Supporting Information Venetz et al. 10.1073/pnas.1416694112 39892 100 % % 100 0 800 0 20000 25000 30000 35000 40000 1000 45000 1200 1400 50000 1600 1800 1900 m/z 55000 60000 m [Da] Fig. S1. Representative ESI-TOF mass spectrum of deglycosylated F8-IL9 (SE batch 1) after PNGase F treatment and protein-A purification. (Inset) Total ion chromatogram. Fig. S2. Amino acid sequence of glycosylated F8-IL9 fusion proteins. F8-based diabody, blue; Gly/Ser-linker, gray; mIL9, green; N-glycosylation sites, red. 2.0 FI [mV] 1.6 1.2 0.8 0.4 0.0 5 6 7 8 9 10 11 12 13 14 15 [GU] Fig. S3. Representative HILIC-HPLC glycoprofile of the 2-AB–labeled glycan pool of deglycosylated (DS) F8-IL9 (TGE batch 3) after PNGase F treatment. Venetz et al. www.pnas.org/cgi/content/short/1416694112 1 of 6 TGE DG DS anti-CD31 anti-IL9 merged SE Fig. S4. Ex vivo immunofluorescence stainings of F8-IL9 obtained from CHO cell cultures SE batch 3 and TGE batch 3 as well as deglycosylated (DG) and desialylated (DS) F8-IL9 derived from TGE batch 3. Mouse IL9 is stained in green; the endothelial marker CD31 is red; overlapping stainings in merged images are yellow. (Scale bars: 100 μm.) 6 F8-IL9 [%ID/g] 5 4 3 2 1 0 tumor liver lung spleen heart kidney intestine blood Fig. S5. Quantitative in vivo biodistribution profiles of desialylated F8-IL9 batch 3 (gray) and deglycosylated F8-IL9 batch 1 (black) 24 h after i.v. injection into F9-tumor–bearing mice. Venetz et al. www.pnas.org/cgi/content/short/1416694112 2 of 6 Fig. S6. MS profiles of tryptic glycopeptides of F8-IL9. (A and B) TGE batch 3 (A) and SE batch 3 (B) show the glycan microheterogeneity of site 2. All observed glycoforms on each site are summarized in Table S2. The gray bar represents the peptide backbone. (C) MS/MS spectrum of m/z 1482.175 (+2) confirming the identity of this glycopeptide. Note: Based on highly abundant glycan oxonium ions, 204.09 for [HexNAc]+ and 366.14 for [HexNAcHex]+, Y1 ion, [peptide+ HexNAc]+, and few b fragmented ions from peptide backbone, the amino acid sequence of this peptide could be confirmed as EGLLQLTNATQK, and its glycan structure was core-fucosylated N-glycan with three GlcNAc. Here, the nomenclature of peptide fragment ions and glycan fragmentation ions were done according to previous studies (1, 2). 1. Roepstorff P, Fohlman J (1984) Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed Mass Spectrom 11(11):601. 2. Dell A, et al. (1994) Mass spectrometry of carbohydrate-containing biopolymers. Methods Enzymol 230:108–132. Venetz et al. www.pnas.org/cgi/content/short/1416694112 3 of 6 Table S1. Summary for observed m/z of permethylated glycans and their corresponding carbohydrate compositions Core represents trimannosyl N-glycan core structure, containing two N-acetylglucosamines and three mannoses. Rows shaded in gray indicate glycan structures with sialic acids (underlined). Fuc, Fucose; Gal, galactose; GlcNAc, N-acetylglucosamine; Man, mannose; NeuAc, N-acetylneuraminic acid. *Base peak in MS spectra. Venetz et al. www.pnas.org/cgi/content/short/1416694112 4 of 6 Table S2. Summary table of site-specific glycopeptide analysis for each site of F8-IL9 Site Observed m/z* Peptide [M+H]+ 1 2 3 4 1,129.4464+ 1,180.2144+ 1,210.4744+ 1,230.9864+ 1,261.2464+ 1,301.7594+ 1,352.5334+ 1,393.0474+ 1,484.3204+ 1,283.2554+ 1,356.0214+ 1,447.3014+ 1,520.0854+ 1,611.3624+ 1,266.5822+ 1,279.0992+ 1,380.6392+ 974.7793+ 1,482.1752+ 1,542.6892+ 1,563.2012+ 1,583.7132+ 1,096.4873+ 1,664.7452+ 