BlotGlyco Glycan purification and labeling kit

Transcription

1 Aug. 14th, 2012 BlotGlyco Glycan purification and labeling kit S-BIO Business Division SUMITOMO BAKELITE CO., LTD. 1

2 BlotGlyco kit Biological sample Glycan Hz BlotGlyco :Label MS HPLC 2

3 BlotGlyco kit is universal platform for glycan sample preparation. Crude sample Trypsin or Surfactant treatment PNGaseF or Hydrazine decomposition Glycan purification by beads On-beads methyl esterification of sialic acid BlotGlyco kit s protocol covers these steps. Original Tag aowr Per- Non labeled methylation * PA 2AB Non labeled APTS * * Under MALDI-TOF MS Monosaccharide HPLC HPAEC CE, development (positive mode) analysis LC-MS -PAD CE-MS 3

4 Conventional method of glycan analysis Crude sample containing released glycan (Ex.glycoprotein, serum, plasma,cell, tissue, treated by PNGase) Column chromatography 2-3days Many steps are required labeling MS HPLC The bottleneck of the conventional method is time-consuming and low accuracy. 4

5 BlotGlyco kit MS Crude sample containing released glycan (Ex.glycoprotein, serum, plasma,cell, tissue, treated by PNGase) BlotGlyco beads 4.5~6 hours HPLC Precise purification and labeling Beads based process enables to obtain highly purified and labeled glycans, easily and speedy. 5

6 BlotGlyco has been reviewed as At last, large-scale functional glycomics in Analytical Chemistry. (p1354, March 1, 2008) 6

7 What is the BlotGlyco beads? polymer beads with high density hydrazide groups Hz HZ:-NHNH 2 Product condition: Dry Powder hydrazide groups : 2 mmol / 1 mg beads (evaluated by TNBS test) 5 mg beads / one assay 10 mmol Hz/ one assay 7

8 How does BlotGlyco work? Applying beads into crude sample Aldehyde of reducing end of glycan binds covalently to hydrazide of BlotGlyco beads. *Hz: hydrazide group (-NHNH 2 ) HO HO HO O OH OH HO HO HO OH OH O HO HO HO OH OH N NH Hz Peptide Glycan Lipid Protein DNA Capturing and Purification Various labeling for MS, HPLC Glycan captured by BlotGlyco beads endure harsh wash. All impurities, even peptides, surfactants, are easily removed! Labeled glycan All the process are carried out in one spin column tube! No special equipment is necessary except a heating block and a small desktop centrifuge. 8

9 Choice of Labeling Step MALDI-TOF MS A. Labeling by on beads imine-exchange reaction B. Labeling with fluorescent amino-compound HPLC, LC-MS Captured glycans C. Non-Labeling (HPAEC-PAD etc.) 9

10 A) Labeling by on-beads imine-exchange -ONH 2 HO HO HO OH OH excess -ONH 2 OH N NH HO HO HO OH N O Glycans are labeled with special reagent named aowr for high sensitive MALDI-TOF MS. Other reagents retaining aminooxy group or hydrazide group can be also applicable to exchange reaction. H 2 N H 2 N HN O NH O O NH O CH 3 O NH NH The orignal labeling reagent aowr is included in the BlotGlyco kit. 10

11 B) Labeling with amino-compound releasing glycan from BlotGlyco beads in mild acidic condition Addition of 2-AB labeling Solution releasing Labeling Free glycans : 2-AB Glycans are labeled by reductive amination. Fluorescent labels such as 2-aminobenzamide (2-AB), 2-aminobenzoic acid (2-AA), 2-aminopyridine (2-AP,PA) are applicable. Existing databases on 2-AB-labeled glycans are applicable. Commercially available 2-AB-labeled glycans can be used as standards. 11

12 BlotGlyco 96 well plate kit --high throughput and easy operation-- New Release 1.Capturing, purifying and labeling glycan on a 96 well filter plate. Using a heat block for heating process. Using a vacuum manifold in stead of centrifuge. 2.Collecting labeled glycan into a original deep well plate. 3. Removing excess labeling compound by original clean up plate. 4. Analysis HPLC High speed HPLC LC-ESI-MS MALDI-TOF MS In about 6 hours, 96 wells can be processed at once. (Using a plate seal, each well can be processed one at a time.)

