Development of virtual two-dimensional gels and application to the detection of the small protein-coding open reading frames in the Saccharomyces cerevisiae genome.

Abstract

The virtual 2-dimensional gel (V2DG) substitutes the size dimension of standard 2-dimensional electrophoretic gels by direct matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis of an immobilized pH gradient (IPG) gel, thereby increasing mass accuracy and resolution, and promising future advantages in automated proteomics. To understand the practical issues in the use of V2DG, this technology was applied to the detection of proteins encoded by the small Open Reading Frames (smORFs) of <italic>S. cerevisiae. </italic> The complete genome of this model organism has been sequenced, and most of the larger protein-coding ORFs have been detected and characterized. The protein products of many smORFs, however, continue to elude detection. The current study uses the identification of small proteins in <italic> S. cerevisiae</italic> as a means of pinpointing and addressing the challenges remaining for V2DG technology in 3 crucial areas: (1) processing of the IPG gel, (2) control of the mass spectrometer, and (3) software processing of mass spectrometric data files. Proteins of <italic>S. cerevisiae </italic> were focused on IPG strips, and the strips were introduced directly into both single MALDI-TOF MS and tandem MALDI-TOF-TOF MS instruments. Single stage MALDI-MS analysis was used to characterize signal quality on the gel surface. Tandem MALDI-MS analysis generated ion fragmentation spectra used to identify protein-coding ORFs in a database containing the full nucleotide sequence of <italic>S. cerevisiae.</italic> The unique contributions made by this research include: (1) showing that tandem MS can be used to make protein identifications directly from V2DGs, (2) development of a set of software programs dedicated specifically to the creation, manipulation, and analysis of V2DGs were developed, and (3) showing how the ion optics parameters of the MALDI mass spectrometer can be tuned to compensate for variation in the surface height of the IPG strip to generate ion fragmentation spectra with sufficient signal strength for database searches. One remaining technical challenge is the need to preserve the sharply focused proteins bands on the strip.