Exploring the O -GlcNAc proteome: Direct identification of O -GlcNAc-modified proteins from the brain

Khidekel et al. 10.1073/pnas.0403471101.

Supporting Information

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Supporting Figure 4

Fig. 4. Annotated MS⁴ spectra of O-GlcNAc-glycosylated peptides identified from rat brain. Approximately 20% of the avidin-enriched peptides from each cation exchange fraction were loaded onto a 360-mm O.D. X 75 mm i.d. precolumn packed with 4 cm of 5-mm Monitor C18 particles (Column Engineering, Ontario, CA) at a flow rate of 4 ml/min. After desalting, the vent was closed and peptides eluted to a 360 mm O.D. ´75 mm i.d. analytical column with integrated emitter tip [10 cm of 5 mm C18, ~5-mm tip, see Martin et al. for more details (1)]. The chromatographic profile was from 100% solvent A (0.1% aqueous AcOH) to 50% solvent B (0.1% AcOH in CH₃CN) in 30 min. The flow rate through the analytical column was »100 nl/min. The mass spectrometer was programmed to record a full-scan electrospray mass spectrum (m/z 500-2000), followed by MS⁴ scans of each targeted peptide (relative collision energy = 35%; 3.5-Da isolation window for each fragmentation). In the first stage of MS/MS, precursor ions were isolated and fragmented to yield O-GlcNAc-modified peptide ions. In the second stage, these ions were isolated and fragmented to yield ions corresponding to the unmodified peptide. In the third stage, unmodified peptide ions were isolated and fragmented, and the fragment ions were detected in the final stage. For all MS experiments, the electrospray voltage was set at 1.6 kV and the heated capillary was maintained at 250°C. For database analysis to identify O-GlcNAc proteins, BIOWORKS BROWSER 3.1SR1 (Thermo Electron) software was used to create files from MS⁴ data. These files were then directly queried, using the SEQUEST algorithm (2), against amino acid sequences in the National Center for Biotechnology Information rat/mouse protein database.

1. Martin, S. E., Shabanowitz, J., Hunt, D. F. & Marto, J. A. (2000) Anal. Chem. 72, 4266-4274.

2. Eng, J. K., McCormack., A. L. &Yates, J. R., III (1994) J. Am. Soc. Mass Spectrom. 5, 976-989.

Supporting Figure 5

Fig. 5. Annotated MS³ (A) and MS⁴ (B) spectra of the tagged O-GlcNAc-glycosylated peptide ¹⁵⁸AIPVSREEKPSSAPSS¹⁷³ from a A-crystallin (m/z 787.4). MS analysis was performed as described in Materials and Methods and Fig. 4.

Fig. 6. Functional classification of the identified O-GlcNAc proteins according to categories described by Schoof et al. (1).

1. Schoof, H., Zaccaria, P., Gundlach, H., Lemcke, K., Rudd, S., Kolesov, G., Arnold, R., Mewes, H. W. & Mayer, K. F. (2002) Nucleic Acids Res. 30, 91-93.

Supporting Figure 7

Fig. 7. MS⁴ analysis of a tagged peptide from the brain that exhibited a hallmark loss of 49 Da ([M + 2H]²⁺), consistent with the loss of H₃PO₄. The spectrum is consistent with a peptide bearing both a phosphate and GlcNAc moiety. Sequencing of the peptide based on the y and b fragmentation patterns was inconclusive.

Fig. 8. Combining the chemoenzymatic and b -elimination approaches to map specific glycosylation sites. (A) Strategy for the formation of a stable sulfide adduct from tagged O-GlcNAc peptides. (B) MS/MS analysis of the sulfide adduct of peptide ³⁶⁰APVGSVVSVPSHSSASSDK³⁷⁸ from HIV-1 Rev binding protein. Several y and b fragment ions containing the sulfide moiety (indicated with the subscript S) were observed, permitting peptide sequencing and identification of Ser-372 or Ser-373 as the glycosylation site. (C) MS/MS spectrum of the corresponding peptide before b -elimination shows the characteristic ketone-biotin signature, indicating that the original peptide was O-GlcNAc glycosylated. MS⁴ analysis of the peptide confirmed its amino acid sequence. (D) Summary of the prominent b and y ions from MS/MS analysis of the b -eliminated peptide. Fragment ions y7, y10, and b15 localized the site of modification to one of two serines within the peptide. An additional ion, consistent with b13, strongly suggested Ser-372 as the specific site of glycosylation.

Supporting Figure 9

Fig. 9. Annotated MS/MS spectrum of the doubly charged (m/z 935.6) (A) and the triply charged (m/z 623.8) (B) sulfide adduct of peptide ³⁶⁰APVGSVVSVPSHSSASSDK³⁷⁸ from HIV-1 Rev binding protein. The spectrum demonstrates the attachment of a butanethiol moiety to Ser-372 or Ser-373. A peak consistent with fragment ion b13 strongly implicates Ser-372 as the specific site of glycosylation.

Supporting Figure 10

Fig. 10. We confirmed the identity of the singly charged peptide VTQHFAK of bassoon by chemically synthesizing the corresponding peptide and comparing its MS/MS spectrum to the MS⁴ spectrum of the tagged peptide isolated from the brain (see Fig. 4). The spectra were nearly identical, verifying the assignment.