Mass Spectrometry of Glycans and Glycoproteins

Transcription

1 Mass Spectrometry of Glycans and Glycoproteins Ron rlando Complex Carbohydrate Research Center University of Georgia Athens, GA Course utline Saturday Morning 7:00 am 8:00 am Continental Breakfast 8:00 am 8:30 am Introduction to Glycans, Glycosylation, Glycobiology Ron rlando 8:30 am 9:00 am Derivatization and Classical Carbohydrate Characterization Ron rlando 9:00 am 10:00 am Issues related to Glycoproteins, and Glycan Biosynthesis Ron rlando 10:00 am 10:30 am Morning Break 10:30 am 12:00 pm Glycoproteins Release and Analyze, and Glycorproteomic Approaches Ron rlando 12:00 pm 1:00 pm Lunch 1

2 Course utline Saturday Afternoon 1:00 pm 2:00 pm Glycopeptides and Methods for Site specific Analysis Ron rlando 2:00 pm 3:00 pm Glycan Quantitation Ron rlando 3:00 pm 3:30 pm Afternoon Break 3:30 pm 4:30 pm Bioinformatics of Glycans and Glycoproteins Marshall Bern Glycobiology: biology of sugars Not just what we eat Sugar metabolism and nutrition are not a major focus most of the time It really focuses on things like cell surface sugar structures (glycans) in complex settings like cell cell recognition 2

3 Glycobiology Carbohydrates in Biological Systems Energy metabolism DNA, RNA Structural elements, e.g. chitin, cellulose, etc. (Cellulose l the most abundant organic compound on Earth) Glycoconjugates Glycolipids GPI anchors Proteoglycans Protein N and linked glycosylation Roles of glycoconjugates gy g Protein stability, folding, trafficking (intra & extracellular) Modulation of protein activity, circulatory half life Signaling Development Infection & Immunity Glycoproteins constitute a large and heterogeneous class of glycoconjugates: most secreted or membrane bound/associate proteins are glycosylated gy y 3

4 Cells are sugar coated proteoglycans, glycolipids, glycoproteins Electron microscopic overview of an Alcian blue 8GX stained rat left ventricular myocardial capillary (bar=1 μm). van den Berg B M et al. Circulation Research 2003;92:

5 We catch the flu because of the sugars that coat our sinuses and bronchial airways Influenza viruses recognize neuraminic acids Tamiflu is a neuraminic acid analogue that inhibits this interaction The AB blood group antigens are carbohydrates The difference between A/B and is the addition of one additional sugar while the difference between A and B is the identity of this extra sugar. 5

6 Glycomics: the sugar version of genomics and proteomics The cell surface landscape is richly decorated with oligosaccharides anchored to proteins ti or lipids id within the plasma membrane. Cell surface oligosaccharides mediate the interactions of cells with each other Science 291:2337 Why is it so hard? Glycosylation is the only protein modification i requiring ii detailed dstructural characterization Requires additional analytical and informatics tools, and requires a systemsbased approach to understand properly. Glycosylation is Complex: Stereoisomers 6 CH 2 H H H H, H 2 3 H D-Galactose 4 H 6 CH 2 H H 5 3 H 1 2 D-Mannose H, H Stereochemical Centers 6

7 Glycosylation is Complex: Anomers Interconversion between the two anomers of galactose CH 2 H CH 2 H H H H H H H H H H H β-d-galactose H CH 2 H H α-d-galactose Anomeric Carbon Branching & multiple linkages and termini an example of different glycosidic linkage sites Galactose β 1-4 (Galactose β 1-2) Galactose α 1-6 Galactose 6 CH 2 H 6 CH 2 H H H 4 1 H CH 2 H 5 H 6CH H H 3 2 H 5 Non-Reducing Termini 4 H H H H, H Reducing Terminus 7

