advances.sciencemag.org/cgi/content/full/2/1/e1500678/dc1 Supplementary Materials for Chemical synthesis of erythropoietin glycoforms for insights into the relationship between glycosylation pattern and bioactivity Masumi Murakami, Tatsuto Kiuchi, Mika Nishihara, Katsunari Tezuka, Ryo Okamoto, Masayuki Izumi, Yasuhiro Kajihara The PDF file includes: Published 15 January 2016, Sci. Adv. 2, e1500678 (2016) DOI: 10.1126/sciadv.1500678 Fig. S1. Acid stability of sialyloligosaccharide phenacyl ester. Fig. S2. General scheme of the synthesis of a sialylglycopeptide-α-thioester by an improved Boc SPPS method. Fig. S3. HPLC profile and ESI mass spectrum of H-[Ala 1 -Gly 28 ]-α-thioester. Fig. S4. HPLC profile and ESI mass spectrum of H-[Cys 29,33 (Acm)-Tyr 49 ]-αthioester. Fig. S5. HPLC profile and ESI mass spectrum of H-[Cys 29,33 (Acm)- Asn 38 (glycan)-tyr 49 ]-α-thioester. Fig. S6. HPLC profile and ESI mass spectrum of H-[Cys 79 (Thz)-Trp 88 -(formyl)- Lys 97 ]-α-thioester. Fig. S7. HPLC profile and ESI mass spectrum of H-[Cys 79 (Thz)-Asn 83 (glycan)- Trp 88 (formyl)-lys 97 ]-α-thioester. Fig. S8. HPLC profile and ESI mass spectrum of H-[Cys 98 (Thz)-Ala 127 ]-αthioester. Fig. S9. HPLC profile and ESI mass spectrum of H-[Cys 50 -Ala 78 ]-α-hydrazide. Fig. S10. HPLC profile and ESI mass spectrum of H-[Ala 1 -Asn 24 (glycan)-gly 28 ]- α-thioester. Fig. S11. Monitoring NCL between H-[Cys 29, 33 (Acm)-Asn 38 (glycan)-tyr 49 ]-αthioester and H-[Cys 50 -Ala 78 ]-α-hydrazine. Fig. S12. Monitoring NCL between H-[Cys 29, 33 (Acm)-Asn 38 (glycan)-ala 78 ]-αhydrazide and H-[Cys 79 -Asn 83 (glycan)-arg 166 ]-OH. Fig. S13. Monitoring the desulfurization reaction of H-[Cys 29, 33, 161 (Acm)-Cys 50, 79, 98, 128 -Asn 38, 83 (glycan)-arg 166 ]-OH. Fig. S14. Monitoring of the removal of Acm group of H-[Cys 29, 33, 161 (Acm)- Asn 38, 83 (glycan)2-arg 166 ]-OH by RP-HPLC and ESI-MS.
Fig. S15. Monitoring the NCL between H-[Ala 1 -Asn 24 (glycan)-gly 28 ]-α-thioester and H-[Cys 29 -Asn 38, 83 (glycan)2-arg 166 ]-OH. Fig. S16. The folding reaction of EPO N24, N38, N83 (polypeptide form of H-[Ala 1 - Asn 24, 38, 83 (glycan)3-arg 166 ]-OH. Fig. S17. The folding reactions of EPO N38, N83 (polypeptide form of H-[Ala 1 - Asn 38, 83 (glycan)2-arg 166 ]-OH) and EPO N24, N83 (polypeptide form of H-[Ala 1 - Asn 24, 83 (glycan)2-arg 166 ]-OH). Fig. S18. The folding reactions of EPO N24, N38 (polypeptide form of H-[Ala 1 - Asn 24, 38 (glycan)2-arg 166 ]-OH) and EPO N83 (polypeptide form of H-[Ala 1 - Asn 83 (glycan)-arg 166 ]-OH). Results of folding experiments Fig. S19. Monitoring of in vitro folding by SDS-PAGE. Fig. S20. Analysis of disulfide bond positions of EPO N24, N38, N83 2 by trypsin digestion. Fig. S21. Analysis of disulfide bond positions of EPO N38, N83 3 by trypsin digestion. Fig. S22. Analysis of disulfide bond positions of EPO N24, N83 4 by trypsin digestion. Fig. S23. Analysis of disulfide bond positions of EPO N24, N38 5 by trypsin digestion. Fig. S24. Analysis of disulfide bond positions of EPO N83 6 by trypsin digestion. Fig. S25. Characterization of misfolded EPO N24, N83 (compound 7). High-resolution mass spectra of EPO glycoforms Fig. S26. High-resolution mass spectrum of EPO N24, N38, N83 2. Fig. S27. High-resolution mass spectrum of EPO N38, N83 3. Fig. S28. High-resolution mass spectrum of EPO N24, N38, 4. Fig. S29. High-resolution mass spectrum of EPO N38, N83 5. Fig. S30. High-resolution mass spectrum of EPO N83 6.
