2,5-Dihydroxybenzoic acid (DHB) is the most commonly used matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF) of oligosaccharides. Because acidic, sialylated oligosaccharides are detected at only the low picomole level with DHB, alternative matrices were screened to identify a matrix with a lower limit of detection. Negative-ion spectra of pure mono-, di-, and trisialylated oligosaccharides were acquired with either 6-aza-2-thiothymine or 2',4',6'-trihydroxyacetophenone (THAP) in the linear mode. Detection limits of less than 50 fmol with signal-to-noise ratios of better than 5:1 were achieved with both matrices. THAP was the preferred matrix because it provided a lower limit of detection and gave less prompt fragmentation. Incorporation of ammonium citrate into the matrix, along with vacuum drying of the sample, was required in order to obtain maximum sensitivity with THAP. No evidence of competition for ionization was found when a mixture of mono-, di-, and trisialylated oligosaccharides was analyzed with THAP. These findings suggest that MALDI/TOF analysis may provide a rapid means to identify changes in carbohydrate composition in glycoproteins. In addition, THAP offered improved sensitivity for detection of acidic glycopeptides over alpha-cyano-4-hydroxycinnamic acid.

We have engineered two Chinese hamster ovary cell lines secreting different recombinant glycoproteins to express high levels of human beta1,4-galactosyltransferase (GT, E.C. 2.4.1.38) and/or alpha2, 3-sialyltransferase (ST, E.C. 2.4.99.6). N-linked oligosaccharide structures synthesized by cells overexpressing the glycosyltransferases showed greater homogeneity compared with control cell lines. When GT was overexpressed, oligosaccharides terminating with GlcNAc were significantly reduced compared with controls, whereas overexpression of ST resulted in sialylation of >/=90% of available branches. As expected, GT overexpression resulted in reduction of oligosaccharides terminating with GlcNAc, whereas overexpression of ST resulted in sialylation of >/=90% of available branches. The more highly sialylated glycoproteins had a significantly longer mean residence time in a rabbit model of pharmacokinetics. These experiments demonstrate the feasibility of genetically engineering cell lines to produce therapeutics with desired glycosylation patterns.

Mps1 is a dual specificity protein kinase that is essential for the bipolar attachment of chromosomes to the mitotic spindle and for maintaining the spindle assembly checkpoint until all chromosomes are properly attached. Mps1 is expressed at high levels during mitosis and is abundantly expressed in cancer cells. Disruption of Mps1 function induces aneuploidy and cell death. We report the identification of MPI-0479605, a potent and selective ATP competitive inhibitor of Mps1. Cells treated with MPI-0479605 undergo aberrant mitosis, resulting in aneuploidy and formation of micronuclei. In cells with wild-type p53, this promotes the induction of a postmitotic checkpoint characterized by the ATM-and RAD3-related-dependent activation of the p53-p21 pathway. In both wild-type and p53 mutant cells lines, there is a growth arrest and inhibition of DNA synthesis. Subsequently, cells undergo mitotic catastrophe and/or an apoptotic response. In xenograft models, MPI-0479605 inhibits tumor growth, suggesting that drugs targeting Mps1 may have utility as novel cancer therapeutics. Mol Cancer Ther; 10(12); 2267-75. Ó2011 AACR.

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Identification of ligands separated with affinity chromatography has been facilitated by direct analysis of the bound ligand using matrix-assisted laser desorption time-of-flight mass spectrometry. The mass spectral detection of analytes separated by immunoaffinity chromatography and immobilized metal ion affinity chromatography is shown. For example, cytochrome c is used as an affinity support to purify the anti-cytochrome c monoclonal antibody from ascites, and the mass spectrum of the anti-cytochrome c monoclonal antibody was obtained by direct analysis of an aliquot of the column bed. Direct analyses of metal binding proteins and phosphopeptides bound to immobilized metal ion affinity columns are also demonstrated.The method is characterized by minimal sample manipulation and high sensitivity with low picomoles and high femtomoles of analytes being readily observed.