Iodoacetamide

Streptozotocin (STZ), urea, thiourea, sodium dodecyl sulfate (SDS), 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS), 2-mercaptoethanol (2-ME), dithiothreitol (DTT), bromophenol blue, iodoacetamide, RNase A, and DNase I were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Source information for all other assay reagents and materials is stated in the Materials and Methods section described below.

We purchased the therapeutic mAb immunoglobulin (IgG) gamma 1 drugs adalimumab and rituximab from Eisai Co., Ltd. (Tokyo, Japan) and Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan), respectively. In addition, we purchased Tris-HCl buffer (pH 8.0), 10% trifluoroacetic acid, 0.1% formic acid, acetonitrile 0.1% formic acid and 7 K Dialysis Casettes from Thermo Fisher Scientific (San Jose, CA, USA). We obtained 8 M guanidine hydrochloride from Sigma-Aldrich (St. Louis, MO), dithiothreitol, iodoacetamide, sodium acetate, 2-morpholinoethanesulfonic acid, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid and deuterium oxide from FUJIFILM Wako Pure Chemical Co., Ltd. (Tokyo, Japan), and MicroSpin G-25 columns from GE Healthcare (Chicago, IL). We purchased trypsin from Promega Co. (Madison, WI); angiotensin II from Peptide Institute, Inc. (Osaka, Japan); and ¹⁸O-water, acetonitrile (LC-MS grade), and ordinary water (LC-MS grade) from Merck (Darmstadt, HE).

Clicked and dissolved protein samples were diluted to 4 M urea with 50 mM ammonium bicarbonate (pH 8, AmBic, Sigma-Aldrich). The proteins were reduced with 4 mM DTT (Sigma-Aldrich) for 60 min at room temperature and alkylated in the dark using 8 mM iodoacetamide (Sigma-Aldrich) for 30 min. Residual iodoacetamide was quenched by adding DTT to a final concentration of 4 mM. Next, samples were diluted 2× with 50 mM AmBic and digested with LysC (1:75 enzyme to protein ratio, Wako) for 4 h at 37 °C. Finally, proteins were digested overnight using trypsin (1:50, enzyme to protein ratio, Sigma-Aldrich) at 37 °C in a final volume of 2 ml. Digested material was desalted using 3 cc C18 Seppak cartridges (Waters) and air dried using a vacuum centrifuge.
For the digestion with pepsin, protease incubation (Porcine, 1:50, enzyme to protein ratio, Sigma-Aldrich) was performed for 4 h at 37 °C in 40 mM HCl in a total volume of 2 ml (pH 2). After incubation, pepsin was irreversible inactivated by adjusting the pH > 6 with 1M AmBic. Digested material was desalted using 3 cc C18 Seppak cartridges and air dried using a vacuum centrifuge.

The proteinase inhibitors Nα-p-tosyl-_L-lysine chloromethyl ketone (TLCK) and iodoacetamide were purchased from Wako (Japan), and leupeptin was obtained from the Peptide Institute (Japan).

AAV samples were mixed with 50 mM ammonium bicarbonate (FUJIFILM Wako Chemicals, Osaka, Japan), 6 M guanidine hydrochloride (FUJIFILM Wako Chemicals), 3 mM tris (2-carboxyethyl) phosphine (FUJIFILM Wako Chemicals), and 3 mM iodoacetamide (FUJIFILM Wako Chemicals). The mixture was incubated at 37°C for 1 h in a dark place for denaturation, reducing, and alkylation. Then, the sample solution was desalted by buffer exchange into acetonitrile using MonoSpin C18 (GL Sciences, Inc., Tokyo, Japan). The collected samples were freeze dried by an Epsilon 2-4 LSCplus Freeze-Dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany). The collected pellet was dissolved in 10% acetonitrile in water and digested by trypsin (Promega, Madison, WI, USA) for 30 min at 37°C. Digested samples were injected into a nanoElute UHPLC (CTC Analytics AG, Zwingen, Switzerland) coupled to a trapped ion mobility spectrometer with time-of-flight instrument (Bruker, Billerica, MA, USA). Data analysis with ion mobility data was carried out by PEAKS software ver 10.6 (Bioinformatics Solutions, Inc., Waterloo, Canada).

The proteinase inhibitors Nα-p-tosyl-l-lysine chloromethyl ketone hydrochloride (TLCK) and leupeptin were purchased from Wako (Osaka, Japan) and Peptide Institute (Osaka, Japan), respectively. Iodoacetamide was purchased from Wako (Osaka, Japan).

Tissues from 3-week-old male chickens were a kind gift from Dr. Toshie Sugiyama of Niigata University. Trypsin and chymoTrypsin were purchased from Sigma-Aldrich (St. Louis, MO, USA), and guanidine hydrochloride, iodoacetamide, and dithiothreitol were purchased from Fujifilm Wako Pure Chemical Co. (Osaka, Japan). Recombinant glycoamidase F from Flavobacterium meningosepticum (GAF, aka N-glycosidase F, PNGase F) was purchased from Roche (Mannheim, Germany). Neuraminidase (α2-3,6,8,9 neuraminidase) from Arthrobacter ureafacience was purchased from Nacalai Tesque (Kyoto, Japan). α1-3,4 Fucosidase from the sweet almond tree and β1-4 galactosidase S from Streptococcus pneumonia were purchased from New England BioLabs (Ipswich, MA, USA). Other materials including reagents, columns for LC, and PA-N-glycans from human γ-globulin, α1-AGP, and bovine fetuin were obtained as described previously^{39 (link),40 (link)}. α2,6-Monosialylated PA-N-glycans were prepared from human transferrin and human γ-globulin, and α2,3-monosialylated PA-N-glycans were prepared by treatment of asialo-biantennary PA-N-glycans with recombinant α2,3-sialyltransferase from Photobacterium phosphoreum^{39 (link)}.