Computer‐assisted mass spectrometric analysis of naturally occurring and artificially introduced cross‐links in proteins and protein complexes

A versatile software tool, virtualmslab, is presented that can perform advanced complex virtual proteomic experiments with mass spectrometric analyses to assist in the characterization of proteins. The virtual experimental results allow rapid, flexible and convenient exploration of sample preparation strategies and are used to generate MS reference databases that can be matched with the real MS data obtained from the equivalent real experiments. Matches between virtual and acquired data reveal the identity and nature of reaction products that may lead to characterization of post‐translational modification patterns, disulfide bond structures, and cross‐linking in proteins or protein complexes. The most important unique feature of this program is the ability to perform multistage experiments in any user‐defined order, thus allowing the researcher to vary experimental approaches that can be conducted in the laboratory. Several features of virtualmslab are demonstrated by mapping both disulfide bonds and artificially introduced protein cross‐links. It is shown that chemical cleavage at aspartate residues in the protease resistant RNase A, followed by tryptic digestion can be optimized so that the rigid protein breaks up into MALDI‐MS detectable fragments, leaving the disulfide bonds intact. We also show the mapping of a number of chemically introduced cross‐links in the NK1 domain of hepatocyte growth factor/scatter factor. The virtualmslab program was used to explore the limitation and potential of mass spectrometry for cross‐link studies of more complex biological assemblies, showing the value of high performance instruments such as a Fourier transform mass spectrometer. The program is freely available upon request.