2.1 Chemicals and materials
Sequencing grade-modified trypsin was purchased from Promega Corp. (Madison, WI). Acrolein (ACR) (≥ 99%) was obtained from Fluka (St. Louis, MO). Aldehyde-reactive probe (ARP, N-aminooxymethylcarbonylhydrazino D-biotin) was purchased from Dojindo Laboratories (Kumamoto, Japan). UltraLink-immobilized monomeric avidin, iodoacetyl-PEG2-Biotin (IPB) and Triton X-100 detergent were obtained from Pierce (Rockford, IL). Bovine serum albumin (BSA) was from Calbiochem (La, CA). Macrospin strong cation exchange (SCX) columns were from Nest Group (Southborough, MA).
2.2 Animal treatment
The experimental protocol for the animal studies was approved by the Institutional Animal Care and Use Committee at Oregon State University (ACUP #3770). Seven Male F344 rats (Harlan, Indianapolis, IN) were housed individually in plastic cages covered with Hepa filters. The animals were allowed free access to standard animal chow and water ad libitum
. After 1 week of acclimatization, one 25-month old rat was sacrificed for an analytical reproducibility study and the other six 3-month old rats were transferred to metabolism cages. These six animals were divided into two groups of three, with one group receiving an intraperitoneal dose of 1 mL/kg CCl4
(dissolved in corn oil), and the other group (control) receiving the vehicle alone. The CCl4
dose of 1 mL/kg was chosen on the basis of literature reports [33
]. The rats were sacrificed 24 h after the treatment.
2.3 Preparation of IPB-labeled and ARP-ACR-modified BSA peptides
A solution of bovine serum albumin (BSA, 2 mg) in 1.0 mL of phosphate buffer (20 mM, pH 8.2) was reacted with 25 µL of DTT (60 mM in 20 mM phosphate buffer, pH 8.3) at 95 °C for 10 min. One half of the reduced BSA solution was then mixed with 200 µL 20 mM IPB in 50 mM phosphate buffer, (pH 8.3). The mixture was kept in the dark at room temperature for 90 min to form IPB-labeled BSA. Excess reagent was removed by adding 30 µL DTT (60 mM in 20 mM phosphate buffer, pH 8.3). The other half of the reduced BSA was reacted with 60 µL of acrolein (80 mM in 20 mM phosphate buffer, pH 8.3) at room temperature for 60 min. The excess of acrolein was removed by adding 40 µL DTT (60 mM in 20 mM phosphate buffer, pH 8.3). The ACR-modified BSA was then reacted with 250 µL ARP (30 mM in H2O) at room temperature for 60 min. Both ARP-ACR-modified and IPB-labeled BSA were digested with trypsin and passed through an ultrafiltration membrane (10 kDa MWCO). The peptide encompassing the residues 286 and 297 of BSA, YIC*DNQDTISSK modified on the cysteine residue (marked with an asterisk) with the ARP-ACR and IPB moiety, respectively, was used for method development.
2.4 Mitochondrial preparations
Mitochondria were isolated from rat hearts and separated by differential centrifugation to obtain subsarcolemmal mitochondria (SSM) [35
]. Mitochondria were stored at −80 °C. Each sample of subsarcolemmal mitochondria (SSM) containing approximately 0.5 mg total protein was washed twice with phosphate buffer (10 mM NaH2
pH 7.4) at 0 °C. The mitochondria were then resuspended in 400 µL of 10 mM NaH2
(pH 7.4) containing 1% Trition X-100 detergent and 3 mM DTT. DTT was added to prevent thiol modifications by reactive species during sample preparation. The mitochondria were sonicated in ice water for 5 min to solubilize the proteins. The soluble protein fraction was obtained by centrifugation at 14,000 × g for 15 min at 4 °C. The supernatant was then filtered through an Amicon Microcon centrifugal filter (10 kDa MWCO) to remove low molecular weight molecules at 4 °C. Mitochondrial proteins were re-suspended in 400 µL of 10 mM NaH2
(pH 7.4) containing 1 mM DTT. Further sample preparation was as follows (Supporting Information Figure S19
- Proteins were labeled with ARP for 1 hr at room temperature. The final ARP concentration was 3 mM. Proteins were digested with a 1:50 ratio of trypsin at 37 °C for 15 h.
- 15 µL DTT (30 mM in 20 mM phosphate buffer, pH 8.0) was added to the tryptic peptides to reduce disulfide bonds. The sample was then filtered through an Amicon Microcon centrifugal filter (10 kDa MWCO) to remove trypsin.
- 40 µL of the sample was added to 120 µL IPB (5 mM, 50 mM Tris buffer, pH 8.3). The alkylation reaction was performed in the dark at room temperature for 90 min to form IPB-labeled peptides. Unreacted IPB was trapped with 80 µL DTT (30 mM in 20 mM phosphate buffer, pH 8.0). The reaction mixture was diluted with water to a volume of 1750 µL.
- 35 µL of the sample from step 3 (IPB-modified peptides) were mixed with 800 µL of the sample from step 2 (ARP-labeled peptides). The mixed sample was then subjected to SCX and affinity chromatography, and stored at −20 °C prior to LC-MS/MS SRM analysis.
