Typing of Blood-Group Antigens on Neutral Oligosaccharides by Negative-Ion Electrospray Ionization Tandem Mass Spectrometry

Blood-group antigens, such as those containing fucose and bearing the ABO­(H)- and Lewis-type determinants expressed on the carbohydrate chains of glycoproteins and glycolipids, and also on unconjugated free oligosaccharides in human milk and other secretions, are associated with various biological functions. We have previously shown the utility of negative-ion electrospay ionization tandem mass spectrometry with collision-induced dissociation (ESI-CID-MS/MS) for typing of Lewis (Le) determinants, for example, Le^a, Le^x, Le^b, and Le^y on neutral and sialylated oligosaccharide chains. In the present report, we extended the strategy to characterization of blood-group A-, B-, and H-determinants on type 1 and type 2 and also on type 4 globoside chains to provide a high sensitivity method for typing of all the major blood-group antigens, including the A, B, H, Le^a, Le^x, Le^b, and Le^y determinants, present in oligosaccharides. Using the principles established, we identified two minor unknown oligosaccharide components present in the products of enzymatic synthesis by bacterial fermentation. We also demonstrated that the unique fragmentations derived from the D- and ^0,2A-type cleavages observed in ESI-CID-MS/MS, which are important for assigning blood-group and chain types, only occur under the negative-ion conditions for reducing sugars but not for reduced alditols or under positive-ion conditions

Profiling of Sialylated Oligosaccharides in Mammalian Milk Using Online Solid Phase Extraction-Hydrophilic Interaction Chromatography Coupled with Negative-Ion Electrospray Mass Spectrometry

Sialylated oligosaccharides are important components in mammalian milk. They play a key role in the health and growth of infants by helping to shape up infant’s gastrointestinal microbiota and defense against infection by various pathogenic agents. A detailed knowledge of the structures, compositions, and quantities of the sialylated milk oligosaccharides (SMOs) is a prerequisite for understanding their biological roles. However, because of the presence of very large amounts of lactose and neutral oligosaccharides, accurate analysis of SMOs is difficult. A pretreatment step is required to remove lactose and neutral oligosaccharides but conventional off-line pretreatment methods are time-consuming and of poor reproducibility. In this presentation, we linked solid-phase extraction (SPE) with hydrophilic interaction chromatography (HILIC) followed by mass spectrometry (MS) identification for the analysis of SMOs. A SPE column with electrostatic repulsion function was used for removal of lactose and neutral oligosaccharides, a HILIC analytical column for separation of the SMOs, and negative-ion electrospray ionization tandem MS was used for their identification and sequencing. The success of the established online SPE-HILIC-MS method was demonstrated by profiling of SMOs in human to investigate detailed SMO changes during lactation period and in animals to compare the difference in SMO contents among the different species

<i>O</i>-GlcNAcylations of endogenous Akt inhibit Akt phosphorylations at Thr 308 and Ser 473.

