Navigation Links
Characterization of Protein Oligosaccharides Using MSn

Andrew S.Weiskopf and Paul Vouros, Northeastern University, Boston, MA
David J. Harvey, University of Oxford, Oxford, UK

Protein Analysis

The data presented here can be acquired using the Thermo Finnigan LCQ Series of ion trap mass spectrometers.

Introduction

This application report provides a brief overview of protein oligosaccharides that present a unique analytical challenge among the families of biomolecules. Peptides and oligonucleotides are linear oligomers, so sequence information is all that is necessary for complete primary structure characterization. Oligosaccharides, on the other hand, are branched structures, with their constituent monosaccharides linked together by a variety of glycosidic bonds. As a result, while six amino acids can yield only 720 different peptides, six unique monosaccharides can be arranged to form many millions of oligosaccharides. Though much is understood about carbohydrate biosynthesis to eliminate most of these isomers from consideration, the permutations that remain still provide a formidable analytical problem.

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) has become a valuable technique in oligosaccharide characterization.1 Permethylation of the oligosaccharide before analysis more effectively directs fragmentation of the ion into various familiar series of product ions: glycosidic-bond fragments which indicate monosaccharide sequence and branching, and crossring fragments which identify the linkages between monosaccharides. However, interpretation is complicated by the relatively low abundance of these crossring fragments, and cleavage of especially labile bonds can suppress the formation of other fragments.

The MSn capabilities of the LCQ Series present a powerful solution to these problems encountered with MS/MS. The removal of monosaccharide and disaccharide substituents by successive stages of MSn not only provides a facile method for obtaining sequence information, but can produce ion species which themselves yield fragments of greater variety and higher relative intensity. As a result, the LCQ Series reveals structural details otherwise lost by traditional tandem MS, and provides a new approach to the mass spectrometric characterization of protein glycans.2

Goal

To demonstrate:
How MSn can be used in oligosaccharide analysis to determine monosaccharide sequence and composition, carbohydrate branching, and monosaccharide linkages.
The ability of MSn to perform separate characterizations of two isobaric oligosaccharides, even when infused into the LCQ Series as an unseparated mixture.
That early stages of MSn can remove labile substituents, producing an ion substrate which yields previously unattainable information when subjected to further stages of MSn.


Figure 1a. Two HexNAc5Hex5 glycans released from chicken ovalbumin.
Figure 1b. GlcNAc8Man3 oligosaccharide from chicken ovalbumin.

Experimental Conditions


Oligosaccharides were released from hen ovalbumin by hydrazinolysis and were used as an unseparated mixture. The oligosaccharides were then dissolved in a slurry of sodium hydroxide and dimethyl sulfoxide, permethylated with methyl iodide, and extracted into chloroform per Ciucanu and Kerek.3

LCQ Conditions


Ionization method: ESI
Sample introduction: direct infusion, 3-5 L/min
with LCQ syringe pump
Sample conditions: 50 ng/L total mixture in
50:50 MeOH/water, 1 mM sodium acetate
Capillary temperature: 200C
Needle voltage: +4.5-5 kV
Sheath gas: nitrogen, 30 unit
Collision energies: 60-65 units, except where noted


Figure 2. MS2 of m/z 1169.5, the doubly-sodiated molecular ion of N5H5-I and N5H5-II.

Results

Figure 1a shows two oligosaccharides from the ovalbumin carbohydrate mixture. Since galactose and mannose are both hexoses, and therefore diastereomers of one another, both permethylated oligosaccharides have the same composition (HexNAc5Hex5) and molecular weight (2293 Da). With these two oligosaccharides infused together, MS2 of their doubly-sodiated molecular ions at m/z 1169.5 (Figure 2) produces fragments corresponding to both N5H5-I and N5H5-II, with each oligosaccharide effectively contaminating the spectrum of the other.

The fragment at m/z 937.7 corresponds to loss of a Hex-HexNAc disaccharide antenna, which is a structural feature only found on N5H5-I. Therefore, selecting the m/z 937.7 ion and subjecting it to MS3 (Figure 3a) yields structural information on N55H5-I, even as N5H55-II is continuously infused into the instrument. Favorable cleavage sites lie adjacent to GlcNAc residues, particularly terminal GlcNAcs, resulting in the preferred fragmentation of glycosidic bonds (noted by B and Y labels) and suppression of cross-ring fragments (A and X-type ions).4 By performing MSn on a fragment which has already undergone these losses in a previous stage of MS (Figure 3b, m/z 1333.7), cross-ring fragment products emerge, revealing the nature of saccharide linkages in the molecule. Similar results can be achieved for N5H5-II by MSn of the ion at m/z 1606.8 from MS2.


