Supporting Information for:
S- to N-Palmitoyl Transfer during Proteomic Sample Preparation
Yuhuan Ji,1,2 Markus M. Bachschmid,3 Catherine E. Costello,1,2 Cheng Lin1,2*
1. Center for Biomedical Mass Spectrometry, 2. Department of Biochemistry, 3. Cardiovascular
Proteomics Center and Vascular Biology Section, Department of Medicine, Boston University
School of Medicine, Boston, MA 02118
*
To whom correspondence should be addressed
Phone: +1 (617) 638 6705
Fax: +1 (617) 638 6761
Email: chenglin@bu.edu
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Table of Contents
Figure S1
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Figure S2
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Figure S3
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Figure S4
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Figure S5
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Figure S6
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Figure S7
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Figure S8
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Figure S9
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Figure S10
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Scheme S1
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Scheme S2
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Scheme S3
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Table S1
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Figure S1. MALDI-TOF mass spectra of the peptide standard, GCLGNAK, (a) before and (b)
after reaction with palmitoyl chloride in 100% TFA. (c) The MALDI-TOF mass spectrum of
sample (b) after subsequent incubation with 500 mM HA/25 mM IAM.
Figure S2. MALDI-TOF mass spectra of the peptide standard, MGCpalmVQCpalmKDKEA, (a)
before and (b) after incubation in 100 mM ABC buffer at 95 oC for 5 minutes.
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Figure S3. MALDI-TOF mass spectra of the S-palmitoyl peptide standard, GCpalmLGNAK, (a)
after incubation in 100 mM ABC buffer at 37 oC for 3 hr, (b) followed by DTT treatment, and (c)
after subsequent reaction with iodoacetamide.
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Figure S4. CID tandem mass spectra of the doubly charged peptide, (GCIAMLGNAK)palm,
produced by a 3-hr incubation of the S-palmitoyl peptide standard, GCpalmLGNAK, in 100 mM
ABC buffer, followed by reduction by DTT and alkylation by iodoacetamide. Fragment ions
labeled in red could be produced from an N-terminus palmitoylated precursor,
GpalmCIAMLGNAK, whereas fragment ions labeled in blue may be formed from a lysine
sidechain palmitoylated precursor, GCIAMLGNAKpalm.
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Figure S5. MALDI-TOF mass spectra of a mixture of GCpalmLGNAK and GCIAMLGNAK (a)
before and (b) after incubation in 100 mM ABC buffer at 37 oC for 3 hr.
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Figure S6. (a) The base peak chromatogram of GCpalmLGNAK after incubation in 50 mM Tris
buffer with 0.1% RapiGest at 37 oC for 3 hr; (b-d) the extracted ion chromatograms of various
modified forms of GCLGNAK.
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Figure S7. The decay of UV absorbance at 230 nm of GCpalmLGNAK over 3-hr incubation in 50
mM Tris buffer either without (a) or with (b) 0.1% RapiGest.
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Figure S8. ETD tandem mass spectra of (a) GCpalmLGNAK, (b) GCLGNAKpalm, and (c)
GpalmCLGNAK.
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Figure S9. CID tandem mass spectra of (a) GCpalmLGNAKpalm, (b) GpalmCpalmLGNAK. *
indicates loss of one palmitoyl group.
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Figure S10. CID tandem mass spectra of the disulfide-linked homo-dimers of (a) GCLGNAKpalm
and (b) GpalmCLGNAK.
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Scheme S1. Formation of the b and y ions via the oxazolone pathway.
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Scheme S2. Proposed mechanism for the formation of b1+palm ion by CID of the S-palmitoyl
peptide, GCpalmLGNAK, via gas-phase palmitoyl migration.
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Scheme S3. Proposed mechanism for the formation of the [M + 2H – C15H31COHS]+• ion from a
doubly charged S-palmitoyl peptide precursor ion by ETD.
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Table S1. The average ion abundances of the peptides resulting from incubation of
GCpalmLGNAK in Tris and Tris-RapiGest and their relative ratios.
Peptide Sequence
Average Ion Abundance
Abundance Ratio
Tris
Tris-RapiGestTM
GCpalmLGNAK
9.16 x 108
1.07 x 1010
Tris /TrisRapiGestTM
0.09
GCpalmLGNAKpalm
5.25 x107
1.88 x 106
27.99
GpalmCpalmLGNAK
3.39 x 108
6.82 x 107
4.97
GCLGNAKpalm (dimer)
2.30 x 105
5.19 x 105
0.44
GpalmCLGNAK (dimer)
3.49 x 105
3.66 x 106
0.10
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