Supplemental Figure 1. Acid stability of sialyloligosaccharide phenacyl ester. (A) The proposed mechanism of intramolecular catalyst for the acceleration of acid hydrolysis of sialyl linkage. (B) Hydrolysis yield of sialyl linkage under acid condition (40 mm HCl) and the structure of released sialic acid (1). Green lozenge indicates the yield of free sialic acid released from Fmoc-Asn- (sialyloligosaccharide)-oh shown in (B). Blue circle in the panel indicates that phenacyl ester interferes the hydrolysis of sialyl linkage. The released sialic acid by acid hydrolysis was estimated from the consumption of Fmoc-Asn-(sialyloligosaccharide)-OH with UV-detected HPLC-mass instrument (1). (1) M. Murakami, et. al. Angew Chem Int Ed Engl 51, 3567-3572 (2012)
Supplemental Figure 2. General scheme of the synthesis of a sialylglycopeptide-α-thioester by an improved Boc SPPS method (1). (1) M. Murakami, et. al. Angew Chem Int Ed Engl 51, 3567-3572 (2012)
Supplemental Figure 3. HPLC profile and ESI mass spectrum of H-[Ala 1 -Cys 7 -Gly 28 ]-α-thioester. (A) Analytical RP-HPLC of purified H-[Ala 1 -Cys 7 -Gly 28 ]-α-thioester. (B) ESI-Mass spectrum of the purified H-[Ala 1 -Cys 7 -Gly 28 ]-α-thioester (m/z calcd. for C139H233N39O46S3: [M+H] + 3283.8, found 3282.4 (deconvoluted)). Purification was performed by preparative HPLC (Proteonavi C4 Φ10 250 mm, 0.1% TFA: 0.1% TFA in 90% MeCN = 95 : 5 over 5 min then 65 : 35 to 45 : 55 over 30 min at 2.5 ml/min). Supplemental Figure 4. HPLC profile and ESI mass spectrum of H-[Cys 29, 33 (Acm)-Tyr 49 ]-αthioester. (A) Analytical RP-HPLC of purified H-[Cys 29, 33 (Acm)-Tyr 49 ]-α-thioester. (B) ESI-Mass spectrum of the purified H-[Cys 29, 33 (Acm)-Tyr 49 ]-α-thioester (m/z calcd. for C111H171N29O39S4: [M+H] + 2665.0, found 2665.4 (deconvoluted)). Purification was performed by preparative HPLC (Proteonavi C4 Φ10 250 mm, 0.1% TFA : 0.1% TFA in 90% MeCN = 95 : 5 over 5 min then 75 : 25 to 50 : 50 over 30 min at 2.5 ml/min).
