Microscale on-membrane PMF analysis can be conducted on proteins transferred to a PVDF membrane utilizing the microdispensing function of a chemical printer (Refer to AXIMA Application 14, 17). Moreover, using the AXIMA-QIT ion trap MS in conjunction with the chemical printer enables MS analysis with high mass accuracy (<0.1Da). In this application, we succeeded in conducting MS/MS analysis of proteins immobilized on PVDF transfer membranes, something which has been difficult up to now due to the irregularities of the membrane surface and electrical charge. Direct on-membrane MS/MS analysis using the chemical printer together with the AXIMA-QIT, in addition to the established methodology for onmembrane PMF analysis, is expected to be extremely effective in identification of unknown proteins.
Protein spots from E. coli separated by 2-dimensional electrophoresis were transferred to a PVDF membrane for use as the sample (Fig. 1). A chemical printer was then used to perform enzymatic digestion (trypsin digestion) on selected protein spots. Following enzymatic digestion, matrix solution was microdispensed on each print position, and onmembrane MS analysis was then conducted using the AXIMA-QIT. The obtained MS spectra are shown in Fig. 2.
Next, the AXIMA-QIT was used to conduct on-membrane MS/MS analysis of precursor ions identified from characteristic signals of protein spots verified through PMF analysis. We were able to identify the respective protein spots by conducting an MS/MS ion search based on the obtained MS/MS spectra.
Fig. 1 shows a portion of the transfer membrane stained with Direct Blue 71 after transfering E. Coli extract separated by 2-dimensional electrophoresis to the PVDF membrane. The MS spectra obtained from microscale on-membrane PMF analysis using the chemical printer are shown i n Fig. 2. Combining the chemical printer with the AXIMA-QIT ion trap mass spectrometer enables, on-membrane MS analysis with high mass accuracy (<0.1Da).
Fig. 3 shows the results obtained from on-membrane MS/MS analysis of the precursor ions identified from characteristic signals (Spot 1: m/z =1610.79, Spot 2: m/z =1708.87, Spot 3: m/z =1568.85) confirmed by PMF analysis. The results of MS/MS ion search using these MS/MS spectra provided identification of these protein spots with convincing scores (Fig. 4).
Literature cited: T. Nakanishi et al., J. Proteome Res ., 2005, 4, 743-747