Iodoacetamide iaa

Electrophoresis bands
were manually excised and transferred to 2.5 mL protein LoBind tubes
(Eppendorf) and then washed twice with distilled water and 50% (v/v)
ACN/25 mM AmBic until no trace of blue color was observed. Afterward,
the gel spots were washed with 25 mM AmBic and dehydrated with pure
ACN. Fifty microliters of 20 mM DTT (Bio-Rad) in 25 mM AmBic was added
for 1 h at 37 °C. After this time, the solution was removed and
washed with 100 μL of 25 mM AmBic. Again, 100 μL of ACN
was added, and once the gel turned white, it was removed. Next, 50
μL of 100 mM iodoacetamide (IAA, Bio-Rad) in 25 mM AmBic was
added and allowed to react in the dark at room temperature for 45
min. Finally, the pellets were washed repeatedly with 25 mM AmBic/50%
ACN solution and dried with 100% ACN.
For protein digestion,
30 μL of trypsin (20 ng/μL in 12.5 mM AmBic/2% (v/v) ACN)
was added to the gel spots and incubated for 60 min at 0 °C.
The nonabsorbed trypsin solution was subsequently removed, and the
gels were covered with 100 μL of 12.5 mM AmBic. The samples
were incubated for 12 h at 37 °C, and then 50 μL of 5%
(v/v) formic acid (FA) was added. The supernatant was transferred
to a fresh LoBind tube, and the peptides were isolated using 3 consecutive
50% (v/v) ACN/0.1% (v/v) trifluoroacetic acid (TFA) extractions and
a final wash with ACN. The samples were dried and stored at −20
°C until use.

Potassium bromide, potassium phosphate, diethylenetriaminepentaacetic acid, sucrose, agar, tris, ammonium bicarbonate, sodium deoxycholate, trifluoroacetic acid (TFA), Luria-Bertani (LB) broth, formic acid and acetonitrile (MS grade) were purchased from Sigma-Aldrich (San Luis, USA). Dithiothreitol (DTT) and iodoacetamide (IAA) were purchased from Bio-Rad. Trypsin was purchased from Promega. Synthetic labeled peptides were purchased from Thermo Scientific. Escherichia coli BL21(DE3) was kindly provided by Professor Shaker Chuck Farah.

Iron(III) chloride hexahydrate (FeCl₃·6H₂O), chicken avidin (CA), human immunoglobulin G (IgG), bovine serum album (BSA), N,N’-methylenebisacrylamide (MBA), γ-methacryloxypropyltrimethoxysilane (MPS), tetraethyl orthosilicate (TEOS), triethylammonium bicarbonate buffer (TEAB), [2-(methacryloyloxy)ethyl]trimethylammonium chloride solution (MAC), 2,5-dihydroxybenzoic acid (DHB), ammonium bicarbonate (NH₄HCO₃), trifluoroacetic acid (TFA), formic acid (FA), formaldehyde (HCHO), formaldehyde-d2 (DCDO), TPCK-treated trypsin and peptide-N-glycosidase (PNGase F) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dithiothreitol (DTT) and iodoacetamide (IAA) were purchased from BioRad (Hercules, CA, USA). HPLC grade acetonitrile (ACN), ethanol and commercial HILIC (SeQuant, ZIC-HILIC, 3.5 μm) were purchased from Merck (Darmstadt, Germany). Sodium acetate (NaAc), ethylene glycol (EG) and ammonium solution (37%) were purchased from Sinopharm Chemical Reagent Company (Shanghai, China). Ultrapure water was prepared from a Millipore purification system (Billerica, MA, USA). All other chemical agents were obtained from Shanghai Chemical Reagent.