High-Throughput and High-Sensitivity Nano-LC/MS and MS/MS for O-Glycan Profiling

Karlsson, Hasse; Larsson, Jessica Holmén; Thomsson, Kristina A.; Härd, Iris van Dijk; Bäckström, Malin; Hansson, Gunnar C.

doi:10.1007/978-1-59745-022-5_9

Cited by 14 publications

(15 citation statements)

References 7 publications

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“…Oligosaccharides were analyzed by LC/MS 2 using a 20 cm × 250 µm i.d. column containing 5 µm porous graphitized carbon (PGC) particles (Thermo Scientific, Waltham, MA, USA) prepared in‐house .…”

Section: Methodsmentioning

confidence: 99%

“…Sulfated oligosaccharides are typically in low abundance in complex oligosaccharide samples and liquid chromatography (LC) capable of separating oligosaccharides will help to target and sequence sulfated oligosaccharides by CID in the presence of high abundant neutral and/or sialylated oligosaccharides. ESI‐MS in conjunction with LC provides a robust and sensitive method for the analysis of native sulfated oligosaccharides …”

mentioning

confidence: 99%

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Sulfate migration in oligosaccharides induced by negative ion mode ion trap collision‐induced dissociation

Kenny

Issa

Karlsson

2011

Rapid Comm Mass Spectrometry

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Migration of sulfate groups between hydroxyl groups was identified after collision-induced dissociation (CID) of sulfated oligosaccharides in an ion trap mass spectrometer in negative ion mode. Analysis of various sulfated oligosaccharides showed that this was a common phenomenon and was particularly prominent in sulfated oligosaccharides also containing sialic acid. It was also shown that the level of migration was increased when the sulfate was positioned on the flexible areas of the oligosaccharides not involved in the pyranose ring, such as the extra-cyclic C-6 carbon of hexoses or N-acetylhexosamines, or on reduced oligosaccharide. This suggested that migration is dependent on the spatial availability of the sulfate in the ion trap during collision. It is proposed that the migration is initiated when the negatively charged -SO3 (-) residue attached to the oligosaccharide precursor becomes protonated by a CID-induced proton transfer. This is supported by the CID fragmentation of precursor ions depleted of acidic protons such as doubly charged [M - 2H](2-) ions or the sodiated [M + Na - 2H](-) ions of oligosaccharides containing one sulfate and one sialic acid in the same molecule. Compared to the CID fragmentation of their monocharged [M - H](-) ions, no migration was observed in CID of proton depleted precursors. Alternative fragmentation parameters to suppress migration of sulfated oligosaccharides also showed that it was not present when sulfated oligosaccharides were fragmented by HCD (High-Energy C-trap Dissociation) in an Orbitrap mass spectrometer.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Sulfate migration in oligosaccharides induced by negative ion mode ion trap collision‐induced dissociation

Kenny

Issa

Karlsson

2011

Rapid Comm Mass Spectrometry

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“…An example of the increased sensitivity is shown in Figure 3; here we can see that the increase in peak height is a fac� tor of 30; in this example a column length of 5 cm was employed to match the previously acquired 2.1�mm internal diameter column data; resulting in reduced retention of 3.6 min. Quantifying the effect of reducing the LC flow rate is beyond the scope of this paper, but it has been widely reported that reducing the LC flow rate increases desolvation efficiency and hence, boosts the MS response [21,22]. The increase in sensitivity observed here obtained by employing a 300�µm LC device was most likely due to a combination of both of these two factors.…”

Section: Resultsmentioning

confidence: 58%

Addressing The Challenge of Limited Sample Volumes in In Vitro Studies with Capillary-Scale Microfluidic LC–MS/MS

et al. 2011

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Miniaturization of chromatographic separation systems provides a means of greatly increasing sensitivity in LC-MS. In this article, we demonstrate the use of an integrated microfluidic chromatographic device for the LC-MS/MS investigation of the in vitro microsomal metabolism of the model drug propranolol using a sample volume of 1 µl of a 1-µM incubation. With such samples the system was capable of obtaining high-quality MS and MS/MS data from the injection of test drug substance containing sufficient information to correctly derive the structure of the drug metabolites. The analytical column was tolerant to the injection of a large percentage of organic solvent in the sample and still delivered a high-quality separation. The data suggest that these types of micro-LC-MS/MS devices are robust enough for routine applications and well suited to the analysis of small samples. Other potential applications include the generation of pharmacokinetic profiles from the reduced sample volumes obtained from serially bled small rodent studies, or the facilitation of analysis of limited-volume samples from neurological studies.

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“…However, existing technologies for mass spectrometrybased analysis of glycans (Morelle and Michalski 2007;Karlsson et al 2009;Bereman and Muddiman 2011) can be used to assay how the repertoire of glycosylation states is affected in deletions of all ER proteins. In addition, novel methodologies of glycan arrays should be used for gathering information on glycan/protein interactions (Blixt et al 2004).…”

Section: Future Directionsmentioning

confidence: 99%

The Contribution of Systematic Approaches to Characterizing the Proteins and Functions of the Endoplasmic Reticulum

Schuldiner

Weissman

2013

Cold Spring Harbor Perspectives in Biology

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The endoplasmic reticulum (ER) is a complex organelle responsible for a range of functions including protein folding and secretion, lipid biosynthesis, and ion homeostasis. Despite its central and essential roles in eukaryotic cells during development, growth, and disease, many ER proteins are poorly characterized. Moreover, the range of biochemical reactions that occur within the ER membranes, let alone how these different activities are coordinated,is not yet defined. In recent years, focused studies on specific ER functions have been complemented by systematic approaches and innovative technologies for high-throughput analysis of the location, levels, and biological impact of given components. This article focuses on the recent progress of these efforts, largely pioneered in the budding yeast Saccharomyces cerevisiae, and also addresses how future systematic studies can be geared to uncover the "dark matter" of uncharted ER functions.

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High-Throughput and High-Sensitivity Nano-LC/MS and MS/MS for O-Glycan Profiling

Cited by 14 publications

References 7 publications

Sulfate migration in oligosaccharides induced by negative ion mode ion trap collision‐induced dissociation

Sulfate migration in oligosaccharides induced by negative ion mode ion trap collision‐induced dissociation

Addressing The Challenge of Limited Sample Volumes in In Vitro Studies with Capillary-Scale Microfluidic LC–MS/MS

The Contribution of Systematic Approaches to Characterizing the Proteins and Functions of the Endoplasmic Reticulum

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