Cyclic DiaminesAs Mass Spectrometry Cleavable Crosslinkers for Protein Interactions
Journal of the American Society for Mass Spectrometry
Billy Clifford-Nunn[1], H.D. Hollis Showalter[2], Philip C. Andrews1,[3],[4]
Supplementary
Table of Contents:
ContentPage
Online Resource1: SDS-PAGE of DC4 CrosslinkedAldolaseS-2
Online Resources 2 through 10: CID Spectra of DC4 CrosslinkedAldolase PeptidesS-3
Online Resources 11 through 19: CID Spectra of ISD fragments from DC4 Crosslinked
Aldolase PeptidesS-8
Online Resource 20: Ribbon Drawing of Aldolase
Online Resource 1. SDS-PAGE of Aldolasecrosslinked with increasing amounts of DC4. DC4 to Lysine ratio is indicated across the top of the gel. Molecular weight standards are indicated in lane 1. Ratios of crosslinker to lysyl residues greater than 25:1 resulted in formation of higher multimers (data not shown).
Online Resource 2. CID of the ion 1851.1 which was assigned to the crosslinked peptide in shown in the black box.(A) This peptide contained both a crosslink and a dead-end which had already fragmented via ISD. The intact doubly modified peptide was only observed with the n-terminal glutamine cyclizedto pyroglutamic acid (B). Similar peaks were observed in the MS spectrum from ISD and CID fragmentation of m/z 651.3 and 959.6 are shown in Online Resource 17 and Online Resource 16, respectively.
Online Resource 3.CID of the ion 2221.2 which was assigned to the crosslinked peptide in shown in the black box. Similar peaks were observed in the MS spectrum from ISD and CID fragmentation of m/z 651.3 and 1346.5 are shown in Online Resource 17 and Online Resource 19, respectively.
Online Resource 4. CID of the ion 2611.3 which was assigned to the crosslinked peptide in shown in the black box. Similar peaks were observed in the MS spectrum from ISD and CID fragmentation of m/z 1153.5 and 1346.5 are shown in Online Resource 13 and Online Resource 19, respectively.
Online Resource 5. CID of the ion 1827.9 which was assigned to the crosslinked peptide in shown in the black box. Similar peaks were observed in the MS spectrum from ISD and CID fragmentation of m/z 829.4 and 887.5 are shown in Online Resource 12 and Online Resource 18, respectively.
Online Resource 6. CID of the ion 1631.9 which was assigned to the crosslinked peptide in shown in the black box. Similar peaks were observed in the MS spectrum from ISD and CID fragmentation of m/z 633.5 and 887.5 are shown in Online Resource 15 and Online Resource 18, respectively.
Online Resource 7. CID of the ion 1970.1 which was assigned to the crosslinked peptide in shown in the black box. Similar peaks were observed in the MS spectrum from ISD and CID fragmentation of m/z 970.5 and 887.5 are shown in Online Resource 11 and Online Resource 18, respectively.
Online Resource 8. CID of the ion 2152.1 which was assigned to the crosslinked peptide in shown in the black box. Similar peaks were observed in the MS spectrum from ISD and CID fragmentation of m/z 887.5 and 1153.5 and are shown in Online Resource 18 and Online Resource 13, respectively.
Online Resource 9. CID of the ion 2184.1 which was assigned to the crosslinked peptide in shown in the black box. Similar peaks were observed in the MS spectrum from ISD and CID fragmentation of m/z 919.5 and 1153.5 are shown in Online Resource 14 and Online Resource 13, respectively.
Online Resource 10. CID of the ion 1507.9 which was assigned to the crosslinked peptide in shown in the black box. Similar peaks were observed in the MS spectrum from ISD and CID fragmentation of m/z 633.5 and 651.3 are shown in Online Resource 15 and Online Resource 17, respectively.
Online Resource 11. CID fragmentation of the ISD peak 970.5 which was identified to be the peptide NLKAAQEE with the lysine modified as shown.
Online Resource 12. CID fragmentation of the ISD peak 829.4 which was identified to be the peptide CVLKIGE with the lysine modified as shown.
Online Resource 13. CID fragmentation of the ISD peak 1153.5 which was identified to be the peptide PHSHPALTPE with the n-terminus modified as shown.
Online Resource 14.CID fragmentation of the ISD peak 919.5 which was identified to be the peptide KDGADFAK with the only the first lysine modified as shown.
Online Resource 15. CID fragmentation of the ISD peak 633.5 which was identified to be the peptide YVKR with the lysine modified as shown.
Online Resource 16. CID fragmentation of the ISD peak 959.5 which was identified to be the peptide KVLAAVYK with the first lysine modified as shown.
Online Resource 17. CID fragmentation of the ISD peak 651.3 which was identified to be the peptide QKKE with the lysines modified as shown and the glutamate pyrolyzed to glutamic acid. Although this species has two modified lysines and is shown as the canonical structure containing two protonated centers, it is still singly charged due to the loss of a proton, most likely from the C-terminus or the glutamic acid side chain to form a probable internal ion pair.
Online Resource 18. CID fragmentation of the ISD peak 887.5 which was identified to be the peptide STGSIAKR with the lysine modified as shown.
Online Resource 19. CID fragmentation of the ISD peak 1346.7 which was identified to be the peptide ILPDGDHDLKR with the lysine modified as shown.
Online Resource 20. Ribbon drawing of the aldolase tetramer. The two lysine residues that were not modified by DC4 are shown in red and are buried.
S-1
[1] Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, MI 48109, United States.
[2]University of Michigan Vahlteich Medicinal Chemistry Core, Department of Medicinal Chemistry, College of Pharmacy, 428 Church Street, Ann Arbor, MI 28109, United States
[3] Department of Biological Chemistry, 1150 West Medical Center Drive, Room 5301 MSRB III, Ann Arbor, MI 48109, United States.
[4] Center for Computational Medicine and Bioinformatics, University of Michigan Medical School, 2017 Palmer Commons Bldg, 100 Washtenaw Avenue, Ann Arbor, MI 48109, United States.
To whom correspondence should be addressed. E-mail: . Fax: 734-647-0951