<p>(<b>A</b>) Schematic representation of the method for characterization of the interplay between <i>O</i>-GlcNAc and <i>O</i>-phosphate of Akt. The proteins in the cell are first divided into two pools by <i>O</i>-GlcNAc immunoprecipitation: the un-GlcNAcylated and <i>O</i>-GlcNAcylated proteins subpopulations. Next, the phosphorylation levels of Akt between the two subpopulations are measured by western blotting. This will directly reflect the effect of <i>O</i>-GlcNAcylations on the phosphorylations of Akt. Here, MCF-7 cells were serum-starved under PUGNAc treatment (<b>B</b> and <b>C</b>) or under normal conditions (<b>D</b> and <b>E</b>), followed by IGF-1 stimulation for the indicated times. The un-GlcNAcylated and <i>O</i>-GlcNAcylated proteins subpopulations were prepared. Akt <i>O</i>-GlcNAcylations under PUGNAc treatment: (<b>B</b>) Determination of the same level of total Akt between the <i>O</i>-GlcNAcylated and un-GlcNAcylated proteins subpopulations. The <i>O</i>-GlcNAcylated proteins subpopulation was compared to a series of known amounts of the un-GlcNAcylated proteins subpopulations by immunoblotting against total Akt. (<b>C</b>) Comparison of the phosphorylation levels of the un-GlcNAcylated and <i>O</i>-GlcNAcylated Akt. The un-GlcNAcylated and <i>O</i>-GlcNAcylated proteins subpopulations were immunoblotted against Akt, <i>O</i>-GlcNAc, and the phosphorylations at Thr 308 and Ser 473. The constitutive <i>O</i>-GlcNAcylations of Akt: (<b>D</b>) Determination of the same level of total Akt between the <i>O</i>-GlcNAcylated and un-GlcNAcylated proteins subpopulations. The <i>O</i>-GlcNAcylated proteins subpopulation was compared to a series of known amounts of the un-GlcNAcylated proteins subpopulations by immunoblotting against total Akt. (<b>E</b>) Comparison of the phosphorylation levels of the un-GlcNAcylated and <i>O</i>-GlcNAcylated Akt. The un-GlcNAcylated and <i>O</i>-GlcNAcylated proteins subpopulations were subjected to immunoblot assay of Akt, <i>O</i>-GlcNAc, and the phosphorylations at Thr 308 and Ser 473. One unit was equal to 0.01 µl of the un-GlcNAcylated proteins. U: un-GlcNAcylated proteins; O: <i>O</i>-GlcNAcylated proteins.</p

Molecular modeling analysis.

<p>(<b>A</b>) Molecular modeling of T305Y mutant (pink) and Akt <i>O</i>-GlcNAcylated at Thr 305 (green). Tyr305, <i>O</i>-GlcNAcylated Thr305 and Asp325 are represented as sticks. (<b>B</b>) Molecular modeling of T312Y mutant (pink) and Akt <i>O</i>-GlcNAcylated at Thr 312 (green). Tyr312, <i>O</i>-GlcNAcylated Thr312, Asp274 and Lys276 are shown as sticks. Additionally, oxygen atoms are shown in red, nitrogen atoms are shown in blue, and the hydrogen bonds are shown in red dot lines.</p

<i>O</i>-GlcNAclyations at Thr 305/312 suppress the Thr308 phosphorylation via disrupting the interaction between Akt and PDK1.

<p>(<b>A</b>) Immunoblot analysis of the phosphorylation levels of wild-type Akt and its mutants. MCF-7 cells were transfected by the indicated plasmids, followed by IGF-1 stimulation. The cell lysates were subjected to immunoblotting analysis of the phosphorylation levels of Akt. (<b>B</b>) Fluorescence images of the subcellular localization of Akt and its mutants in COS-7 cells. COS-7 cells were transfected by the indicated plasmids and serum-starved overnight, followed by IGF-1 stimulation. Cells were fixed and probed by primary Akt antibody. (<b>C</b>) Immunoblot analysis of subcellular fractions of COS-7 cells expressing wild-type Akt and its mutants in response to IGF-1 stimulation. Na/K ATPase and actin were used as the plasma membrane and cytoplasmic markers, respectively. PM, plasma membrane; CYT, cytosol. (<b>D</b>) Coimmunoprecipitation assay for the interaction between PDK1 and Akt or its mutants. MCF-7 cells were transfected by the indicated plasmids and treated by IGF-1. Akt and its mutants were immunoprecipitated with anti-Flag agarose and the precipitate were probed against Akt and PDK1. (<b>E</b>) <i>In vitro</i> Kinase assay of PDK1. Akt and its mutants were immunoprecipitated from overexpressed MCF-7 cells and incubated with PDK1 at room temperature. The reaction mixture were immunoblotted against the Thr308 phosphorylation and total Akt.</p