Identification and separation of protein, contaminant and substrate peaks using gentle-secondary ion mass spectrometry and the g-ogram.

RATIONALE: Secondary ion mass spectrometry (SIMS) is an important technique for the characterization of proteins at surfaces. However, interpretation of the mass spectra is complicated owing to confusion with peaks from contaminants and the substrate which is further compounded by complex fragmentation mechanisms. We test a new development of the G-SIMS method called the g-ogram to separate out spectral components without a priori information about which peaks to include in the analysis and which peaks relate to each component.

METHODS

The effectiveness of the g-ogram method is investigated using a model system of lysozyme adsorbed onto a silicon wafer and indium tin oxide substrates. In the method, two SIMS spectra are acquired using Bi(+) and Mn(+) primary ions which create lower and higher fragmentation in the spectra, respectively. The g-ogram separates out components using a separation parameter that is related to the fragmentation energy.

RESULTS

The g-ogram separates the spectrum of lysozyme adsorbed onto a silicon wafer into three components: (i) the substrate and PDMS contamination; (ii) a second, but unexpected, contaminant; and (iii) peaks from the protein amino acids. Similar results are achieved for the indium tin oxide substrate. In addition, evidence of fragments from plural amino acids with two candidate peaks at 140.12 Da and 185.08 Da is observed.

CONCLUSIONS

The g-ogram method effectively separates out mass peaks relating to the substrate, contamination and protein without any a priori information or subjective decisions about which peaks to include in the analysis (so called 'peak picking'). This is a great help to analysts. We find two possible peaks from plural amino acids but no evidence of pluralities is found for peaks above 240 Da that are generated from when using Bi or Mn primary ions.