PROCESS AND ITS APPLICATION FOR IMPROVING REPRODUCIBILITY IN MALDI-TOF GLYCAN PROFILING OF HUMAN SERUM: EXPERIMENTAL PROCEDURE AND APPLICATION TO THE SCREENING FOR OVARIAN TUMORS

摘要

Disclosed is that the most crucial point in the quantitative analysis of MALDI-TOF MS is to minimize quantitative variances (RSDs, relative standard deviations) of peak intensities of obtained spectrum data. The minimized RSD is translated into improved reproducibility of the data and finally leads to high accuracy of the results. Several technical procedures based on the concept of automatization to reduce the RSDs (to less than 10%) in the MALDI-TOF MS of serum glycans were proposed for the preparation steps of extracting the targeted glycans from human blood and sample loading processes onto the plates. The presented techniques described in the examples proved the contributions of the present invention to enhanced reproducibility of the MALDI-TOF MS and consequently to improved screening accuracy for the differentiation of the benign ovarian tumor patients from the borderline ovarian tumor patients in comparison with accuracy of differentiation based on other analysis methods.

主权项

1. A method of glycan analysis comprising the steps of:
(a) injecting individual human serum samples into each area of a Plate #1 having a plurality of sample-loading areas; (b) extracting at least one glycan from the sample by introducing glycan-releasing enzyme into each area of the Plate #1; (c) preparing a Plate #2 having a plurality of sample-loading areas by spreading a matrix solution for MALDI-TOF mass spectrometry at least two times onto each area of the Plate #2 at 40 to 65° C., and subsequently drying and crystallizing the matrix; (d) loading a solution comprising at least one glycan extracted from the step (b) onto each area of the Plate #2; (e) drying the Plate #2 under reduced pressure; and (f) obtaining a mass spectrum of at least one glycan from each area in the Plate #2 using MALDI-TOF, wherein an average coefficient of variation of intensity of each obtained peak from the mass spectrometry is less than 10%.