A novel fluorescent probe, 3-(acetylamino)-6-aminoacridine (AA-Ac), has been synthesized and its applicability to the analysis of picomole levels of N-linked glycans investigated. AA-Ac was found to be an excellent derivatization reagent for N-linked glycans, giving at least twice the intensity of fluorescence as its predecessor 2-aminoacridone. AA-Ac-labeled glycans were analyzed by both normal and reversed-phase HPLC. They were also amenable to enzymatic sequencing and analysis by MALDI-TOF mass spectrometry, free zone capillary electrophoresis, and capillary electrophoresis/electrospray ionization mass spectrometry.
A protocol has been developed involving the derivatization of glycan mixtures with 2-aminoacridone and co-injection with a dextran ladder derivatized with methyl 4-aminobenzoate (M-4AB). These two derivatizing agents have very different ultraviolet absorbance and fluorescence characteristics. A chromatographic separation using a normal-phase column support followed by in-series UV and fluorescence detection allowed simultaneous analysis of the two mixtures of the separately derivatized carbohydrates without any interference. This new approach uses the M-4AB dextran ladder derivatives as internal standards spanning the whole chromatogram, allowing an accurate and detailed comparison of glycosylation profiles. It also saves much time by avoiding the necessity of "sandwiching" an unknown glycan mixture between two chromatographic runs of a dextran ladder. The use of this technique has been demonstrated in the case of glycans released from ribonuclease B and human IgG.
A 1-mm microbore hydrophilic interaction column has been used for the separation of 2-aminoacridone (2-AMAC)-derivatized glycan mixtures, released from naturally occurring and recombinant proteins. Primary structure identification of the 2-AMAC glycan derivatives was carried out by HPLC using fluorescence and mass spectrometric detection. In some cases, enzymatic digestion of the 2-AMAC glycans was applied to confirm glycan structure. This strategy is considerably more rapid than methods normally used in glycan analysis, which involves manual collection of separated oligosaccharide derivatives and analysis of individual fractions by mass spectrometry.
A single dose of puromycin aminonucleoside (PAN) given parenterally to rats induces ultrastructural glomerular changes and a nephrotic syndrome similar in many respects to human minimal change nephropathy. The exact aetiologies of both the human and the experimental syndromes are unknown, and are probably multifactorial. However, among the observed consequences in humans and rats is increased plasma protein excretion in urine, beginning in the latter typically 3—6 days after PAN administration. In view of this, two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) has been used to profile urinary proteins during PAN‐induced nephrotoxicity and subsequent recovery in the rat. In addition, urinary high performance liquid chromatography (HPLC) profiles and high resolution proton nuclear magnetic resonance (NMR) spectroscopy has been utilised to simultaneously detect toxin‐induced changes in the relative concentrations of a number of metabolites. The proteomic approach, in conjunction with these other techniques, has the potential to provide significantly more mechanistic information than is provided readily by traditional clinical chemistry.
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