8 Stereochemistry and Anomeric Configuration have a huge impact CH 2 H H CH 2 H H H CH 2 H H H poly β-1,4-d-glucose Cellulose H CH 2 H H CH 2 H H CH 2 H H poly α-1,4-d-glucose Amylose, starch H H H Decoding Sugar symbols What does mean? Shape designate type of sugar, i.e., hexoses circles HexNAcs squares hexosamines squares divided diagonally Color denotes stereochemistry, i.e., Galactose Yellow Glucose Blue Mannose Green Acidic sugars Diamonds Neu5Ac purple Neu5Gc light blue 8

9 Detailed Glycan Characterization Information needed to solve structures: Monomer ID Linkage positions (and dbranching points) Anomeric configurations Sequence Two basic routes: Bottom up break it apart, and characterize the pieces Top down keep it together, take it apart systematically ften both approaches are needed Classical Methods Composition Analysis how much of which sugars are present Problems with sialic acids decomposed by strong acid Linkage Analysis Identify which hydroxyl on each glycan is involved in a glycosidic bond Data is for monomers no sequence only information on linkages to each sugars Different experiments needed for neutral and amino sugars (HexNAc) 9

10 Dete e 2/22/2013 Composition Analysis Preparation of Alditol Acetates H H H H Deuterium used to disrupt the symmetry i.e., allow C1 to be differentiated from C6 H H H H H NaBD 4 CHDAc CAc AcCH CAc CHAc Ac 2 /Pyr. H H H H CHH H D CH 2 Ac Composition Analysis GLC Profile of Alditol Acetates (Supelco SP2330 Column) Man Gal Rh Fu ha c b a Rib Ara Xy yl c Glc ector Response Inos. Hep 5 10 Minutes 10

11 De nse Ara Rha Fuc 2/22/2013 Composition Analysis samples with acidic sugars Preparation of Trimethylsilyl (TMS) Methyl Glycosides* H ( C H) H H ( C CH 3 ) H H H H CH 3 H H CH 3 ( C CH 3 ) H H H H CH 3 ( C CH 3 ) TMS TMS TMS CH 3 TMS (CH 3 ) 3 SiX ( C CH 3 ) TMS TMS TMS TMS CH 3 furanose forms *allows detection of acidic sugars GLC of TMS Methyl Glycosides (J&W Scientific DB1 column) etector Respon Xyl Man Glc Hep GalA Gal al Ga Glc ca Hep Inos. GalA GlcA Glc Minutes 11

12 De nse Ara Rha Fuc 2/22/2013 Q: Why do the monosaccharides appear as multiple GC peaks? GLC of TMS Methyl Glycosides (J&W Scientific DB1 column) etector Respon Xyl Man Glc Hep GalA Gal al Ga Glc ca Hep Inos. GalA GlcA Glc Minutes A: There are multiple forms of each monosaccharide 12

13 Composition Analysis samples with acidic sugars Preparation of Trimethylsilyl (TMS) Methyl Glycosides H ( C H) H H ( C CH 3 ) H H H H CH 3 H H CH 3 ( C CH 3 ) H H H H CH 3 ( C CH 3 ) TMS TMS TMS CH 3 TMS (CH 3 ) 3 SiX ( C CH 3 ) TMS TMS TMS TMS CH 3 furanose forms Composition Analysis Preparation of Alditol Acetates H H H H H H H H H NaBD 4 CHDAc CAc AcCH CAc CHAc Ac 2 /Pyr. H H H H CHH H D CH 2 Ac 13

