4.3.1 Materials
E. coli cell lysate was obtained from McLab (San Francisco, CA in USA).
Guanidination kit, dithiothreitol (DTT) and iodoacetamide (IAM) were purchased from Sigma Aldrich (St. Louis, MO in USA). iTRAQ 4-plex was obtained from Applied Biosystems (Framingham, MA in USA). Trypsin Gold (mass spectrometry grade) was obtained from Promega (Madison, WI in USA). Endoproteinase Asp-N (sequencing grade) was purchased from Roche (Indianapolis, IL in USA). Zeba spin desalting column (7K MWCO) and the NHS-activated agarose spin column were obtained from Pierce Biotechnology (Rockford, IL in USA).
4.3.2 Reduction and alkylation
2 mg of E. coli cell lysate was dissolved with 200 µL of Urea (6 M, with 0.1
M phosphate buffer, pH 7.5). The solution was incubated at 37 °C for 30 min. A 40
µL aliquot of DTT (0.5M) was added and the mixture was incubated at 37 °C for
30 min. A 100 µL aliquot of IAM (0.5 M) was added and the sample was
incubated at room temperature in the dark for 30 min. Another 10 µL aliquot of
DTT was added to quench the alkylation reaction.
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The guanidination kit was obtained from Sigma-Aldrich. The concentration of o-methylisourea hemisulfate was prepared at 6 mg/mL in water. The protein sample was adjusted to pH 10 by NaOH (1N) after reduction and alkylation and 100-fold molar excess of o-methylisourea hemisulfate was added. The mixture was then incubated at 65 °C for 30 min.
A 50 µL aliquot of urea (6M at pH 7.5) was added to make a 5 mg/mL
protein solution. A Zeba spin desalting column was first buffer exchanged with 100 mM sodium phosphate buffer (pH 7.5) (300 µL of buffer each time, spin down
for 1 min at 1000 g, 4 times in total for each spin column). Two separate Zeba spin columns were used, with each loaded with 120 µL of protein sample. The
flow-through of one sample was used for Asp-N digestion, while the flow-through of the other was labeled with iTRAQ directly as a control.
4.3.4 AspN-digestion
6 µg of endoproteinase Asp-N was dissolved in 120 µL of water. The
enzyme was mixed with 60 µg of cell lysate (after guanidination and Zeba spin
desalting clean-up), and the mixture was incubated at 37 °C for 18 h. The sample was freeze-dried prior to iTRAQ labeling.
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Two 25 µg control portions of cell lysate (after guanidination and Zeba spin
desalting clean-up) were each diluted with 20 µL of urea (6M, with 0.1 M
phosphate buffer, pH 8.5) in separate vials. Each cell lysate vial was mixed with
the iTRAQ reagent dissolved in 70 µL ethanol; one vial was reacted with iTRAQ
tag 114 and the other with iTRAQ tag 115. The mixture was incubated for 2 hr at room temperature.
Two replicates of 25 µg of AspN digested cell lysate were each diluted with
20 µL of phosphate buffer (pH 8.5) in separate vials. Each digested sample vial
was mixed with the iTRAQ reagent dissolved in 70 µL ethanol; one vial was
reacted with iTRAQ tag 116 and the other with iTRAQ tag 117. The mixture was incubated for 2 hr at room temperature.
All the iTRAQ labeled samples were pooled into a single vial and dried by centrifuging. The sample was re-suspended in 196 µL of sodium bicarbonate (0.1
M).
4.3.6 Trypsin digestion
4 µg of trypsin (1 µg/µL in water) was added to the pooled iTRAQ solution.
The solution was incubated at 37 °C overnight.
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PBS buffer (200 µL, 0.1 M sodium phosphate and 0.3 M sodium chloride,
pH 7.2) was added to the trypsin-digested solution, which was directly added to NHS-activated agarose dry resin. After mixing the sample end over end for 1 hr, the spin column was centrifuged at 1000 g for 1 min and the flow through was collected; this flow-through contained the N-termini enriched portion. The resin was further washed twice with 400 µL of PBS buffer, with collection of the flow
through. These three flow-through portions were combined and desalted by C18 cartridge. The salt was washed out with 0.1% formic acid in water and the N- terminal peptides were eluted with 0.1% formic acid in 80% acetonitrile in water. After drying the solvent by centrifuge, the N-terminal peptides were re-dissolved
with 40 µL of water and subjected to immediate LC-MS/MS analysis or stored at
−20 °C for further analysis.
4.3.8 LC-MS/MS
LC-MS/MS was performed on a capillary Dionex U3000 HPLC system (Thermo Scientific, San Jose, CA) coupled to a QExactive Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA). A Targa C18 column from Higgins Analytics (150 cm* 300 µm, 3 µm particle) was used with mobile phase A
(0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile). Peptides were eluted over 90 min by increasing mobile phase B from 0 to 30% linearly. Mass spectrometry data were acquired automatically using Xcalibur
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software (Thermo Scientific) in data dependent acquisitions, with both survey scans and MS2 scans in the FT mode (resolution set at 35000 for parent ions and 17500 for fragment ions). Each survey scan was followed by 12 MS/MS scans. MS/MS spectra were acquired in HCD mode with normalized collision energy (NCE) of 25.
Peptides were identified by the Thermo Proteome Discovery 1.3 software package (Thermo Scientific). Peptide and protein identifications were obtained using Sequest and Mascot algorithms. The E. coli K12 strain database prepared in a fashion similar to that described by Dormeyer et al23 was used for searching. Specifically, all protein sequences in the database were cut at arginine (C- terminal); for each resulting peptide (i.e., the series of neo-N-terminal peptides created as a result of these cleavages), multiple sequence database entries were generated that represented sequential truncations of each N-terminal amino acids. Only sequences 6 amino acids or longer were retained in the database. The mass tolerance for parent ions was set to be 10 ppm and 0.1 Dalton for fragment ions. Trypsin cleavage was applied with up to 1 missed cleavage in the search. Carboamidomethyl of cysteine (+57.021Da) was set as a static modification. Dynamic modification included guanidination for lysine (+42.022 Da) and N- terminal modification with iTRAQ (+144.102 Da). Percolator (Thermo Scientific) was used for peptide filtering, with a peptide FDR threshold set to 0.01.
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