Figure 3a-b. MS3 and MS4 for isolation and characterization of glycan N5H5-I.

The oligosaccharide in Figure 1b is a heavily GlcNAcylated oligosaccharide of ovalbumin. The MS2 spectrum of the doubly-sodiated ion (Figure 4) is dominated by ions corresponding to the loss of non-reducing terminal GlcNAc sugars. No other information is obtainable due to the sup-pression of other bond cleavages. By reducing the collision energies to 35 units and using MS3-6, one can not only count terminal GlcNAc residues in a facile manner, but can produce a species upon MS7 which is no longer susceptible to suppression effects (Figure 5). MS8 of this ion subsequently reveals the locations of the previously attached GlcNAc residues, indicating three acetylaminosugars on one mannose antenna, a fourth and fifth on the other arm, and a sixth residing on the bisected mannose of the core (Figure 6).


Figure 4. MS2 of the ovalbumin glycan GlcNAc8Man3.

The use of higher collision energies to remove two or three residues per stage of MSn can also produce the m/z 1087.8 ion in fewer steps. These alternate MSn experiments yield the same structural information by MS5 or MS6, suggesting that perhaps the structural integrity of the m/z 1087.8 ion is maintained regardless of its pathway of formation.


Figure 5. MS2-7 for stepwise removal of terminal GlcNAc residues and formation of the more stable GlcNAc2Man3(OH)6 core substrate, m/z 1087.7.



Figure 6. Determination of GlcNAc8Man3 glycan branching patterns by MS6.

Conclusions

Electrospray ion trap mass spectrometry is a particularly effective method for detailed characterization of protein oligosaccharides. The MSn capabilities of the LCQ Series overcome the significant limitations often encountered with traditional triple quadrupole techniques. By exploiting unique saccharide losses, MSn enables the isolation and analysis of individual glycan species, even in the presence of an isobaric contaminant. The removal of especially labile substituents in early stages of MSn produces stable ion substrates which, upon further MSn analysis, yield spectra that are far more structurally informative than those obtained by MS/MS.

References

1. Reinhold, V.N., Reinhold, B.B., Chan, S., Methods Enzymol., 271, 377, (1996).
2. Weiskopf, A.S.; Vouros, P.; Harvey, D.J. Rapid Commun. Mass Spectrom., 11, 1493,(1997).
3. Ciucanu, I.; Kerek, F. Carbohydr. Res., 131, 209, (1984).
4. Nomenclature system from Domon, B.; Costello C.E. Glycoconj, J. 5, 397, (1988).




'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. Purification and Characterization of beta-Lactoglobulin Genetic Variants A and B Using Preparative Electrophoresis and Isoelectric Focusing
2. Isolation and Characterization of Anti-Viral Nucleoside Compounds from the Chinese Herb Taraxacum Mongolicum
3. A Robust LC/MS/MS Method for the Quantitation of Very Long Chain Fatty Acids for Research into the Characterization of Peroxisomal Disorders
4. Characterization of tooth decay
5. Sciclone ALH 3000 Liquid Handler: Functional Characterization of the High Volume Head - Part 1: Plate-to-Plate Precision Testing
6. A New C-Terminal GST Vector for Protein Production in S. pombe
7. New Mammalian Two-Hybrid System Detects Protein-Protein Interactions
8. Antibodies for Studying NMDA Receptor Protein Expression and Synapse-Specific Immunolabeling
9. Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins
10. New Yeast Cloning System for Producing Proteins with Native Amino Acid Sequences
11. Optimized Imaging of Protein Gels Stained with Coomassie Brilliant Blue Dye
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: Characterization Protein Oligosaccharides Using MSn