2.5 SCX Cleanup
Modified peptides were purified by strong cation exchange (SCX) chromatography using Macrospin SCX columns (Nest Group, Inc., Southborough, MA). Acetonitrile was used to activate the column. An elution buffer consisting of 20 % acetonitrile in 10 mM potassium phosphate buffer/0.6 M KCl (adjusted to pH 3 with H3PO4) was applied to condition the column. A washing buffer (10 mM potassium phosphate, 10 mM KCl, pH 3.0) containing 20% acetonitrile was used to equilibrate the column. Peptide samples (~pH 3.0, adjusted with H3PO4) were applied to the conditioned column and rinsed with washing buffer three times to remove Triton X-100 detergent and unbound components. Finally, 350 µL elution buffer was applied to the columns to release the peptides.
2.6 Affinity chromatography
Ultralink monomeric avidin (200 µL, Pierce, Rockford, IL) was packed into Handee Mini Spin Columns (Pierce Rockford, IL) following the manufacturer’s protocol. Columns were washed with 1.5 mL of 10 mM NaH2PO4,pH 7.4. Irreversible binding sites, consisting of tetrameric avidin, were blocked by washing with 600 µL of 2 mM D-biotin. To remove excess D-biotin, columns were washed with 1 mL of 2 M glycine-HCl (pH 2.8). The columns were then re-equilibrated by washing twice with 2 mL of phosphate buffered saline (PBS, 20 mM NaH2PO4, 300 mM NaCl). The peptide samples were then slowly added to the affinity columns. To remove non-labeled and non-specifically bound peptides the column was washed twice with 1 mL PBS followed by 1 mL of 10 mM NaH2PO4 (pH 7.4) and finally 1.5 mL of 50 mM NH4HCO3 containing 20 % CH3OH. The columns were then rinsed with 1 mL of MilliQ H2O before eluting the ARP-labeled peptides with 0.4 % aqueous formic acid containing 20 % acetonitrile. Collected fractions were concentrated using a freeze dryer and stored at −20 °C prior to mass spectrometric analysis.
An Ultimate LC Packing system (Dionex, Sunnyvale, CA) was used. Peptide samples were loaded onto a 5 mm × 0.50 mm C18 trap cartridge (Dionex, Sunnyvale, CA) at a flow rate of 20 µL/min. After 4 min the trap cartridge was automatically switched in-line to a 75 µm i.d. ×15 cm C18 PepMap 100 column (Dionex, Sunnyvale, CA). Peptides were eluted using a gradient from 9 % to 18 % solvent B in A over 90 min at 0.260 µL/min. Solvent A was 1% aqueous acetonitrile containing 0.1% formic acid and solvent B was acetonitrile containing 0.1% formic acid.
2.8 Mass Spectrometry
All LC-MS/MS analyses were carried out on a 4000 Q-Trap hybrid tandem mass spectrometer (AB/MDS SCIEX, Concord, Ontario, Canada) equipped with a nano-ESI source. The electrospray voltage was set to 2300 V and the declustering potential was 60 V. For the identification of ARP-labeled peptides, precursor ion scanning was performed over a mass range of 400–1300 amu at 500 amu/s (Q1 and Q3 with unit resolution).
An enhanced product ion scan (MS/MS) was performed if the intensity of any of the precursors of m/z 227 exceeded the threshold value of 1000 counts/s (cps). The scan rate for MS/MS was set to 4000 amu/s. Tandem mass spectral data were analyzed using MASCOT v2.1 (Matrix Science, London, UK) as described previously [21
]. The Swiss Prot database v50 (270778 sequences, 99412397 residues) was searched using the following parameters: taxonomy rodentia (20991 sequences), ± 0.5 Da mass tolerances for the precursor and fragment ions, possibility of 2 missed proteolytic cleavage sites, with trypsin/P or semitrypsin selected as the digesting enzyme, and ARP-Acrolein (CHK), ARP-HNE (CHK), ARP-ONE (CHK), ARP-HHE (CHK), ARP-MDA (KR), ARP-β-hydroxyacrolein, ARP-crotonaldehyde selected as variable modifications at the residues specified in parenthesis. Fragment ion assignments were verified and probe-specific ions were annotated manually.
The SRM analyses were conducted with Q1 and Q3 set at unit resolution. Each SRM transition period was 30 ms. SRM collision energies were 50 eV and 51 eV for ARP-ACR-modified and IPB-labeled model peptides. For the mitochondrial peptides the collision energies used for the SRM analyses are listed in .
SRM parameters and retention times for ARP-ACR (*) and IPB (#) labeled peptides.
MALDI mass spectrometry was performed with an ABI 4700 Proteomics Analyzer with TOF/TOF optics and equipped with a 200-Hz frequency-tripled Nd:YAG laser operating at a wavelength of 355 nm (Applied Biosystems, Inc., Framingham, MA). Mass Spectra were obtained over a range of m/z 700–4000 in the reflectron mode. External mass calibration was applied using the ABI 4700 calibration mixture consisting of the following peptides des-Arg1-bradykinin ([M+H]+, m/zcalc 904.4675), angiotensin I ([M+H]+, m/zcalc 1296.6847), Glu1-fibrinopeptide B ([M+H]+, m/zcalc 1570.6768), and ACTH 18–39 ([M+H]+, m/zcalc 2465.1983). Peptide samples were clean up prior to MS analyses with C18 ZipTips (Millipore, Billerica, MA) following the manufacture’s protocol. Peptides were mixed with α-cyano-4-hydroxycinnamic acid (2 mg/mL in 50 % acetonitrile containing 0.1 % TFA) and 0.5 µL of the mixture was spotted onto a 144-spot stainless steel target plate.