13 Fundamental data HPLC analysis of glycans labeled with 2AB* O C NH 2 NH 2 *Labeling compound 2-aminobenzamide Measurement device Waters HPLC system 2695 Alliance separations module 2475 fluorescence detector Ex: 330 nm, Em: 420 nm 13

14 Comparison of BlotGlyco with liquid phase reaction HPLC analysis of 2-AB labeled N-glycans from fetuin of bovine serum (A) Liquid phase 2-AB labeling (B) BlotGlyco one tube 2-AB labeling (A) Liquid phase reaction (B) BlotGlyco method Same results as liquid phase reaction are available by using BlotGlyco. 14

15 Comparison of BlotGlyco with liquid phase reaction HPLC analysis of 2-AB labeled N-glycans from IgG of bovine serum (A) Liquid phase 2-AB labeling (B) BlotGlyco one tube 2-AB labeling (A) neutral Liquid phase reaction Sialic acid (B) BlotGlyco method Same results as liquid phase reaction are available by using BlotGlyco. 15

16 Fluorescence Fluorescence Sensitivity N-glycans were released from each amount of bovine serum IgG. N-glycans were labeled with 2-AB by BlotGlyco and detected by HPLC Black: 10 mg IgG Red: 1 mg IgG Blue: 100 ng IgG Close up Red: 1 mg IgG Blue: 100 ng IgG mg of IgG is sufficient to obtain glycan profile by HPLC. 16

17 Peak area Peak area Peak area Quantitative Reliability of BlotGlyco 8.0E+09 Correlation between concentration of glycan and HPLC peak area *Maltoheptaose solution was used mm - 5 mm 2.0E E E E E E E E E Conc. [mm] 0.0E Conc. [mm] 4.0E E E E+06 Low concentration area: under 10 mm *Detectable from 0.1 mm solution (2 pmol Maltoheptaose) *Standard curve shows linearity in the range of 0.1 mm to 5 mm. 0.0E Conc. [mm] Excellent Standard curve, wide range of linearity. 17

18 Peak area Effect of glycan size Variously-sized glycans *1) were labeled with 2-AB by BlotGlyco and detected by HPLC. Peak areas were compared with each other. 1.6E+08 (Glc) (Glc) 2 (Glc) 5 (Glc) 1 (Glc) 4 (Glc) 6 (Glc) E E E E+00 (Glc)1 (Glc)2 (Glc)3 (Glc)4 (Glc)5 (Glc)6 (Glc)7 *1) Maltooligosaccharide solution was used. Possible to label glycans at a constant rate regardless of their size *2). *2) Monosaccharide was partially lost through cleanup process. 18

19 No effect of fucose at reducing end (HPLC) Sample a,b,and c were 2AB labeled through BlotGlyco protocol and detected by HPLC. a)na2 100% b)na2f 100% Peak area Ratio c)mixture of NA2 and NA2F (NA2 50%, NA2F 50%) 分 Good quantitation. No effect of fucose. 19

20 Purification Efficiency AU Peptides are not removed by conventional purification methods AB-labeled N-glycan (IgG) HPLC (UV, l=245 nm) Red: conventional method Blue:purified with BlotGlyco Impurities (peptides) 2AB-glycan RT As a result of trypsin digestion, sample includes peptides as impurity. Using a conventional purification method, peptides are not observed in fluorescent chromatogram, however, they does exist in background as shown in UV chromatogram (above). BlotGlyco is the key technology for an accurate glycan analysis. 20

