Protein identification by 3D OrbiSIMS to facilitate in situ imaging and depth profiling

Label-free protein characterization at surfaces is commonly achieved using digestion and/or matrix application prior to mass spectrometry. We report the assignment of undigested proteins at surfaces in situ using secondary ion mass spectrometry (SIMS). Ballistic fragmentation of proteins induced by a gas cluster ion beam (GCIB) leads to peptide cleavage producing fragments for subsequent Orbitrap(TM) analysis. In this work we annotate 16 example proteins (up to 272kDa) by de novo peptide sequencing and illustrate the advantages of this approach by characterizing a protein monolayer biochip and the depth distribution of proteins in human skin. Label-free protein characterization at surfaces requires digestion or matrix application prior to mass spectrometry. Here, the authors report the assignment of undigested proteins at surfaces by de novo sequencing and apply the methodology to a protein monolayer biochip and for in situ depth profiling of proteins through human skin.