Mass microscopy : imaging biomolecules on surfaces

Abstract

Now, that mass spectrometry (MS) has been established as the analytical tool for the analysis of proteins a new challenge awaits: the development of spatial resolved mass spectrometry directly on biological tissue and cells at single cell level or beyond.

The desire to study the complexity of biological systems and their diseased states requires high lateral resolution, molecule specific imaging tools capable of visualizing the distributions of a wide range of known and unknown molecules in biological samples (cells / tissue). Especially, the identity, distributions and modifications of proteins are of great interest since they can serve as biomarkers for specific diseases. In contrast to other molecular imaging techniques, imaging mass spectrometry combines lateral resolving power with mass spectrometry's chemical specificity. The combination of these characteristics makes imaging mass spectrometry especially attractive for application as research tool in the areas of cell biochemistry, biomarker discovery and drug development and potentially as diagnostic tool in medical sciences.

We have implemented a stigmatic or microscope mode ion imaging methodology for UV-MALDI MS to improve lateral resolution and speed of operation. For this purpose a time-of-flight mass spectrometer with stigmatic ion optics was equipped with an UV- MALDI source. In contrast to the conventional scanning microprobe MALDI imaging methodology the microscope mode approach uses spatial resolved ion detection instead of spatial resolved ion generation to record the distributions of biomolecules. The resulting apparatus is capable of projecting magnified ion-optical images of distributions of macromolecules from within the illuminated area (~200 ìm in diameter) onto a detector. It is shown that these ion images can be recorded with 4 ìm lateral resolution. One cycle of the experiment is completed within 1 ms; in this time ion images across the entire mass range are created. In comparison with the conventional scanning microprobe methodology the microscope mode imaging MS offers a reduction in acquisition time of three orders of magnitude.

The microscopic nature of the presented technique removes the demand for a highly focused laser spot to achieve high spatial resolution. This offers a greater versatility in choice of ionization sources for high resolution macromolecular imaging MS. This is demonstrated in experiments in which a 2.94 ìm IR laser was used for ionization purposes (IR-MALDI). The spatial resolution that was achieved in these experiments was equal to the resolution obtained for UV-MALDI microscope mode imaging.

To improve protein identification possibilities and feasible mass range in microscope mode imaging MS it is combined with the molecular scanner methodology. This protein identification technique uses one-step-digestion-transfer and peptide mass fingerprinting to rapidly identify proteins from complex mixtures. It is shown that in this manner high mass proteins (> 100,000 Da) can be detected and identified.

Another ionization technique, which is frequently used in imaging MS, is secondary ion mass spectrometry (SIMS). It is capable of submicron spatial resolution but the chemical information that is obtained is limited: in the energetic SIMS ion formation process nearly all molecular species are extensively fragmented. Multiple sample preparation protocols have been developed to improve the intact molecular ion yield in SIMS. One of these is termed: matrix-enhanced SIMS (ME-SIMS). We have studied the effects of matrix application in imaging MS studies and shown that one of the factors that improves the molecular ionization yields in ME-SIMS is reduction of the internal energy of the molecular ions created in the ionization process.