A significant challenge is validating our list of putative small genes with short ORFs or transcripts with potentially short ORFs that, based on all available data, we cannot confidently assign as protein coding. For example, if a region has an ORF of 200 amino acids and is conserved with C. briggsae, we can be confident it is a gene. However, if we have a 75 amino acid ORF not confirmed in other nematodes, then we need another type of confirmation. For this we employ the hSRM approach, prioritizing testing of candidate genes by their length, conservation in other nematodes and expression level. The candidate protein coding genes are analyzed as described below.
Method Overview. We will confirm the translation of selected protein sequences using a targeted mass spectrometry approach. Using the putative protein sequences provided for mass spectrometry confirmation, we will i) derive proteotypic tryptic peptide sequences for analysis (Kuster et al. 2005), ii) generate a standard for each peptide, and iii) monitor the precursor and a product ion for each proteotypic peptide using hSRM on a triple quadrupole mass spectrometer. With an hSRM based assay, only molecules that have the same precursor m/z (mass of the peptide divided by charge) and product m/z (a sequence specific fragment) will be transmitted to the detector ? producing a very selective measurement that minimizes contaminating matrix interferences. The presence of the peptide within the C. elegans lysate is confirmed by the detection of a peak in the hSRM chromatogram with signal-to-noise >5 within a retention time of <1% relative error of the peak observed using the peptide standard.
The steps are as follows:
1) Prediction of proteotypic peptide sequences
2) Generation of proteotypic peptide standards
3) Sample Preparation
4) Measurement of Proteotypic Peptides in Complex mixtures by nanoLC-MS/MS using highly selective reaction monitoring (hSRM)
5) Data analysis of hSRM Chromatograms