14 D-Fuc L-Fuc L-Fuc D-Fuc L-Xyl D-Xyl D-Xyl STD L-Xyl L-Man D-Man D-Gal L-Gal L-Gal D-Gal L-Glc D-Glc L-Gal LD-Glc 2/22/2013 Determination of the Stereochemical Configuration of Glycosyl Residues CH 2 TMS T MS Me Me CH 2 T MS TMS TMS TMS TMS TMS 2,3,4,6-tetra- -trimethylsilylmethyl 2,3,4,6-tetra- -trimethylsilyl- -D-glucoside methyl -L-glucoside Enantiomers: Identical physical properties, unable to separate. CH 2 TMS CH 3 CH 2 TMS T MS CH 3 CH 2 TMS TMS TMS CH 2 CH 3 2,3,4,6-tetra- -trimethylsilyl- 2-(-)-butyl -D-glucoside CH 3 T MS T MS 2,3,4,6-tetra- -trimethylsilyl- 2-(-)-butyl -L-glucoside Diastereomers: Different physical properties, able to separate. GC Separation of Trimethylsilyl (-)-2-Butyl Glycosides Detector Response Minutes Gerwig, G.J., Kamerling,J.P., and Vliegenthart, J.F.G Carbohydr. Res. 62:

15 2-lin nked-araf 3-2,3-linke ed-araf t-linked-arap 2-linked-Arap 2/22/2013 Glycosyl Linkages by Preparing Partially Methylated Alditol Acetates (PMAAs) CH 3SCH 3 H H H H H H H H H H H H H R KH - K CH2SCH3 CH 3I H 3C H 3C H 3C H 3C H 3C CH 3 CH 3 CH 3 H 3C H 3C CH 3 CH 3 R1H B - R1 - R2I R1R2 Ether synthesis CH 3 R 2 M TFA CHDAc CHDAc CHDAc CHDAc Me Me Me Me Me Me Me Ac Ac Me Ac Me Ac2/Pyr NaBD4 Ac Ac Ac Ac CH 2Me CH 2Me CH 2Me CH 2Me Merkle and Poppe (1994) Methods Enzymol. 230: 1 15; York, et al. (1985) Methods Enzymol. 118:3 40. GLC Profile of Arabinosyl PMAAs ector Response e t-linked-araf -linked-araf 4-linked-Arap 3-linked-Arap 3,4-linked-Arap or 3,5 2,3-linked-Arap 2,5-linked-Araf an 5-linked-Araf nd 2,4-linked Arap 2,3,5-linked-Araf 2,3,4-linked-Arap and Det Minutes 15

16 Problems with Glycans/Glycoproteins Poor Ionization efficiency large mass increase no sites for protonation sometimes has a negative charge hydrophobicity Transparent Heterogeneity Large size ther useful Derivatizations Depolarizing Derivatization Reactions for MS Peracetylation Pyridine/Acetic Anhydride, RT/overnight Pertrimethylsilylation Pyridine/BSTFA/TMCS, RT/half hour Permethylation Suspension of NaH in dry DMS add to dry glycan(s) Methyl Iodide add after ~2 hrs Chloroform/Water extraction, several washes, dry 16

17 Why Permethylate the ligosaccharide? The mass increase is not too much to shift the mass to higher mass range and decrease sensitivity. It increase the sensitivity of oligosaccharides for subsequent MS analysis. Equalizes the MS response for different glycans It allows for diagnostic molecular ions which are easier to interpret than the native oligosaccharides. Stabilizes negatively charged sugars (sialic acids for example) Makes tandem mass spectra more interpretable ther useful Derivatizations: Reductive Amination 17

18 Heterogeneity: the quality or state of being heterogeneous Heterogeneous: consisting of dissimilar or diverse ingredients No two cars are ever exactly the same, and the sequence in which they are produced must be carefully controlled to minimize resource utilization. For some of the larger options, the structure of the assembly must be adapted. M. Dincbas, H. Simonis, and P. van Hentenryck. Solving the car sequencing problem in constraint logic programming. In Y. Kodratoff, editor, Proceedings ECAI 88, pp , 1988 Heterogeneity: the quality or state of being heterogeneous Heterogeneous: consisting of dissimilar or diverse ingredients 18

19 Large Size Schematic illustration of a proteoglycan macromolecule aggregate in which multiple aggrecan molecules with glycosaminoglycans (GAG) side chains attach to a hyaluronan chain. X. Lux Lu, Chester Miller, Faye H. Chen, X. Edward Guo, Van C. Mow, J. Biomechanics, Vol 40 (11), 2007, Pg Questions? 19