10 Figure S4. Deconvoluted EThcD (a) and HCD (b) spectra of peptide pair containing disulfide bond 2 of lysozyme (Cys30-Cys115) (Table S1) (3+). Fragment ions denoted with a blue dot contain the intact mass of the B-chain. Fragment ions denoted with a red dot contain the intact mass of the A-chain. S 10

11 Figure S5. Deconvoluted UVPD spectrum of non-reduced tryptic peptide pair (z = 4+) from lysozyme containing disulfide bonds 3 (C64-C80) and 4 (C76-C94) (Table S1). Fragment ions denoted with a blue dot contain the intact mass of the B-chain. Fragment ions denoted with a red dot contain the intact mass of the A-chain. S 11

12 Figure S6. Deconvoluted EThcD (a) and HCD (b) spectra of partially reduced peptide pair from lysozyme digest (4+). Disulfide bond 4 (Cys76-Cys94) was reduced and alkylated, while disulfide bond 3 (Cys64-Cys80) remained intact prior to activation (Table S1). Fragment ions denoted with a blue dot contain the intact mass of the B-chain. Fragment ions denoted with a red dot contain the intact mass of the A-chain. A star denotes an NEM alkylation. S 12

13 Figure S7. Sequence of human serotransferrin (M r 75 kda) with expected disulfide bonds represented by blue lines and numbered. The signal peptide is not shown. S 13

14 Figure S8. Base peak chromatogram (gray trace) of partially reduced (60 minute reduction) and alkylated serotransferrin tryptic digest. Representative extracted ion chromatograms are overlaid in color, and the identities are summarized in the table. Peptides found containing disulfide bonds 4, 8, 17, and 18 contain a missed cleavage. S 14

Figure S9. Deconvoluted UVPD spectrum of non-reduced enzymatic peptide pair (z = 4+) from serotransferrin containing disulfide bond 1 (C9-C48) (Table S2).

15 Figure S9. Deconvoluted UVPD spectrum of non-reduced enzymatic peptide pair (z = 4+) from serotransferrin containing disulfide bond 1 (C9-C48) (Table S2). Fragment ions denoted with a blue dot contain the intact mass of the B-chain. Fragment ions denoted with a red dot contain the intact mass of the A-chain. S 15

Figure S10. Deconvoluted UVPD spectrum of non-reduced enzymatic peptide pair (z = 3+) from serotransferrin containing disulfide bond 2 (C19-C39) (Table S2).

16 Figure S10. Deconvoluted UVPD spectrum of non-reduced enzymatic peptide pair (z = 3+) from serotransferrin containing disulfide bond 2 (C19-C39) (Table S2). Fragment ions denoted with a blue dot contain the intact mass of the B-chain. Fragment ions denoted with a red dot contain the intact mass of the A-chain. S 16

Figure S11. Deconvoluted UVPD spectrum of non-reduced enzymatic peptide pair (z = 4+) from serotransferrin containing disulfide bond 3 (C118-C194) (Table S2).

17 Figure S11. Deconvoluted UVPD spectrum of non-reduced enzymatic peptide pair (z = 4+) from serotransferrin containing disulfide bond 3 (C118-C194) (Table S2). Fragment ions denoted with a blue dot contain the intact mass of the B-chain. Fragment ions denoted with a red dot contain the intact mass of the A-chain. S 17

serotransferrin containing disulfide bond 9 (C227-C241) (Table S2).

18 Figure S12. Deconvoluted UVPD spectrum of non-reduced enzymatic peptide pair (z = 5+) from serotransferrin containing disulfide bond 9 (C227-C241) (Table S2). Fragment ions denoted with a blue dot contain the intact mass of the B-chain. Fragment ions denoted with a red dot contain the intact mass of the A-chain. S 18

19 Figure S13. Deconvoluted UVPD spectrum of non-reduced enzymatic peptide pair (z = 5+) from serotransferrin containing disulfide bond 14 (C418-C637) (Table S2). Fragment ions denoted with a blue dot contain the intact mass of the B-chain. Fragment ions denoted with a red dot contain the intact mass of the A-chain. S 19

20 Figure S14. Deconvoluted UVPD spectrum of non-reduced enzymatic peptide pair (z = 4+) from serotransferrin containing disulfide bond 15 (C ) (Table S2). Fragment ions denoted with a blue dot contain the intact mass of the B-chain. Fragment ions denoted with a red dot contain the intact mass of the A-chain. S 20

Fragment ions denoted with a blue dot contain the

21 Figure S15. Deconvoluted UVPD spectrum of non-reduced enzymatic peptide pair (z = 4+) from serotransferrin containing disulfide bond 18 (C563-C577) (Table S2). Fragment ions denoted with a blue dot contain the intact mass of the B-chain. Fragment ions denoted with a red dot contain the intact mass of the A-chain. S 21

22 Figure S16. Deconvoluted UVPD spectrum of non-reduced enzymatic peptide (z = 3+) from serotransferrin containing disulfide bond 19 (C615-C620) (Table S2). S 22

from serotransferrin containing disulfide bond 4 (C137-C331) (Table S2).