Supplemental Figure 5. HPLC profile and ESI mass spectrum of H-[Cys 29, 33 (Acm)-Asn 38 (glycan)- Tyr 49 ]-α-thioester. (A) Analytical RP-HPLC of purified H-[Cys 29, 33 (Acm)-Asn 38 (glycan)-tyr 49 ]-αthioester. (B) ESI-Mass spectrum of the purified H-[Cys 29, 33 (Acm)-Asn 38 (glycan)-tyr 49 ]-α-thioester (m/z calcd. for C211H319N35O102S4: [M+H] + 5107.2, found 5106.4 (deconvoluted). Purification was performed by preparative HPLC (Proteonavi C4 Φ10 250 mm, 0.1% TFA : 0.1% TFA in 90% MeCN = 95 : 5 over 5 min then 75 : 25 to 55 : 45 over 30 min at 2.5 ml/min). Supplemental Figure 6. HPLC profile and ESI mass spectrum of H-[Cys 79 (Thz)-Trp 88 (formyl)- Lys 97 ]-α-thioester. (A) Analytical RP-HPLC of purified H-[Cys 79 (Thz)-Trp 88 (formyl)-lys 97 ]-αthioester. (B) ESI-Mass spectrum of the purified H-[Cys 79 (Thz)-Trp 88 (formyl)-lys 97 ]-α-thioester (m/z calcd. for C103H160N26O32S3: [M+H] + 2371.7, found 2371.4 (deconvoluted). Purification was performed by preparative HPLC (Proteonavi C4 Φ10 250 mm, 0.1% TFA : 0.1% TFA in 90% MeCN = 95 : 5 over 5 min then 75 : 25 to 50 : 50 over 30 min at 2.5 ml/min). Another peak beside main mass peak in B is the desired H-[Cys 79 (Thz)-Trp 88 (formyl)-lys 97 ]-α-thioester, but formyl group of tryptophan removed. This formyl group would be removed during synthesis of EPO-full peptide by NCL and therefore this segment was used for NCL without further purification.
Supplemental Figure 7. HPLC profile and ESI mass spectrum of H-[Cys 79 (Thz)-Asn 83 (glycan)- Trp 88 (formyl)-lys 97 ]-α-thioester. (A) Analytical RP-HPLC of purified H-[Cys 79 (Thz)-Asn 83 (glycan)- Trp 88 (formyl)-lys 97 ]-α-thioester. (B) ESI-Mass spectrum of the purified H-[Cys 79 (Thz)-Asn 83 (glycan)- Trp 88 (formyl)-lys 97 ]-α-thioester (m/z calcd. for C203H308N32O95S3: [M+H] + 4814.0, found for 4814.1 (deconvoluted). Purification was performed by preparative HPLC (Proteonavi C4 Φ10 250 mm, 0.1% TFA : 0.1% TFA in 90% MeCN = 95 : 5 over 5 min then 75 : 25 to 50 : 50 over 30 min at 2.5 ml/min). Supplemental Figure 8. HPLC profile and ESI mass spectrum of H-[Cys 98 (Thz)-Ala 127 ]-αthioester. (A) Analytical RP-HPLC of purified H-[Cys 98 (Thz)-Ala 127 ]-α-thioester. (B) ESI-MS spectrum of the purified H-[Cys 98 (Thz)-Ala 127 ]-α-thioester (m/z calcd. for C131H226N38O44S3: [M+H] + 3134.6, found 3134.8 (deconvoluted)). Purification was performed by preparative HPLC (Proteonavi C4 Φ10 250 mm, 0.1% TFA : 0.1% TFA in 90% MeCN = 95 : 5 over 5 min then 75 : 25 to 50 : 50 over 30 min at 2.5 ml/min).
Supplemental Figure 9. HPLC profile and ESI mass spectrum of H-[Cys 50 -Ala 78 ]-α-hydrazide. (A) Analytical RP-HPLC of purified H-[Cys 50 -Ala 78 ]-α-hydrazide. (B) ESI-Mass spectrum of the purified H-[Cys 50 -Ala 78 ]-α-hydrazide (m/z calcd. for C143H233N43O39S2: [M+H] + 3243.8, found 3244.1 (deconvoluted). Purification was performed by preparative HPLC (Proteonavi C4 Φ10 250 mm, 0.1% TFA : 0.1% TFA in 90% MeCN = 95 : 5 over 5 min then 75 : 25 to 50 : 50 over 30 min at 2.5 ml/min). Supplemental Figure 10. HPLC profile and ESI mass spectrum of H-[Ala 1 -Cys 7 -Asn 24 (glycan)- Gly 28 ]-α-thioester. (A) Analytical RP-HPLC of purified H-[Ala 1 -Cys 7 -Asn 24 (glycan)-gly 28 ]-α-thioester. (B) ESI-Mass spectrum of the purified H-[Ala 1 -Cys 7 -Asn 24 (glycan)-gly 28 ]-α-thioester (m/z calcd. for C239H381N45O109S3: [M+H] + 5726.0, found 5725.7 (deconvoluted). Purification was performed by using a linear gradient (Proteonavi C4 Φ10 250 mm, 0.1% TFA : 0.1% TFA in 90% MeCN = 95 : 5 over 5 min then 75 : 25 to 50 : 50 over 30 min at 2.5 ml/min).
