MASS SPECTROMETRY OF PROTEINS DERIVED FROM SHORT OPEN READING FRAME

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. For almost 20 years, computational efforts to define open reading frames (ORFs) have relied heavily on length of the ORF as a metric for separating the dubious from the genuine. The confidence of these prediction algorithms is proportional to the length of the putative ORF; thus an ORF has been operationally defined as a genetic element that is ultimately translated into a polypeptide of at least 100 amino acids. We have recently developed a sequencing-based method that allows us to determine, in a way that is independent of prior definitions of ORFs, which parts of the genome are being transcribed and translated. This method therefore gives us the ability to experimentally annotate all of the ORFs in any given genome, including those that are shorter than 100 codons, so-called short ORFs (sORFs). Application of this method to yeast undergoing meiosis reveals that there are thousands of sORFs being translated, almost all of which are currently unannotated. We are interested in using mass spectrometry to demonstrate the presence of peptides produced from these sORFs.