23 Figure S17. Deconvoluted UVPD spectrum of partially reduced-reduced enzymatic peptide pair (z = 4+) from serotransferrin containing disulfide bond 4 (C137-C331) (Table S2). Fragment ions denoted with a blue dot contain the intact mass of the B-chain. Fragment ions denoted with a red dot contain the intact mass of the A-chain. A star denotes an NEM alkylation. S 23

24 Figure S18. Deconvoluted UVPD spectrum of partially reduced-reduced enzymatic peptide pair (z = 5+) from serotransferrin containing disulfide bond 5 (C339-C596) (Table S2). Fragment ions denoted with a blue dot contain the intact mass of the B-chain. Fragment ions denoted with a red dot contain the intact mass of the A-chain. A star denotes an NEM alkylation. S 24

from serotransferrin containing disulfide bond 10 (C345-C377) (Table S2).

25 Figure S19. Deconvoluted UVPD spectrum of partially reduced-reduced enzymatic peptide pair (z = 4+) from serotransferrin containing disulfide bond 10 (C345-C377) (Table S2). Fragment ions denoted with a blue dot contain the intact mass of the B-chain. Fragment ions denoted with a red dot contain the intact mass of the A-chain. A star denotes an NEM alkylation. S 25

from serotransferrin containing disulfide bond 11 (C355-C368) (Table S2).

26 Figure S20. Deconvoluted UVPD spectrum of partially reduced-reduced enzymatic peptide pair (z = 4+) from serotransferrin containing disulfide bond 11 (C355-C368) (Table S2). Fragment ions denoted with a blue dot contain the intact mass of the B-chain. Fragment ions denoted with a red dot contain the intact mass of the A-chain. A star denotes an NEM alkylation. S 26

27 Figure S21. Deconvoluted UVPD spectrum of partially reduced-reduced enzymatic peptide pair (z = 3+) from serotransferrin containing disulfide bond 12 (C402-C674) (Table S2). Fragment ions denoted with a blue dot contain the intact mass of the B-chain. Fragment ions denoted with a red dot contain the intact mass of the A-chain. A star denotes an NEM alkylation. S 27

28 Figure S22. Deconvoluted UVPD spectrum of partially reduced-reduced enzymatic peptide pair (z = 4+) from serotransferrin containing disulfide bond 13 (C474-C665) (Table S2). Fragment ions denoted with a blue dot contain the intact mass of the B-chain. Fragment ions denoted with a red dot contain the intact mass of the A-chain. A star denotes an NEM alkylation. S 28

29 Figure S23. Deconvoluted UVPD spectrum of partially reduced-reduced enzymatic peptide pair (z = 4+) from serotransferrin containing disulfide bond 16 (C484-C498) (Table S2). Fragment ions denoted with a blue dot contain the intact mass of the B-chain. Fragment ions denoted with a red dot contain the intact mass of the A-chain. A star denotes an NEM alkylation. S 29

Figure S24. Deconvoluted UVPD spectrum of partially reduced-reduced enzymatic peptide pair (z = 4+) from serotransferrin containing disulfide bond 17 (C495-C506) (Table S2).

30 Figure S24. Deconvoluted UVPD spectrum of partially reduced-reduced enzymatic peptide pair (z = 4+) from serotransferrin containing disulfide bond 17 (C495-C506) (Table S2). Fragment ions denoted with a blue dot contain the intact mass of the B-chain. Fragment ions denoted with a red dot contain the intact mass of the A-chain. A star denotes an NEM alkylation. S 30

31 Scheme S1. Workflow diagram for the elucidation of disulfide bonds. S 31

32 Scheme S2. Amino acid sequence of insulin with predicted disulfide bonds. S 32

33 Scheme S3. Reaction pathway for TCEP reduction and NEM alkylation. The NEM alkylation adds a +125 mass tag. S 33

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