Supplemental Figure 11. Monitoring NCL between H-[Cys 29,33 (Acm)-Asn 38 (glycan)-tyr 49 ]-αthioester and H-[Cys 50 -Ala 78 ]-α-hydrazine. (A) RP-HPLC analysis of reaction mixture at a) starting point (t<1 min) and b) after 3 h, c) deprotection of Pac group completed after 2 h under the basic condition (ph 9.3), and d) after purification of the product (H-[Cys 29, 33 (Acm)-Asn 38 (glycan)-ala 78 ]-αhydrazide). (B) ESI-MS analysis of the product (H-[Cys 29, 33 (Acm)-Asn 38 (glycan)-ala 78 ]-α-hydrazide). m/z calcd. for C336H534N78O136S4: [M+H] + 7971.6, found 7971.1 (deconvoluted).
Supplemental Figure 12. Monitoring NCL between H-[Cys 29, 33 (Acm)-Asn 38 (glycan)-ala 78 ]-αhydrazide and H-[Cys 79 -Asn 83 (glycan)-arg 166 ]-OH. (A) RP-HPLC analysis of reaction mixture a) after 5 h and b) after purification of the product (H-[Cys 29, 33 (Acm)-Asn 38, 83 (glycan)2-arg 166 ]-OH). (B) ESI-MS analysis of the product (H-[Cys 29, 33 (Acm)-Asn 38,83 (glycan)2-arg 166 ]-OH). m/z calcd. for C853H1374N208O322S8: [M+H] + 19952.8, found 19952.8 (deconvoluted).
Supplemental Figure 13. Monitoring the desulfurization reaction of H-[Cys 29, 33, 161 (Acm)-Cys50, 79, 98, 128 -Asn 38, 83 (glycan)2-arg 166 ]-OH (A) RP-HPLC analysis of reaction mixture at a) starting point (t<1 29, 33, min), b) after 2 h and c) the product after purification. (B) ESI-MS analysis of the product (H-[Cys 161 (Acm)-Asn 38, 83 (glycan)2-arg 166 ]-OH). m/z calcd. for C853H1374N208O322S4: [M+H] + 19824.6, found 19823.9 (deconvoluted).
Supplemental Figure 14. Monitoring of the removal of Acm group of H-[Cys 29, 33, 161 (Acm)-Asn 38, 83 (glycan)2-arg 166 ]-OH by RP-HPLC and ESI-MS. (A) RP-HPLC analysis of reaction mixture at a) starting point (t<1 min), b) after 2 h and c) the product after purification. (B) ESI-MS analysis of the product (H-[Cys 29, 33, 161 -Asn 38, 83 (glycan)2-arg 166 ]-OH). m/z calcd. for C844H1359N205O319S4: [M+H] + 19611.3, found 19611.3 (deconvoluted).
Supplemental Figure 15. Monitoring the NCL between H-[Ala 1 -Asn 24 (glycan)-gly 28 ]- -thioester and H-[Cys 29 -Asn 38, 83 (glycan)2-arg 166 ]-OH. (A) RP-HPLC analysis of reaction mixture at a) starting point (t<1 min) and b) after 3 h; c) deprotection of Pac group completed after 2 h under the basic condition (ph 9.3). d) The product after purification. (B) ESI-Mass analysis of the product (H-[Ala 1 - Asn 24, 38, 83 (glycan)3-arg 166 ]-OH). m/z calcd. for C1065H1722N250O423S5: [M+H] + 24957.9, found 24957.0 (deconvoluted).