21 総面積値 Total peak area Reproducibility of BlotGlyco method Assay variability 1.4E+07 Total glycan recovery (HPLC) 総面積値の比較 #1 #2 #3 1.2E E E+06 Sample: human IgG 2AB-labeled N-glycan Measured by HPLC 6.0E E E E+00 Day 1 Day 2 Day 3 1 日目 2 日目 3 日目 Day 1 Day 2 Day 3 Total Peak area 1.1E7 1.2E7 1.3E7 C.V. (in the day) C.V. (day-to-day) 7.0 C.V.: coefficient of variation Variability is less than 10% (both day-to-day and in the day ) 21

22 Relative ピーク相対値 peak area [%] (%) Peak pattern variability 45 Glycan ピークパターンの比較 peak pattern ( アッセイ間 (HPLC) ) Day 日目 1 2 Day 日目 2 3 Day 日目 Peak ピーク No. # Peak pattern remains high reproducibility. 22

23 Total 総面積値 peak area BlotGlyco s Lot-to-Lot variability Total ロット間比較 glycan recovery ( 回収量 ) (HPLC) 1.4E E E E E E E E+00 88BH01 94BH02 97BH01 Lot numbers ヒ ース ロット of BlotGlyco beads Lot-to-Lot variability is also less than 10% 23

24 Relative 相対比 peak [%] area (%) Peak pattern variability (Lot-to-Lot) Glycan ロット間比較 peak pattern ( ピークパターン (HPLC) ) 88BH01 Lot 1 94BH02 Lot 2 97BH01 Lot Peak ピーク No. # Peak pattern remains high reproducibility. 24

25 Fundamental data MALDI-TOF MS analysis of glycans labeled with special reagent for high-sensitive MALDI-TOF MS. H 2 N O O O NH NH Measurement device Bruker Daltonics Autoflex III TOF/TOF NH H 2 N HN O O CH 3 NH aowr labeling reagent 25

26 Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] Sensitivity N-glycans were released from each amount of bovine serum IgG. Released N-glycans were purified and labeled using BlotGlyco. x N-glycans 1 pmol 1 mg IgG was treated by BlotGlyco. 1/10 was spotted N-glycans 100 fmol 100 ng IgG was treated by BlotGlyco. 1/10 was spotted. N-glycans 10 fmol 10 ng IgG was treated by BlotGlyco. 1/10 was spotted N-glycans from 100 ng IgG are detectable by MALDI-TOF MS. m/z 26

27 Detection of Glycan with Sialic Acid Sialic acid groups has been protected by methylation on BlotGlyco beads. Fetuin (contains sialic acid) Asialofetuin (contains no sialic acid) Man Gal GlcNAc GalNAc NeuAc Fuc m/z Glycan with sialic acid can be detected. 27

28 No effect of fucose at reducing end (MALDI-TOF MS) Sample a,b,and c were aowr labeled through BlotGlyco protocol and detected by MALDI-TOF-MS. a)na2 100% H 2 N O O O NH NH b)na2f 100% H 2 N HN O NH O CH 3 NH aowr label Peak area ratio c)mixture of NA2 and NA2F (NA2 50%, NA2F 50%) Good quantitation. No effect of fucose m/z 28

29 Applications 29

30 Identification of IgG Glycans BlotGlyco +2AB label +HPLC Glycosylation of antibody medicine can be characterized. 30

31 693.6(2) 743.6(7) 774.6(1) 790.6(6) 858.6(4) 883.5(6) 928.5(7) 968.5(3) (6) (4) (6) (4) (8) (1) (3) (4) (9) (3) 984.7(3) (3) (2) (2) (2) Identification of Bovine Fetuin Glycans BlotGlyco + PA label + LC-ESI-MS mv(x100) 2.0 RF-20AXS:Ex:320nm,Em:400nm min Inten. (x10,000) Glycan with four sialic acid were detected m/z MS Spectrum (integration of min)

32 (4) (2) (2) 985.3(6) (3) (2) (2) (3) mv(x100) 2.0 RF-20AXS:Ex:320nm,Em:400nm min Inten. (x100,000) MS m/z MS Spectrum Inten. (x100,000) 2.00 MS m/z MS/MS Spectrum LCMS-IT-TOF SHIMADZU Corporation (Kyoto, Japan)

