Limited Acid Hydrolysis as a Means of Fragmenting Proteins Isolated upon Proteinchip® Array Surfaces

ProteinChip Array technology enables protein purification, protein profiling and biomarker discovery on a convenient biochip platform. Traditional proteomic approaches towards protein identification rely upon the generation of peptides through the use of specific proteases. However, for a variety of reasons, the digestion of proteins bound to planar arrays using specific proteases, such as trypsin, has proven to be difficult, at times providing little or no protein digestion at all. Additionally, should more than one protein be present on the array surface, the digestion product consists of peptides from different proteins, adding another dimension of complexity to database mining approaches. These factors have driven our group to explore alternative means of on-chip protein digestion. In this article, we describe an approach to generating peptide maps by limited acid hydrolysis. Depending upon the adsorbed protein, this method requires between 500 femtomol to 5 picomol of protein for on-chip hydrolysis. Besides generating several internal peptide fragments, limited acid hydrolysis also has the advantage of generating peptide ladders from the N- or C-terminus of the protein. From these ladders, a partial primary sequence of the protein can be directly derived using analysis by a simple laser desorption/ionization mass spectrometer. Furthermore, tandem mass spectrometry (MS/MS) can be performed on several internal peptide fragments, thus facilitating the identification of several proteins within a mixture. Based upon the preliminary results of this work, we continue to explore the possibility of using limited acid hydrolysis to identify unknown proteins captured on ProteinChip Array surfaces.