Fluorescence-monitored zero dead-volume nanoLC-microESI-QIT-TOF MS for analysis of fluorescently tagged glycosphingolipids

Abstract

An analysis of the glycan processing event is of particular importance to understand the nontemplate dependent synthetic mechanism of the multiple glycosylation reactions taking place in the Golgi apparatus in connection with the post-translational modification of biomolecules. In our efforts to address the issue, we constructed an analysis platform using nano-liquid chromatography (LC), which also worked as a spray tip, with an optical-fiber-based blue (470 nm) light emitting diode (LED)-induced fluorescence (520 nm) detector coupled with a microelectrospray ionization (ESI)-quadrupole ion trap (QIT)-time of flight (TOF) mass spectrometer (MS). This system was designed to enable both quantitative and qualitative analyses of fluorescently tagged molecules such as BODIPY-tagged lactosylceramide. Owing to the zero dead volume after LC separation, an extremely high sensitivity was achieved for the quantitative analysis (260 amol). It was also shown that a simultaneous online structural analysis based on MS could be achieved for the same quantity of analyte. To further demonstrate its potential, an enzymatic reaction of fluorescently tagged lactosylceramide using sialyltransferase was carried out, and the conversion yield was obtained on the basis of fluorescence detection. In addition, the structural details of a product, sialyl lactosylceramide, were obtained by MS and MS/MS analyses.

Profiling Aglycon-Recognizing Sites of UDP-glucose:glycoprotein Glucosyltransferase by Means of Squarate-Mediated Labeling

“…As a result, three fluorescent fractions (designated fractions 1−3 in Figure 4a) were obtained, which were analyzed by a nanoliquid reverse phase C18 chromatography (LC) system equipped with an LED-induced fluorescent detector (FLD) connected to a nanoelectrospray-tandem mass spectrometry (nano-ESI-MS n ) system. 33 Among the fluorescent peaks observed by LC-FLD chromatograms (Figure 4b), only peak 1, which was eluted in fraction 3 of the reverse-phase C4 chromatogram, gave an assignable MS signal that exhibits a characteristic spectrum of the glycopeptide following MS/MS analyses (Figure 4c,d). As other peaks did not show any signal characteristic of glycopeptide (Figure S4 of the Supporting Information), they were not analyzed further.…”

“…To determine the site that was covalently modified by 1 , labeled HUGT1 was digested with trypsin and the resulting fragments were fractionated on a reverse phase C4 column using gradient elution with acetonitrile and water (Figure a). As a result, three fluorescent fractions (designated fractions 1–3 in Figure a) were obtained, which were analyzed by a nanoliquid reverse phase C18 chromatography (LC) system equipped with an LED-induced fluorescent detector (FLD) connected to a nanoelectrospray-tandem mass spectrometry (nano-ESI-MS n ) system . Among the fluorescent peaks observed by LC-FLD chromatograms (Figure b), only peak 1, which was eluted in fraction 3 of the reverse-phase C4 chromatogram, gave an assignable MS signal that exhibits a characteristic spectrum of the glycopeptide following MS/MS analyses (Figure c,d).…”

Nano-LC/NSI MS Refines Lipidomics by Enhancing Lipid Coverage, Measurement Sensitivity, and Linear Dynamic Range

“…All in all, nLC/NSI systems provide a strategy to come closer toward the goal of comprehensive lipidome coverage even when sample availability is limited. However, despite the apparent advantages of nLC-MS systems for lipid analyses, this promising technology has not yet been established in the lipidomics community even though several laboratories already applied nano/capillary chromatographic systems for (often class restricted) lipid analyses. ,− Nanoscale chromatography for lipidomics appears only to be routinely used by the Moon lab so far. ,,− …”

Fluorescently labelled glycans and their applications

“…When coupled with laser-induced fluorescence or mass spectroscopic detection, this system allowed for the study of glycosphingolipid metabolites at a very broad dynamic range from mM to pM for laser-induced fluorescence detection and down to yotomoles for mass spectroscopic detection [41][42][43]. The extremely high sensitivity in detecting BODIPY labeled lactosylceramide (amol) involved in glycan processing was also demonstrated by a fluorescence monitored mass spectrometry setup using nanoLCmicroelectrospray-quadrupole ion trap-time of flight analysis [44].…”