Gas chromatography and mass spectrometry of trimethylsilyl sugar phosphates

Zinbo, M.; Sherman, William R.

doi:10.1021/ja00710a052

Cited by 72 publications

(23 citation statements)

References 5 publications

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“…This problem is particularly problematic in the analysis of larger phosphorylated oligomers where several stages of tandem mass spectrometry must be utilized for full structural characterization, and thus phosphate cleavage is even more prevalent. Previous methods of characterizing sugar phosphate monosaccharides have utilized trimethylsilyl derivatization coupled to GC/MS [14,15], HPLC/MS/MS [16], and tandem mass spectrometry [17]. However, until recently a purely mass spectrometric method for the characterization of phosphate positioning within monosaccharides and phosphorylated oligomers had not been developed.…”

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confidence: 99%

Probing isomeric differences of phosphorylated carbohydrates through the use of ion/molecule reactions and FT-ICR MS

Leavell

Leary

2003

J. Am. Soc. Mass Spectrom.

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Through the use of ion/molecule reactions and tandem mass spectrometry, phosphate position is assigned in both phosphorylated monosaccharides and oligosaccharides. In previous work [1, 2] phosphate moieties of monosaccharides were stabilized under collisional activation, by first derivatizing the deprotonated monosaccharide with trimethyl borate through an ion/molecule reaction, and the phosphate position determined through marker ions generated in tandem mass spectra. In this work, the methodology is extended to larger phosphorylated oligomers employing chlorotrimethylsilane (TMSCl) as the ion/molecule reagent. Phosphorylated monosaccharides were first investigated to determine diagnostic ions for phosphate linkage in monomeric standards. It was observed that the diagnostic ions showed both linkage and some monosaccharide stereochemical information. Furthermore, it was observed that TMS addition stabilized the phosphate moiety under collisionally activated conditions. Upon identification of the diagnostic ions, the methodology was applied to lactose-1-phosphate. It was found that TMSCl, stabilized the phosphate moiety upon collisional activation, and furthermore, the phosphate linkage could be determined through tandem mass spectrometric analysis. As a further extrapolation to biologically relevant problems, the methodology was applied to a lipophosphoglycan analog from the protozoan parasite Leishmania. This sample contains bridging phosphates which were converted to terminal phosphates through collision induced dissociation. The sample was then analyzed in the same manner as lactose-1-phosphate, yielding phosphate linkage information and stereochemical information. This study showed that, using the developed methodology, phosphate linkage can be determined from both monosaccharides and larger oligosaccharides; furthermore it is applicable to samples in which the phosphates are either terminating or bridging. (J Am Soc Mass Spectrom 2003, 14, 323-331)

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confidence: 99%

Probing isomeric differences of phosphorylated carbohydrates through the use of ion/molecule reactions and FT-ICR MS

Leavell

Leary

2003

J. Am. Soc. Mass Spectrom.

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“…The MS/MS spectra of the putative rhamnose phosphate (S2 Fig, panel C) shows these characteristic ions and additional ions of 116, 180, and 296 m/ z . The ions at m/ z 116 and 180 have been shown to be characteristic of a sugar phosphate [41]. The ion at m/ z 296 is likely specific for rhamnose phosphate.…”

Section: Resultsmentioning

confidence: 99%

“…The ion at m/ z 296 is likely specific for rhamnose phosphate. Derivatization of glucose phosphate yields a major peak at 299 m/ z [41]. The 296-m/ z peak seen in panel C might result from the replacement of the oxygen atom of glucose with the CH 3 group of the methylpentose (rhamnose) (S2 Fig, panel D).…”

Section: Resultsmentioning

confidence: 99%

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Rhamnose Links Moonlighting Proteins to Membrane Phospholipid in Mycoplasmas

2016

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Many proteins that have a primary function as a cytoplasmic protein are known to have the ability to moonlight on the surface of nearly all organisms. An example is the glycolytic enzyme enolase, which can be found on the surface of many types of cells from bacteria to human. Surface enolase is not enzymatic because it is monomeric and oligomerization is required for glycolytic activity. It can bind various molecules and activate plasminogen. Enolase lacks a signal peptide and the mechanism by which it attaches to the surface is unknown. We found that treatment of whole cells of the murine pathogen Mycoplasma pulmonis with phospholipase D released enolase and other common moonlighting proteins. Glycostaining suggested that the released proteins were glycosylated. Cytoplasmic and membrane-bound enolase was isolated by immunoprecipitation. No post-translational modification was detected on cytoplasmic enolase, but membrane enolase was associated with lipid, phosphate and rhamnose. Treatment with phospholipase released the lipid and phosphate from enolase but not the rhamnose. The site of rhamnosylation was identified as a glutamine residue near the C-terminus of the protein. Rhamnose has been found in all species of mycoplasma examined but its function was previously unknown. Mycoplasmas are small bacteria with have no peptidoglycan, and rhamnose in these organisms is also not associated with polysaccharide. We suggest that rhamnose has a central role in anchoring proteins to the membrane by linkage to phospholipid, which may be a general mechanism for the membrane association of moonlighting proteins in mycoplasmas and perhaps other bacteria.

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“…The EI mass spectra of these compounds are dominated by ions formed by cleavages around the derivatized phosphate group and are characterized by extensive migrations of the TMS moieties [94][95][96]. Major ions are derived from tri-and tetra-(trimethylsilyl)-phosphate and fragments derived by losses of CH 3 • The ion at m/z…”

Section: Sugar Phosphatesmentioning

confidence: 99%