33 Detection of glycolylneuraminic acid (NeuGc) BlotGlyco + aowr label + MALDI-TOF-MS N-acetylneuraminic acid (NeuAc) N-glycolylneuraminic acid (NeuGc) does not exist in human cells NeuGc is considered to be immunogenic for humans. 33

34 MALDI-TOF MS Human IgG Mixture of Human IgG and bovine IgG Bovine IgG Purification On-BlotGlyco beads methylesterification of sialic acids (only 1 hour process) Labeling with aowr m/z enlargement MALDI-TOF MS Human Human + bovine NeuAc NeuGc Bovine m/z NeuAc / NeuGc discrimination is achieved easily using BlotGlyco and MALDI-TOF MS 34

35 BlotGlyco with proteomics study Gel electrophoresis (SDS-PAGE) 1. Cutting off the band which is of interest. 2. Release of glycans from gel. (for example, by PNGase.) 3. Purification and labeling of glycans by BlotGlyco. 4. Analysis of labeled glycans. Identification of glycan which are linked to the focused protein. 35

36 Glycan profiling of serum BlotGlyco + aowr label +MALDI-TOF-MS N-glycan profile obtained from 5mL human serum Minor components detected: Human serum (5 ml) Estimated structures from mass data (not yet confirmed). Mannose Galactose N-Acetylglucosamine N-Acetylgalactosamine Neuramic acid (sialic acid) Fucose m/z 49 kinds of N-glycans were detected from only 5mL human serum. 36

37 Progression free survival Potential of biomarker discovery Antibody therapy specific glycan p< 0.05 High group Low group (days) Time (days) Specific Plasma glycan predict clinical response and PFS for antibody therapy. J. Proteome Res., 2009, 8(2), pp Identification of predictive biomarkers for response to trastuzumab using plasma FUCA activity and N-glycan identified by MALDI-TOF-MS. Matsumoto K, Shimizu C, Arao T, Andoh M, Katsumata N, Kohno T, Yonemori K, Koizumi F, Yokote H, Aogi K, Tamura K, Nishio K, Fujiwara Y. For details, please see another handout. 37

38 Intens. [a.u.] x Glycan profiling of Cell BlotGlyco + aowr + MALDI-TOF-MS N-glycan profile obtained from 1 X 10 6 HeLa cells Na + Na + GlcNAc Man Fuc Gal Sia Next slide 0.0?? * m/z 38

39 Intens. [a.u.] N-glycan profile obtained from 1 X 10 6 HeLa cells * * x10 4 * In the range of m/z 2700~5400 * * * 0.5???? * m/z 39

40 Potential of differentiation marker discovery Threshold in Stage-specific embryonic Glycotypes Uncovered by a Full Portrait of Dynamic N-Glycan Expression during Cell Differentiation Molecular & Cellular Proteomics 9, pp , 2010 Maho Amano et al. 40

41 O-linked glycan analysis Hydrazine decomposition + BlotGlyco(labeled with O-benzylhydroxylamine)+MALDI-TOF MS Mucin from Bovine submaxillary gland Porcine stomach mucin m/z

42 Rapid sample preparation from fermentation sample (Under development) IgG 培養上清 Fermentation sample 1.Capturing IgG by Protein A Gel 2.Without releasing IgG from Protein A by acidifying, denaturing protein by surfactants or trypsin. 3.Releasing glycan by PNGaseF 4.Purifying and Labeling Glycan with BlotGlyco Advantage To eliminate time consuming neutralization and desalting process To analyze glycan structure without effect of acid 42

43 Sumitomo Bakelite Co., Ltd. is a total solution provider based on polymer material, plastic processing and precise quality control. We can also provide customized products according to your needs. BlotGlyco beads Hz Various Products Glycan Array Affinity beads Multi well Plate Micro Fluidics chip Cell Culturing laboratory wares Contact Information S-BIO Business Division Sumitomo Bakelite Co., Ltd. s-bio@sumibe.co.jp TEL , FAX