1,110.1683+ 1,150.5112+ 1,164.1823+ 1,218.2003+ 1,272.2173+ 1,393.9323+ 1,125.8303+ 1,193.5233+ 1,222.8603+ 1,247.5443+ 1,344.5713+ 1,369.2523+ 1,441.6043+ 1,466.2853+ 1,563.3133+ 1,660.3483+ 1,265.4922+ 1,346.5222+ 1,367.0352+ 1,427.5452+ 1,448.0602+ 1,468.5722+ 1,529.0852+ 1,549.5982+ 1,610.1122+ 1,630.6272+ 1,711.6512+ 1,128.4983+ 1,182.5203+ 1,196.1963+ 1,236.5383+ 1,250.2133+ 1,263.8843+ 1,304.2303+ 1,317.9073+ 1,358.2453+ 1,371.9253+ 1,425.9423+ 1,479.9573+ 3,070.086 1,315.722 1,085.439 1,938.920 Venetz et al. www.pnas.org/cgi/content/short/1416694112 Peptide sequence Glycan composition† K.CSCSGNVTSCLCLSVPTDDCTTPCYR.E‡ CoreFucGlcNAc2 CoreFucGlcNAc3 CoreFuc(GlcNAcGal)2 CoreFucGlcNAc4 CoreFuc(GlcNAcGal)2GlcNAc CoreFuc(GlcNAcGal)3 CoreFuc(GlcNAcGal)3GlcNAc CoreFuc(GlcNAcGal)4 CoreFuc(GlcNAcGal)5 CoreFuc(GlcNAcGal)2NeuAc CoreFuc(GlcNAcGal)2NeuAc2 CoreFuc(GlcNAcGal)3NeuAc2 CoreFuc(GlcNAcGal)3NeuAc3 CoreFuc(GlcNAcGal)4NeuAc3 R.EGLLQLTNATQK.S‡ CoreHex2 CoreFucGlcNAc CoreFucGlcNAc2 CoreFuc(GlcNAcGal)GlcNAc CoreFucGlcNAc3 CoreFuc(GlcNAcGal)2 CoreFuc(GlcNAcGal)1GlcNAc2 CoreFucGlcNAc4 CoreFuc(GlcNAcGal)2GlcNAc CoreFuc(GlcNAcGal)GlcNAc3 CoreFuc(GlcNAcGal)GlcNAc3 CoreFuc(GlcNAcGal)3 CoreFuc(GlcNAcGal)2GlcNAc2 CoreFuc(GlcNAcGal)3GlcNAc CoreFuc(GlcNAcGal)4 CoreFuc(GlcNAcGal)5 CoreFuc(GlcNAcGal)2NeuAc CoreFuc(GlcNAcGal)2NeuAcGlcNAc CoreFuc(GlcNAcGal)2NeuAc2 CoreFuc(GlcNAcGal)3NeuAc CoreFuc(GlcNAcGal)3NeuAc2 CoreFuc(GlcNAcGal)4NeuAc CoreFuc(GlcNAcGal)3NeuAc3 CoreFuc(GlcNAcGal)4NeuAc2 CoreFuc(GlcNAcGal)4NeuAc3 CoreFuc(GlcNAcGal)4NeuAc4 K.NITCPSFSC.E§ CoreFucGlcNAc2 CoreFuc(GlcNAcGal)GlcNAc CoreFucGlcNAc3 CoreFuc(GlcNAcGal)2 CoreFuc(GlcNAcGal)GlcNAc2 CoreFucGlcNAc4 CoreFuc(GlcNAcGal)2GlcNAc CoreFuc(GlcNAcGal)GlcNAc3 CoreFuc(GlcNAcGal)3 CoreFuc(GlcNAcGal)2GlcNAc2 CoreFuc(GlcNAcGal)3GlcNAc1 EKPCNQTMAGNTLSFLK§ CoreFucGlcNAc2 CoreFuc(GlcNAcGal)GlcNAc CoreFucGlcNAc3 CoreFuc(GlcNAcGal)2 CoreFuc(GlcNAcGal)GlcNAc2 CoreFucGlcNAc4 CoreFuc(GlcNAcGal)2GlcNAc CoreFuc(GlcNAcGal)GlcNAc3 CoreFuc(GlcNAcGal)3 CoreFuc(GlcNAcGal)2GlcNAc2 CoreFuc(GlcNAcGal)3GlcNAc CoreFuc(GlcNAcGal)4 TGE SE × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × 5 of 6 Table S2. Cont. Site Observed m/z* Peptide [M+H]+ 3+ 1,333.565 1,414.9333+ 1,430.6043+ 1,455.2813+ 1,468.9553+ 1,522.9753+ 1,552.3073+ 1,576.9923+ Peptide sequence Glycan composition† CoreFuc(GlcNAcGal)2NeuAc CoreFuc(GlcNAcGal)NeuAcGlcNAc3 CoreFuc(GlcNAcGal)2NeuAc2 CoreFuc(GlcNAcGal)3NeuAc CoreFuc(GlcNAcGal)2NeuAcGlcNAc2 CoreFuc(GlcNAcGal)3NeuAcGlcNAc1 CoreFuc(GlcNAcGal)3NeuAc2 CoreFuc(GlcNAcGal)4NeuAc TGE SE × × × × × × × × × × × × × Putative glycan composition based on m/z values of N-linked glycans and corresponding peptide sequences after peptidase treatment are given. *Each assignment had at least one MS/MS spectrum to support its identity. † To confirm our assignments, all samples were treated with neuraminidase and analyzed. ‡ After trypsin digestion, glycopeptides for the first two sites could be observed. The putative glycosylation sites were marked with an underlined N. All cysteines here were carbamidomethylated. § Because the third and fourth glycosylation sites were located on the same tryptic peptide, the tryptic (glyco)peptide pools from SEand TGE-derived F8-IL9 were further digested by either AspN or GluC endopeptidase. The glycan structures obtained from both treatments were the same. Here, the results from trypsin/AspN were demonstrated. Venetz et al. www.pnas.org/cgi/content/short/1416694112 6 of 6