Protein phosphorylation is known to be involved in the regulation of various in-vivo cell functions such as signal transmission, differentiation and proliferation. In recent years, a mass spectrometer, particularly a MALDI mass spectrometer, has been utilized as an effective tool for analyzing phosphorylated sites in proteins in a living organism. However, an analysis using a mass spectrometer of this kind has a problem that, since the ionization efficiency of phosphopeptide is low, and the strength of the ions derived from phosphopeptide is suppressed by a relatively large amount of non-phosphopeptide, the highly-sensitive detection of phosphopeptide is difficult.
In order to solve this problem, research and development has been carried out in various places mainly on the following three aspects:
(i) specific enrichment of phosphopeptide;
(ii) desorption and chemical modification of the phosphate group of phosphopeptide; and
(iii) selection of a matrix or a matrix additive for sample preparation.
The methods (i) and (ii) out of the three methods have a problem that the highly sensitive detection of phosphopeptide is not assuredly achieved even though the processes and operations are complex and cumbersome. On the other hand, the method (iii) does not require a special process or operation unlike the methods (i) and (ii), and thus the method (iii) can be said to be the most convenient method, with which the analysis throughput is easily improved.
As a conventional technique for the method (iii), a non-patent document “Phosphoric Acid as a Matrix Additive for MALDI MS Analysis of Phosphopeptide and Phosphoproteins” (Sven Kjellstrom et al. Analytical Chemistry, 2004, 76, pp. 5109-5117) discloses that the phosphopeptide-detection efficiency can be improved by using 2,5-dihydroxybenzoic acid (DHBA) as a matrix and adding a phosphoric acid in a sample solution during the preparation of the sample.
Although application of the conventional method improves the phosphopeptide-detection efficiency to some extent, the extent of the improvement is insufficient. Moreover, results of a test conducted by the present inventors revealed that, when a phosphoric acid is used as a matrix additive, some peptides were not easily ionized and specific kinds of non-phosphopeptide were almost not at all detected. Accordingly, the presence of undetectable peptides itself causes a significant problem, and further leads to a lower sequence coverage in protein identification by, in particular, peptide mass fingerprinting (PMF). Hence, credibility of the test result may be greatly deteriorated.