Evolutionary proteomics uncovers ciliary signaling components

Monika Abedin Sigg; Tabea Menchen; Jeffery Johnson; Chanjae Lee; Semil P. Choksi; Galo Garcia; Henriette Busengdal; Gerard Dougherty; Petra Pennekamp; Claudius Werner; Fabian Rentzsch; Nevan Krogan; John B. Wallingford; Heymut Omran; Jeremy F. Reiter

To identify the ciliary proteomes of these species, we analyzed the isolated cilia and 154 flagella by mass spectrometry. We performed mass spectrometric protein profiling on 155 unfractionated purified sea urchin cilia (whole cilia), the isolated axonemal fraction (axonemes) 156 and the non-axonemal fraction (membrane plus matrix). Due to the tractability of obtaining large 157 quantities of sea urchin cilia, we were able analyze cilia isolated from three separate embryo 158 cultures at two different developmental stages. We separated the peptides from unfractionated 159 whole cilia using two distinct methods of chromatography: pre-fractionated by hydrophilic 160 interaction chromatography followed by C18 LC-MS/MS or LC-MS/MS using a high resolution 161 C18 column and an extended reverse phase gradient. For the mass spectrometry data analyses, 162 we chose a low-stringency cut-off to capture signaling proteins: proteins with a unique peptide 163 count of 2 or more were included for future analysis (Table S1) . 164