1. Peptide-assisted annotation of the Mlp genome Philippe Tanguay Nicolas Feau David Joly Richard Hamelin

2. Objective Use peptide libraries to validate the in silico prediction of gene models  Mapping peptides on a translated genome sequence = provides « correct frames of translation » Assumption : « if a peptide protein is detected, then there must be a gene that encodes it »

3. Methodology (hardware) Urediniospores (3729) Protein extraction 1D SDS-PAGE Gel slicing (64) Trypsin digestion LC-MS/MS Bioinformatics Waters MassPREP station LTQ ThermoElectron ExtractionSlicing Digestion Elution Peptide MS/MS data acquisition

4. Methodology (Bioinformatic) Spectral identification by sequence database searching Statistical validation of peptide identifications Protein databases built from… 1 - Comparison of results from both db 2- Comparison of peptides and GM (validation/correction of genome annotations) 6 frames translation of the genome Gene catalog (16694 GM) Mascot Sequest Mascot Sequest

5. MLP proteomic results so far 691 000 MS/MS spectra obtained from the total proteins 10980 3524699 Gene catalog 6-frame translation Mascot + Sequest Only Mascot 352 unique peptides obtained from the 6-frames translation db have do not match GM of the Gene catalog Unique peptides: False discovery rate below 1.6%

6. Peptide frequency distribution on GM 0 50 100 150 200 250 300 024681012141618202224262830323437394143454749515355575961636567697173757779 No. peptide/gene model No. gene model Mean  9 peptides covering 134 AA / GM The 10980 + 4699 peptides represent assignments for nearly 10% of the Gene catalog e.g. 1659 GM

7. Automated classification of peptides with no hit (352) on the Gene catalog 5’ extension of a predicted GM –If peptide (s) located within the 1000 bp upstream the predicted GM start codon 3’ extension of a predicted GM –If peptide (s) located within the 1000 bp downstream the predicted GM stop codon 5’ and 3’ extension of a predicted GM –If peptides located within the 1000 bp upstream the start codon and within the 1000 bp downstream the predicted GM stop codon Internal extension of a predicted GM –If peptide (s) located in the GM New GM –If no predicted GM in the vicinity of the peptide (s)

8. Corrections-Additions to the Gene catalog ModificationNumber of GM 5’ extension44 Internal exon extension31 3’ extension22 5’ and 3’ extension5 New GM73 Total172 Mapping of the peptides with no hit on the genome allowed the following modifications

9. Manual curation- Internal extension

10. EuGene’s prediction is OK

11. Manual curation- New GM

13. Summary – Peptide-assisted genome annotation –Validated 10 % of the predicted GM –Corrected/found > 170 GM According the manual curation accomplished so far, it appears that EuGene had predicted most of the corrected/found > 170 GM With little resources (6000 $ worth of materials and services, and a few weeks worth of labour) our proteomic analysis:

14. A quantitative proteomic approach (iTRAQ) will be used to compare urediniospores, germinated urediniospores and haustoria protein complexes Perspectives Analysing the Sequest output obtained from the 6-frames translation 5051 peptides identified with Mascot (352 with no hits on the Gene catalog) Sequest ?

15. Available material Our set of peptide spectra from urediniospores proteins is available to validate new GM predictions The peptides GFF files will be made available to the Melampsora community

16. Finding the peptides on the different model prediction sets Gene Catalog1669416599,9% EuGene12386134810,9% Genewise1140879776,9% Genewise1Plus1416210467,4% fgenesh1_pg1576011407,2% fgenesh2_pg1783313777,7%  Do we need to perform a new spectra search on the whole model prediction sets ? Total GMModel prediction setGM validated %