It is said that more than half of the proteins which compose living organisms are glycosylated. Glycosylation plays important roles in the structural and functional control of proteins. Recent studies have also revealed that some kinds of diseases (e.g. immunity disorder) are associated with abnormalities in sugar-chain structures or saccharification. With such a technical background, structural analysis of glycoproteins and glycopeptides has been significantly important in bioscience, medical treatment, drug development and various other fields.
Due to the rapid progress in the matrix-assisted laser desorption/ionization ion-trap mass spectrometer (MALDI-IT MS) or matrix-assisted laser desorption/ionization ion-trap time-of-flight mass spectrometer (MALDI-IT-TOF MS) in recent years, accompanied by the advancement in the analytical techniques using such devices, it has become possible to analyze the structure of glycopeptides or similar complexes of molecules having different physical and/or chemical properties (e.g. sugar chain and peptide in the case of glycopeptide). For example, Non Patent Literature 1 discloses an automatic glycopeptide analyzer system using a MALDI-IT-TOF MS. This system is capable of analyzing the structure of a glycopeptide by combining various techniques, such as the MS2 and MS3 analyses, the deduction of sugar-chain structures using de novo sequencing, and the identification of peptides by database search (the deduction of amino-acid sequences).
A MALDI linear TOF MS, which uses no ion trap and allows ions generated from a sample to be directly sent into a flight space, is suitable for a measurement which requires high sensitivity, high quantitative determination accuracy and high reproducibility, since the system has the characteristic that, even if post source decay (the phenomenon in which ions generated from a sample decay in the middle of their flight) occurs, the ions resulting from the decay reach the ion detector without being lost. Another advantage of the MALDI linear TOF MS is that it can perform an analysis with high throughput and over a wide range of mass-to-charge ratios since it does not capture ions with an ion trap. To utilize those advantages, MALDI linear TOF MSs are often used for quantitative analysis of glycopeptides.
For example, Non Patent Literature 2 discloses a technique for determining the quantities of glycoforms in a glycoprotein using a MALDI linear TOF MS. The quantity determination of glycoforms according to this literature is performed as follows:
(1) A glycoprotein is purified.
(2) The glycopeptide is broken into glycopeptides by enzymatic digestion.
(3) The glycopeptides are condensed.
(4) By a reverse-phase liquid chromatograph, the glycopeptides are separated into groups each of which consists of glycopeptides having the same glycosylation site.
(5) The glycoform mixtures separated according to their glycosylation sites are individually subjected to mass spectrometry (MS1 analysis) using a MALDI linear TOF-MS.
(6) The relative quantities of the glycoforms having different sugar-chain structures corresponding to their glycosylation sites are determined using the intensities of the peaks on mass spectra obtained through the MS1 analysis.