(Date:9/22/2014)... , Sept. 22, 2014  WIRB-Copernicus Group (WCG), ... review services for clinical research, announced today that ... has been reaccredited by the Association for the ... endeavors to improve safeguards for clinical research participants. ... A. Deieso , Ph.D., commented, "It is of ...
(Date:9/22/2014)... Olso, Norway (PRWEB) September 22, 2014 ... convenient new parcel and pallet measuring device ... ScanTape™ eliminates manual data entry, offering greater ease ... time savings that result can significantly enhance productivity—an ... in industries where processing volume equals profits. ...
(Date:9/22/2014)... , Sept. 22, 2014  Spherix Incorporated (SPEX) -- an ... of intellectual property, today announced that the United States Patent ... Company in the month of September that is part of ... issued patent is: USRE45,121 , Application number: US ... Filing date: Dec 27, 2012 The listed ...
(Date:9/21/2014)... For the first time, scientists have discovered how to ... strength and stiffness greater than that of today,s strongest ... a research team led by John V. Badding, a ... published in the 21 September 2014 issue of the ... point of view, our discovery is intriguing because the ...
Breaking Biology Technology:Western IRB (WIRB) Awarded AAHRPP Reaccreditation 2ScanTape™ Handheld Dimensioner Increases Processing Volume 2Spherix Notified by United States Patent & Trademark Office of New Standard Essential Patent Issued to Spherix 2Spherix Notified by United States Patent & Trademark Office of New Standard Essential Patent Issued to Spherix 3Smallest possible diamonds form ultra-thin nanothreads 2Smallest possible diamonds form ultra-thin nanothreads 3Smallest possible diamonds form ultra-thin nanothreads 4
... -- China Sky One,Medical, Inc. (OTC Bulletin Board: ... manufacturer, marketer and distributor of pharmaceutical,medicinal and diagnostic ... changes were made in conjunction with the Company,filing ... American Stock Exchange., As of May 7, ...
... ANNAPOLIS, Md., May 13 PharmAthene, Inc.,(Amex: ... against,biological and chemical threats, today reported financial results ... "The first quarter was a particularly productive and ... Avecia,s biodefense vaccines,portfolio, completed on April 2, 2008, ...
... Pa., May 13 Particle Sciences has expanded,its production ... Class 1000 (ISO 6),clean room. The space also contains ... added to the existing 15,000 sq ft facility in ... that of semisolids, suspensions and dispersions. "This,investment is the ...
Cached Biology Technology:China Sky One Medical, Inc. Changes Management Team 2China Sky One Medical, Inc. Changes Management Team 3PharmAthene Reports First Quarter 2008 Financial Results 2PharmAthene Reports First Quarter 2008 Financial Results 3PharmAthene Reports First Quarter 2008 Financial Results 4PharmAthene Reports First Quarter 2008 Financial Results 5PharmAthene Reports First Quarter 2008 Financial Results 6PharmAthene Reports First Quarter 2008 Financial Results 7
(Date:9/22/2014)... is one of seven institutions in the country ... to receive this year,s BEST Award a ... the university,s most promising biomedical sciences graduate trainees ... path that they have traditionally taken. , The ... Opportunities for Biomedical Scientists (iJOBs) program. It includes ...
(Date:9/22/2014)... image burn in Khabarovsk Krai, a territory occupying the coastline ... by plumes of smoke mark active fires. The smoke, which ... the Sea of Okhotsk. Taiga and tundra are ... the central depression, and deciduous forests are the natural vegetation ... Russia in the summer, the 2014 wildfire season appears to ...
(Date:9/22/2014)... could be much more useful in homes, if they ... see the world with cameras and lasers, which have ... are hidden in clutter. A complementary way robots can ... of small ultra-high frequency radio-frequency identification (UHF RFID) tags. ... an RFID-equipped robot to search a room for the ...
Breaking Biology News(10 mins):Rutgers receives $2 million grant to prepare biomedical students for roles in industry 2Rutgers receives $2 million grant to prepare biomedical students for roles in industry 3New RFID technology helps robots find household objects 2New RFID technology helps robots find household objects 3
... [Brown University] From a biological point of view, the ... by fungi. As a kingdom, they are full of surprises, ... seems sure to titillate the intellects of those who ... chromosomes in each cell. Fungal fecundity is a seemingly ...
... the United States National Museum of Natural History and ... researchers have found 16 new species of wasps from ... The new species are described and illustrated in ... Key of the Charipinae (Hymenoptera: Cynipoidea: Figitidae) From the ...
... activities "diluted" in the cell nucleus. In this state it ... tangle of thread, in actual fact its structure is governed ... out its functions. Biologists have studied DNA by observing it ... been supplemented by research in silico, that is to say, ...
Cached Biology News:Yeast's lifestyle couples mating with meiosis 2Yeast's lifestyle couples mating with meiosis 325 years of DNA on the computer 2
Anti-human C3a/C3a des-Arg (neo-epitope), Clone 2991, Monoclonal Antibody...
Rabbit polyclonal to Warfarin ( Abpromise for all tested applications). Antigen: Chemical / Small Molecule conjugated to KLH...
Rabbit polyclonal to MA2 ( Abpromise for all tested applications). entrezGeneID: 10687 SwissProtID: Q9UL42...
Mouse monoclonal [YPF19] to Yersinia pestis ( Abpromise for all tested applications)....
Biology Products: