Blood collection and processing.
Blood samples were obtained from normal and diabetic volunteers at the Johns Hopkins Diabetes Center with written consent. The research was approved by the institutional review board, consistent with the Helsinki Declaration. Subjects gave written informed consent. The identity of subjects was masked to those doing assays and analyzing data, but all authors had access to the primary data. Blood samples were drawn and collected into a vial containing EDTA. O-GlcNAcase inhibitor PUGNAc was added into the vial directly before blood collection to yield a final concentration of ~10 μmol/l. Blood cells were fractionated to isolate erythrocytes using Histopaque-1077 (Sigma-Aldrich) according to the manufacturer's instruction. Erythrocytes were lysed by sonication and centrifuged. Supernatant was recovered, and hemoglobin was partially depleted by HemogloBind resin (Biotech Support Group) following the manufacturer's instructions.
Immunoblotting and immunoprecipitation.
Fifty micrograms of hemoglobin-depleted (partially) erythrocyte proteins were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and blotted by O-GlcNAc antibody (CTD 110.6) (1:5,000) (17
). Signals were visualized by enhanced chemiluminescence (Amersham, Piscataway, NJ). For immunoprecipitation, 1 mg lysates was incubated overnight with protein A/G beads (Santa Cruz Biotechnology) and antibodies against band 3, catalase, peroxiredoxin 2, or HSP90 α (Abcam, Cambridge, MA). After 5× washing with the lysis buffer, bound proteins were eluted by boiling for 5 min in 2× Laemmili sample buffer.
Chemoenzymatic tagging and enrichment of O-GlcNAcylated proteins.
A previously described protocol was modified and followed to isolate O-GlcNAc–modified proteins from erythrocytic lysates (18
). Briefly, labeling of terminal O-GlcNAc by mutant galactose transferase (GalT1) (19
) was performed overnight at 4°C in the presence of 5 mmol/l MnCl2
, 0.5 mmol/l UDP-Gal-ketone, and 2,000 units/ml PNGase F (New England Biolabs, Ipswich, MA). The reaction mixture was then dialyzed into denaturing buffer (5 mol/l urea, 50 mmol/l NH4
, and 100 mmol/l NaCl, pH 7.8). The pH was adjusted to 4.8 by 0.3 mol/l NaOAc. After removing the insoluble by centrifugation, 3 mmol/l aminoxy biotin (Dojindo, Gaithersburg, MD) was added to the supernatant and incubated for 24 h at room temperature. The reaction was quenched by adjusting the pH to 7.9. The reaction buffer was again dialyzed into denaturing buffer, followed by 50 mmol/l NH4
and 10 mmol/l NaCl, pH 7.8. After preclearing with Sepharose 6B beads, the mixture was incubated with agarose-conjugated streptavidin (Pierce, Rockford, IL) for 2 h. The beads were extensively washed by low-salt buffer (0.1 mol/l Na2
, 0.15 mol/l NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS, pH7.5) and high-salt buffer (0.1 mol/l Na2
, 0.5 mol/l NaCl, and 0.2% Triton X-100, pH 7.5). Bound proteins were eluted by boiling the beads in 50 mmol/l Tris-HCl, 2.5% SDS, 100 mmol/l dithiothreitol (DTT), 10% glycerol, and 5 mmol/l biotin. O-GlcNAc proteins were resolved in SDS-PAGE and in-gel digested by trypsin as previously described (20
). Peptides were extracted for mass spectrometric analysis.
Chemoenzymatic tagging and enrichment of O-GlcNAc peptides.
Normal and diabetic erythrocytic lysates (1 mg each) were in-solution digested overnight at 37°C by 40 μg trypsin. Trypsin was removed by filtering the solution through a 5-kDa cutoff membrane (Millipore, Billerica, MA). Fifty units of calf intestine phosphatase (New England Biolabs) were added and incubated for 4 h in the presence of 1 mmol/l MgCl2. UDP-GalNAz (Invitrogen, Carlsbad, CA) was added (~2× in excess) and incubated overnight with mutant GalT1 and 2,000 units/ml PNGase F in 50 mmol/l NH4HCO3. After reaction, excess UDP-GalNAz was removed by passing the mixture through a C18 spin column (Nestgroup, Southborough, MA). Peptides were eluted in 80% acetonitrile and lyophilized. Cycloaddition reaction was performed in a volume of 20 μl containing biotin–polyethylene glycol (PEG)–alkyne (~3× in excess, dissolved in DMSO; Invitrogen), 2 mmol/l Tris (2-carboxyethyl) phosphine hydrochloride, 2 mmol/l Tris [(1-benzyl-1H-1,2,3-triazol-4-yl) methyl] amine, and 2 mmol/l CuSO4. The reaction mixture was incubated for 12 h at room temperature with gentle shaking. The mixture was diluted into cation exchange loading buffer. Cation exchange was performed on a strong cation exchange (SCX) spin column (Nestgroup) according to the manufacturer's instruction. Peptides were eluted in one fraction by high-salt buffer (5 mmol/l KH2PO4, 10% acetonitrile, and 300 mmol/l KCl, pH 3.0). The elutant was allowed to bind to agarose-conjugated streptavidin for 2 h at room temperature, followed by extensive washing.
