This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Introduction: Most classes of animal glycans are acidic, and the use of negative ionization is a natural choice.
The aim of this work is to explain product ion fragmentation patterns for negatively charged acidic glycans. Such an understanding is important to develop tandem MS strategies in glycomics. This work shows how the presence of uronic acid residues influences the product ion patterns of sulfated glycosaminoglycan oligosaccharides. Uronic acid protons becomes mobile during the CID process and destabilize glycosidic bonds. In the absence of such mobile protons, the ions are significantly more stable and require more energy for glycosidic cleavage. Both charge location and potentially mobile protons strongly influence product ion pattern for acidic oligosaccharides. Methods: Oligosaccharides were produced by digesting chondroitin sulfate with chondroitinase ABC or testicular hyaluronidase and purified by size exclusion chromatography using a Superdex Peptide column (Amersham Pharmacia). Oligosaccharides were methyl esterified using methanolic HCl. All mass spectra were acquired in nano-electrospray mode using a Applied Biosystems/Sciex Qstar Pulsar-i mass spectrometer. Samples were dissolved at a concentration of ~1 micromolar in 30% methanol and sprayed through uncoated borosilicate glass tips pulled to a 1 micrometer diameter orifice. Steady signals were typically observed with -1100 V spray potentials. Results: The presence of acidic groups (sulfate, phosphate, sialic acid) strongly influences the pattern of product ions resulting from CID of negative oligosaccharide ions. Tandem MS of chondroitin sulfate oligosaccharides of the form (?HexA)(HexA)n-1(GalNAcSulfate)n, where ?HexA = 4,5-unsaturated HexA, were acquired before and after methyl esterification of uronic acid residues. The native structures fragment under significantly less energetic conditions than do the methyl esterified glycans. Abundances of cross-ring cleavage ions increase for the methyl esterified relative to the native glycans. Chondroitin sulfate oligosaccharides lacking a ?-unsaturated uronic acid residue do not have cross-ring cleavage as a fragmentation channel. In their methyl esterified forms, these ions require approximately -22 V collision energy to reduce the precursor ion intensity by 50%. For the native structures, only -15 V collision energy is required. These results are consistent with the conclusion that, despite the deprotonated precursor ion, remaining carboxyl protons become delocalized as the ion temperature rises during the CID process and associate with glycosidic oxygen atoms. This association predisposes the glycosidic bond to scission, in a manner similar to that described for positively charged glycans. Methyl esterified precursor ions, lacking carboxylic protons, require significantly higher fragmentation energies than do native ions. The abundances of cross-ring cleavages increase because of the lack of mobile protons to destabilize glycosidic bonds. Both the location of negative charge and the presence of protons that become mobilized during the CID process strongly influence the observed product ion patterns. In the absence of mobile protons, acidic glycans resist glycosidic bond cleavage because charge is located distant from the glycosidic bonds. These results have clear implications regarding the design of on-line LC MS/MS conditions for glycomics experiments.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-BECM (03))
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Boston University
Schools of Medicine
United States
Zip Code
Lu, Yanyan; Jiang, Yan; Prokaeva, Tatiana et al. (2017) Oxidative Post-Translational Modifications of an Amyloidogenic Immunoglobulin Light Chain Protein. Int J Mass Spectrom 416:71-79
Sethi, Manveen K; Zaia, Joseph (2017) Extracellular matrix proteomics in schizophrenia and Alzheimer's disease. Anal Bioanal Chem 409:379-394
Hu, Han; Khatri, Kshitij; Zaia, Joseph (2017) Algorithms and design strategies towards automated glycoproteomics analysis. Mass Spectrom Rev 36:475-498
Ji, Yuhuan; Bachschmid, Markus M; Costello, Catherine E et al. (2016) S- to N-Palmitoyl Transfer During Proteomic Sample Preparation. J Am Soc Mass Spectrom 27:677-85
Hu, Han; Khatri, Kshitij; Klein, Joshua et al. (2016) A review of methods for interpretation of glycopeptide tandem mass spectral data. Glycoconj J 33:285-96
Pu, Yi; Ridgeway, Mark E; Glaskin, Rebecca S et al. (2016) Separation and Identification of Isomeric Glycans by Selected Accumulation-Trapped Ion Mobility Spectrometry-Electron Activated Dissociation Tandem Mass Spectrometry. Anal Chem 88:3440-3
Wang, Yun Hwa Walter; Meyer, Rosana D; Bondzie, Philip A et al. (2016) IGPR-1 Is Required for Endothelial Cell-Cell Adhesion and Barrier Function. J Mol Biol 428:5019-5033
Srinivasan, Srimathi; Chitalia, Vipul; Meyer, Rosana D et al. (2015) Hypoxia-induced expression of phosducin-like 3 regulates expression of VEGFR-2 and promotes angiogenesis. Angiogenesis 18:449-62
Yu, Xiang; Sargaeva, Nadezda P; Thompson, Christopher J et al. (2015) In-Source Decay Characterization of Isoaspartate and ?-Peptides. Int J Mass Spectrom 390:101-109
Steinhorn, Benjamin S; Loscalzo, Joseph; Michel, Thomas (2015) Nitroglycerin and Nitric Oxide--A Rondo of Themes in Cardiovascular Therapeutics. N Engl J Med 373:277-80

Showing the most recent 10 out of 253 publications