Travelling-wave ion mobility and negative ion fragmentation of high-mannose N-glycans.

The isomeric structure of high-mannose N-glycans can significantly impact biological recognition events. Here, the utility of travelling-wave ion mobility mass spectrometry for isomer separation of high-mannose N-glycans is investigated. Negative ion fragmentation using collision-induced dissociation gave more informative spectra than positive ion spectra with mass-different fragment ions characterizing many of the isomers. Isomer separation by ion mobility in both ionization modes was generally limited, with the arrival time distributions (ATD) often showing little sign of isomers. However, isomers could be partially resolved by plotting extracted fragment ATDs of the diagnostic fragment ions from the negative ion spectra, and the fragmentation spectra of the isomers could be extracted by using ions from limited areas of the ATD peak. In some cases, asymmetric ATDs were observed, but no isomers could be detected by fragmentation. In these cases, it was assumed that conformers or anomers were being separated. Collision cross sections of the isomers in positive and negative fragmentation mode were estimated from travelling-wave ion mobility mass spectrometry data using dextran glycans as calibrant. More complete collision cross section data were achieved in negative ion mode by utilizing the diagnostic fragment ions. Examples of isomer separations are shown for N-glycans released from the well-characterized glycoproteins chicken ovalbumin, porcine thyroglobulin and gp120 from the human immunodeficiency virus. In addition to the cross-sectional data, details of the negative ion collision-induced dissociation spectra of all resolved isomers are discussed.

Ion Mobility Mass Spectrometry for Ion Recovery and Clean-Up of MS and MS/MS Spectra Obtained from Low Abundance Viral Samples.

Many samples of complex mixtures of N-glycans released from small amounts of material, such as glycoproteins from viruses, present problems for mass spectrometric analysis because of the presence of contaminating material that is difficult to remove by conventional methods without involving sample loss. This study describes the use of ion mobility for extraction of glycan profiles from such samples and for obtaining clean CID spectra when targeted m/z values capture additional ions from those of the target compound. N-glycans were released enzymatically from within SDS-PAGE gels, from the representative recombinant glycoprotein, gp120 of the human immunodeficiency virus, and examined by direct infusion electrospray in negative mode followed by ion mobility with a Waters Synapt G2 mass spectrometer (Waters MS-Technologies, Manchester, UK). Clean profiles of singly, doubly, and triply charged N-glycans were obtained from samples in cases where the raw electrospray spectra displayed only a few glycan ions as the result of low sample concentration or the presence of contamination. Ion mobility also enabled uncontaminated CID spectra to be obtained from glycans when their molecular ions displayed coincidence with ions from fragments or multiply charged ions with similar m/z values. This technique proved to be invaluable for removing extraneous ions from many CID spectra. The presence of such ions often produces spectra that are difficult to interpret. Most CID spectra, even those from abundant glycan constituents, benefited from such clean-up, showing that the extra dimension provided by ion mobility was invaluable for studies of this type.