Chemical derivatization and fractionation of enriched peptides.
Eight times the bead volume of BEMAD buffer (1.5% triethylamine and 20 mmol/l DTT, pH adjusted to 12.0–12.5 by NaOH) was added to the washed avidin beads and allowed to incubate at 52–54°C for 4 h with shaking. The reaction was quenched by neutralizing the pH by 2% trifluoroacetic acid. The supernatant was desalted by C18 spin column as described above. The derivatized peptides were fractionated by SCX using a polysulfoethyl A column (0.32 × 100 mm, 5 μm, 300 Å; Column Technology, Fremont, CA) coupled to an Agilent 1100 series high-performance liquid chromatography (HPLC) (Agilent Technology, Santa Clara, CA). Fractionation was performed with a 40-min linear gradient of 0–350 mmol/l KCl (10 mmol/l KH2PO4 and 25% acetonitrile, pH 2.8) at a flow rate of 5 μl/min. 15 fractions (10 μl each) were collected.
iTRAQ labeling and fractionation of peptides.
Peptides from the flow-through and three washes of the avidin columns were pooled, desalted, and dried down by speed vacuum. The peptides were resuspended and differentially labeled by iTRAQ reagents (Applied Biosystems, Foster City, CA) according to the manufacturer's instruction. After labeling, the peptides were combined and fractionated similarly by SCX as described above except that a different polysulfoethyl A column (2.1 × 100 mm, 5 μm, 300 Å; PolyLC, Columbia, MD) was used instead.
Enriched O-GlcNAcylated proteins were identified by analysis on an LCQ ion trap mass spectrometer coupled to Magic 2002 HPLC (Michrom BioResources) and nanospray interface (Proxeon). The instrument was set in a information-dependent acquisition mode with three MS/MS (tandem mass spectrometry) followed by one full survey scan. Derivatized O-GlcNAc peptides were analyzed either on a Qstar Pulsar mass spectrometer (Applied Biosystems-MDS Sciex, Foster City, CA) or an LTQ-Orbitrap XL, both coupled with an Eksigent nano–liquid chromatography system (Dublin, CA). Peptides were desalted on a precolumn (75 μm inner diameter, 3 cm length, packed with irregular size particles 5–15 μm, 120 Å), and separated on an RF analytical column packed with 10 cm of C18 beads (5 μm, 120 Å; YMC ODS-AQ; Wather, Milford, MA). The main HPLC gradient was 5–40% solvent B (A, 0.1% formic acid; B, 90% acetonitrile and 0.1% formic acid) in 60 min at a flow rate of 300 nl/min. For Qstar, each survey scan was acquired from m/z 350–1,200 followed by MS/MS of up to three most intense precursors. For LTQ-Orbitrap, each survey scan (Fourier transform-MS, 60,000 resolution) of m/z 400–2,000 was followed by collision-assisted dissociation (CAD) MS/MS (ion trap-MS) of up to five most intense precursor ions. Dynamic exclusion was enabled with a repeat count of 2 and exclusion duration of 60 s.
Mass spectrometric data analysis.
For protein identification, peak lists of LCQ raw files were extracted and submitted to the Mascot search engine (version 2.2.0) with the following parameters: SwissProt as database, human as species, trypsin as enzyme with up to one missed cut, carbamidomethyl (C) as fixed modification, and oxidation (M) as variable modification. Mass tolerance was set at 1.2 amu (atomic mass units) for precursors and 0.8 amu for fragment ions. Raw data from derivatized O-GlcNAc peptides were similarly searched against SwissProt database using Mascot except that DTT (ST), DTT-H6(ST), deamination, and oxidation (M) were used as variable modification, and no fixed modification was selected. Precursor and fragment ion mass tolerances were 0.3 and 0.15 amu for Qstar and 0.1 and 0.8 amu for LTQ-Orbitrap, respectively. Quantitation was performed manually by averaging peak areas over the time of elution of given ion pairs. Mass spectrometry spectra originating from iTRAQ-labeled samples were extracted and searched against SwissProt database using ProteinPilot software (version 2.0; Applied Biosystems) with Paragon algorithm. Peptide identifications were further processed by the Pro Group algorithm (Applied Biosystems), which determines the minimal set of proteins that can be reported. Protein abundance ratios were automatically calculated based on ratios of reporter ions originating from peptides that are distinct to each protein isoform. Relative occupancy ratios (RORs) of O-GlcNAc between diabetic (D) and normal (N) samples were calculated using the following equation.