IMPROVED HILIC LC/MS ANALYSIS OF HEPARINOIDS USING A MAKEUP FLOW CHIP

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Heparin and heparan sulfate (HS) are glycosaminoglycans (GAGs) comprised of alternating glucuronic acid and N-acetylglucosamine units. These molecules are most widely recognized for their ability to bind proteins and modulate cellular events such as differentiation, blood coagulation, pathogenesis, and proliferation. Full understanding of these events requires knowledge of GAG structure. Although chip-based hydrophilic interaction chromatography (HILIC) is a powerful means of separating GAGs for on-line LC/MS, the negative-ion spray becomes unstable in high percent aqueous mobile phases. This work demonstrates the effectiveness of a chip designed with post-column addition of makeup LC flow to enable stable electrospray throughout the HILIC gradient. The result is a system with substantially increased spray stability and range of analytes that may be effectively analyzed. The mass spectrometric approach of analyzing heparan sulfate using CID has been limited by the dominant neutral loss of SO3 and consequently, the lack of glycosidic and cross-ring cleavage of the precursor ions. Since the loss of the sulfate is facilitated by mobile protons in the precursor ions, one possible solution would be to enhance the charge states during the ESI process to reduce available protons. Sulfolane and other agents have been shown to increase the charge states of protein complexes in the positive mode. In this study we demonstrate that sulfolane is able to increase both the ESI efficiency and charge states of HS oligosaccharide in the negative mode. We accomplish this by pulsing sulfolane via a novel post-column flow chip at designated time period during an online LC/MS run. Porcine intestine mucosa heparan sulfate was partially digested by heparin lyase I, II and III together to give random composition of oligosaccharides of a certain polymer range. The degree of polymerization (dp) 4-6 fraction was collected by applying the digest products to SEC column. The dp4-6 oligosaccharides were analyzed by online hydrophilic interaction chromatography (HILIC)-LC/MS (Agilent QTOF with Chip cube interface) using a customized pulsed makeup flow chip. 105 mM sulfolane in acetonitrile was pulsed for 1 min at the elution time of most dp4-6 through a makeup flow on a customized Agilent HPLC-chip that has an extra 100 nl reservoir loop for additive pulsing after the analytical column and before spraying. Pulsing of sulfolane increased the ionization response for heparin sulfate oligosaccharides by a factor ranging from 5 to 10. The effect appears to be most pronounced for the most highly sulfated oligosaccharides. 105 mM sulfolane addition increased the intensity of highly sulfated oligosaccharides at higher charge state by approximately 20-fold. The dominant charge state was shifted from 2- to 4-. A maximum charge state 5- for dp6 oligosaccharides was also observed, a charge state that has not been observed when using HILIC LC/MS without sulfolane addition. In addition to the increase in charge state, ions containing ammonium adduction were significantly lower in abundance. The increase in the charge state of highly sulfated oligosaccharides also served to separate them in m/z from less highly sulfated oligosaccharides whose charge state were not increased. As a result, the incidence of overlapping charge states is reduced. The data show potential to enable more effective tandem MS of heparan sulfated oligosaccharides because higher charge states are likely to produce more abundant backbone and cross ring cleavage product ions. Progress update: We are putting together a publication on the use of the pulsed MUF chip for improved tandem MS of GAG oligosaccharides. Going forward, we expect to use the MUF chip to add sulfolane to facilitate HS oligosaccharide tandem MS. This, in combination with HS oligosaccharide derivatization, is likely to maximize the information produced using collisional induced dissociation on this compound class.