S- to N-Palmitoyl Transfer Supporting InformationJi et al.

Supporting Information for:

S- to N-Palmitoyl Transfer during Proteomic Sample Preparation

Yuhuan Ji,1,2Markus M. Bachschmid,3 Catherine E. Costello,1,2Cheng 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)6386705

Fax: +1 (617) 638 6761

Email:

Table of Contents

Figure S1 / S-3
Figure S2 / S-3
Figure S3 / S-4
Figure S4 / S-5
Figure S5 / S-6
Figure S6 / S-7
Figure S7 / S-8
Figure S8 / S-9
Figure S9 / S-10
Figure S10 / S-11
Scheme S1 / S-12
Scheme S2 / S-13
Scheme S3 / S-14
Table S1 / S-15

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.

Figure S3. MALDI-TOF mass spectra of the S-palmitoyl peptide standard,GCpalmLGNAK, (a) after incubation in 100mMABC buffer at 37 oC for 3 hr, (b) followed by DTT treatment, and (c) after subsequent reaction with iodoacetamide.

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 mMABC 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.

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.

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.

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.

Figure S8. ETD tandem mass spectra of (a) GCpalmLGNAK, (b) GCLGNAKpalm, and (c) GpalmCLGNAK.

Figure S9. CID tandem mass spectra of (a) GCpalmLGNAKpalm, (b) GpalmCpalmLGNAK. * indicates loss of one palmitoyl group.

Figure S10. CID tandem mass spectra of the disulfide-linked homo-dimers of (a) GCLGNAKpalm and (b) GpalmCLGNAK.

Scheme S1. Formation of the b and y ions via the oxazolone pathway.

Scheme S2. Proposed mechanism for the formation of b1+palm ion by CID of the S-palmitoyl peptide, GCpalmLGNAK, via gas-phase palmitoyl migration.

Scheme S3. Proposed mechanism for the formation of the [M + 2H – C15H31COHS]+• ion from a doubly charged S-palmitoyl peptide precursor ion by ETD.

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 / Tris /Tris-RapiGestTM
GCpalmLGNAK / 9.16 x 108 / 1.07 x 1010 / 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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