Supplemental Figure 16. The folding reaction of EPO N24, N38, N83 (polypeptide form of H-[Ala 1 - Asn 24, 38, 83 (glycan)3-arg 166 ]-OH. (A) Scheme of EPO N24, N38, N83 2 folding with redox conditions. (B) RP-HPLC profiles of folding intermediate; 0.1 mg/ml sample a) in dissolving buffer; b) in buffer A for 20 h; c) in buffer B for 1 h and d) for 20 h; e) in buffer C for 15 h, f) 0.01mg/mL sample in buffer C for 15 h. A large peak at 14.5 min in each HPLC profiles is due to change the gradient of solution. (C) SDS- PAGE of the folding intermediates. All sample were loaded and analyzed without dithiothreitol (DTT) treatment.
Supplemental Figure 17. The folding reactions of EPO N38, N83 (polypeptide form of H-[Ala 1 -Asn 38, 83 (glycan)2-arg 166 ]-OH) and EPO N24, N83 (polypeptide form of H-[Ala 1 -Asn 24, 83 (glycan)2-arg 166 ]- OH). ESI-Mass spectrum of (A) EPO N38, N83 3 and (B) EPO N24, N83 4. RP-HPLC profiles of folding intermediate (C) EPO N38, N83 3 and (D) EPO N24, N83 4; 0.1 mg/ml sample a) in dissolving buffer; b) in buffer A for 20 h; c) in buffer B for 1 h and d) for 20 h; e) in buffer C for 15 h, f) 0.01 mg/ml sample in buffer C for 15 h. A large peak at 14.5 min in each HPLC profiles is due to change the gradient of solution.
Supplemental Figure 18. The folding reactions of EPO N24, N38 (polypeptide form of H-[Ala 1 -Asn 24, 38 (glycan)2-arg 166 ]-OH) and EPO N83 (polypeptide form of H-[Ala 1 -Asn 83 (glycan)-arg 166 ]-OH). ESI- Mass data of (A) EPO N24, N38 5 and (B) EPO N83 6. RP-HPLC profiles of folding intermediate (C) EPO N24, N38 5 and (D) EPO N83 6; 0.1 mg/ml sample a) in dissolving buffer; b) in buffer A for 20 h; c) in buffer B for 1 h and d) for 20 h; e) in buffer C for 15 h, f) 0.01 mg/ml sample in buffer C for 15 h. A large peak at 14.5 min in each HPLC profiles is due to change the gradient of solution. Results of folding experiments HPLC-Analytical yield was estimated by the calculation: area of folded EPO / (area of folded EPO + misfolded EPO). Folded EPO N24, N38, N83 2: The yield of compound 2 was estimated to be 86% by HPLC. ESI-MS: m/z calcd. for C1065H1718N250O423S5: [M+H] + 24953.8, found 24953 (deconvoluted). Folded EPO N38, N83 3: The yield of compound 3 was estimated to be 90% by HPLC. ESI-MS: m/z calcd. for C981H1582N244O362S5: [M+H] + 22747.9, found 22747 (deconvoluted). Folded EPO N24, N83 4: The yield of compound 4 was estimated to be 66% by HPLC. ESI-MS: m/z calcd. for C981H1582N244O362S5: [M+H] + 22747.9, found 22747 (deconvoluted). Folded EPO N24, N38 5: The yield of compound 5 was estimated to be 76% by HPLC. ESI-MS: m/z calcd. for C981H1582N244O362S5: [M+H] + 22747.9, found 22747 (deconvoluted). Folded EPO N83 6: The yield of compound 6 was estimated to be 63% by HPLC. ESI-MS: m/z calcd. for C897H1446N238O301S5: [M+H] + 20541.9, found 20541 (deconvoluted).
Supplemental Figure 19. Monitoring of in vitro folding by SDS-PAGE. Results of (A) EPO N38, N83 3, (B) EPO N24, N83 4, (C) EPO N24, N38 5, (D) EPO N83 6. All sample were loaded and analyzed without DTT treatment. In terms of EPO N24, N38, N83 2 folding is shown in fig. S16.
Supplemental Figure 20. Analysis of disulfide bond positions of EPO N24, N38, N83 2 by trypsin digestion. (A) HPLC data of a) after trypsin digestion (12 h incubation) and b) after trypsin digestion (12 h incubation) followed by the treatment with TCEP. The blue dotted line indicates a disulfide bond. (B) ESI-Mass spectra of peak (1) (6). Based on the mass data, we determined peptide and glycopeptide sequence as well as the number of disulfide bond. Peptide of peak (1) was found to have a disulfide bond and this peak (1) disappeared after reduction with TCEP. This observation indicated peptide of peak (1) had a disulfide bond. The products reduced could not be found in the same analytical conditions. A peak (4) was newly observed after TCEP treatment and this peak did not include peptideproducts, so we concluded this peak (4) might be derived from reagent. Peak (5) identical with the structure of the reduced peptide of peak (2). Peak (3) and (6) were found to be an identical peptide. The asterisk (*) means from an unspecific compound.
Supplemental Figure 21. Analysis of disulfide bond positions of EPO N38, N83 3 by trypsin digestion. (A) HPLC data of a) after trypsin digestion (12 h incubation) and b) after trypsin digestion (12 h incubation) followed by the treatment with TCEP. The blue dotted line indicates a disulfide bond. (B) ESI-Mass spectra of peak (1) (6). Analysis of these peptide fragments was performed with the same protocol in the analysis of EPO N24, N38, N83 2 (fig. S20). The asterisk (*) means from an unspecific compound. A peak (4) was newly observed after TCEP treatment and this peak did not include peptideproducts, so we concluded this peak (4) might be derived from reagent.
Supplemental Figure 22. Analysis of disulfide bond positions of EPO N24, N83 4 by trypsin digestion. (A) HPLC data of a) after trypsin digestion (12 h incubation) and b) after trypsin digestion (12 h incubation) followed by the treatment with TCEP. The blue dotted line indicates a disulfide bond. (B) ESI-Mass spectra of peak (1) (6). Analysis of these peptide fragments was performed with the same protocol in the analysis of EPO N24, N38, N83 2 (fig. S20). The asterisk (*) means from an unspecific compound. A peak (4) was newly observed after TCEP treatment and this peak did not include peptideproducts, so we concluded this peak (4) might be derived from reagent.
Supplemental Figure 23. Analysis of disulfide bond positions of EPO N24, N38 5 by trypsin digestion. (A) HPLC data of a) after trypsin digestion (12 h incubation) and b) after trypsin digestion (12 h incubation) followed by the treatment with TCEP. The blue dotted line indicates a disulfide bond. (B) ESI-Mass spectra of peak (1) (6). Analysis of these peptide fragments was performed with the same protocol in the analysis of EPO N24, N38, N83 2 (fig. S20). The asterisk (*) means from an unspecific compound. A peak (4) was newly observed after TCEP treatment and this peak did not include peptideproducts, so we concluded this peak (4) might be derived from reagent.
Supplemental Figure 24. Analysis of disulfide bond positions of EPO N83 6 by trypsin digestion. (A) HPLC profile of a) after trypsin digestion (12 h incubation) and b) after trypsin digestion (12 h incubation) followed by the treatment with TCEP. The blue dotted line indicates a disulfide bond. (B) ESI-Mass spectra of peak (1) (6). Analysis of these peptide fragments was performed with the same protocol in the analysis of EPO N24, N38, N83 2 (fig. S20). The asterisk (*) means from an unspecific compound. A peak (4) was newly observed after TCEP treatment and this peak did not include peptideproducts, so we concluded this peak (4) might be derived from reagent.
Supplemental Figure 25. Characterization of misfolded EPO N24, N83 (compound 7). (A) Structure of the resultant glycopeptide and peptide fragments (1), (2) and (3) after trypsin digestion. These structures of (1)-(3) were determined based on mass analysis. The blue dotted line between Cys 29 and Cys 33 indicate disulfide bond. HPLC profile of a) after trypsin digestion (12 h incubation) and b) after trypsin digestion (12 h incubation) followed by the treatment with TCEP. Peak (2) is the reduced sialylglycopeptide fragment of peak (1). (B) SDS-PAGE; line a) without treatment of TCEP; b) treated with 10 mm TCEP and c) with 40 mm TCEP; d) treated with DTT, e) folded EPO N24, N83 (compound 3) treated with DTT. Line a) shows several smear bands from 50 KDa to ca. 70 KDa (See fig. S19 B). (C) CD spectrum of compound 7.
High-resolution mass spectra of EPO glycoforms All measurements were performed with Bruker SolariX (9.4 Tesla). Samples were dissolved into a solution of 50% MeOH containing 1% acetic acid and subjected into an instrument as direct infusion. An internal standard (ESI tuning mix: Agilent Technology ) was used (1521.971475 and 2121.933152). Ionization condition employed electrospray ionization under the 200 C and therefore the small amount of the terminal sialic acid was removed during ionization, although conventional ESI mass instrument did not show the considerable ion peaks derived from desialylation (Bruker Amazon ETD shown Fig. 4, and fig. S17 and S18). The amounts of desialylated ion peaks were automatically increased dependent on the number of sialyloligosaccharides in EPO molecules. This increasing in the intensity of ion peaks should achieve to ca 20% amount in the case of EPO having three glycans, even though an individual sialyloligosaccharide includes 3-5% desialylated oligosaccharide. The amount of population of desialylated oligosaccharides can be estimated by a simple equation (a glycoform having a desialylated oligosaccharide at the 83 position = 100% N24 +100% N38 +95% N83. Another example: a glycoform having two desialylated oligosaccharide at the 24 and 83 position = 95% N24 +100% N38 +95% N83. Total population of desialylated oligosaccharide in high-resolution mass spectrometry was observed according to the addition of these total possible calculations. The marks shown (* and ) in fig. S26-30 are desialylated EPO glycoforms and internal standard ion peak position, respectively. The large ion peaks ( ) of the internal standard were erased by Photoshop CS3 and other ion peaks observed are shown as it is. The most largest monoisotopic ion peak was selected for the determination of correct mass and these values are shown in figure comparing computational simulation pattern.
Supplemental Figure 26. High-resolution mass spectrum of EPO N24, N38, N83 2. The marks shown (* and ) in Figure are desialylated EPO glycoforms and internal standard ion peak position, respectively. The observed monoisotopic high resolution mass was 1664.4700 (the most potent monoisotopic peak: calcd 1664.4712). The estimation (deconvolution) of mass for C1065H1718N250O423S5 is 24952.0500 (based on the most potent monoisotopic peak: calcd 24952.0680).
Supplemental Figure 27. High-resolution mass spectrum of EPO N38, N83 3. The marks shown (* and ) in Figure are desialylated EPO glycoforms and internal standard ion peak position, respectively. The observed monoisotopic high resolution mass was 1517.4114 (the most potent monoisotopic peak: calcd 1517.4195). The estimation (deconvolution) of mass for C981H1582N244O362S5 is 22746.1710 (based on the most potent monoisotopic peak: calcd 22746.2925).
Supplemental Figure 28. High-resolution mass spectrum of EPO N24, N38, 4. The marks shown (* and ) in Figure are desialylated EPO glycoforms and internal standard ion peak position, respectively. The observed monoisotopic high resolution mass was 1517.4121 (the most potent monoisotopic peak: calcd 1517.4195). The estimation (deconvolution) of mass for C981H1582N244O362S5 is 22746.1815 (based on the most potent monoisotopic peak: calcd 22746.2925).
Supplemental Figure 29. High-resolution mass spectrum of EPO N38, N83 5. The marks shown (* and ) in Figure are desialylated EPO glycoforms and internal standard ion peak position, respectively. The observed monoisotopic high resolution mass was 1517.4182 (the most potent monoisotopic peak: calcd 1517.4195). The estimation (deconvolution) of mass for C981H1582N244O362S5 is 22746.2733 (based on the most potent monoisotopic peak: calcd 22746.2928). A slight disorder of observed peak pattern between found and simulation might be due to measurement with slight high concentration of glycoprotein 5 sample.
Supplemental Figure 30. High-resolution mass spectrum of EPO N83 6. The marks shown (* and ) in Figure are desialylated EPO glycoforms and internal standard ion peak position, respectively. The observed monoisotopic high resolution mass was 1468.1803 (the most potent monoisotopic peak: calcd 1468.1792). The estimation (deconvolution) of mass for C897H1446N238O301S5 is 20540.5253 (based on the most potent monoisotopic peak: calcd 20540.5099).