Top-down characterization of proteins in bacteria with unsequenced genomes

1. Top-down characterization of proteins in bacteria with unsequenced genomes Colin WynneCatherine Fenselau University of Maryland, College Park Nathan Edwards Georgetown University Medical Center

2. Microorganism Identification • Important application of mass spectrometry • Match spectra with sequence for identity • Many bacteria will never be sequenced... • Pathogen simulants, for example • ...but many have – about 1000 to date. • Can we use the available sequence to identify proteins from unsequenced bacteria? • Yes, for some proteins in some organisms! • Yersinia rohdei, Erwinia herbicola, Enterobacter cloacae

3. Crude cell lysate Capilary HPLC C8 column LTQ-Orbitrap XL Precursor scan: 30,000 @ 400 m/z Data-dependent precursor selection: 5 most abundant ions 10 second dynamic exclusion Charge-state +3 or greater CID product ion scan 15,000 @ 400 m/z Intact protein LC-MS/MS

4. CID Protein Fragmentation Spectrum from Y. rohdei

5. Enterobacteriaceae Protein Sequences • Exhaustive set of all Enterobacteriaceae protein sequences from • Swiss-Prot, TrEMBL, RefSeq, Genbank, and CMR • Plus, Glimmer3 predictions on Enterobacteriaceae genomes from RefSeq • Primary and alternative translation start-sites • Filter for intact mass in range 1 kDa – 20 kDa • 253,626 distinct protein sequences, 256 species • Derived from "Rapid Microorganism Identification Database" (RMIDb.org) infrastructure.

6. ProSightPC 2.0 • Product ion scan decharging • Enabled by high-resolution fragment ion measurements • THRASH algorithm implementation • Absolute mass search mode • 15 ppm fragment ion match tolerance • 250 Da precursor ion match tolerance • "Single-click" analysis of entire LC-MS/MS datafile.

7. CID Protein Fragmentation Spectrum from Y. rohdei Match to Y. pestis 50S Ribosomal Protein L32

8. Identified E. herbicola proteins • 30S Ribosomal Protein S19 • m/z 686.39, z 15+, E-value 1.96e-16, Δ 0.007 • Six proteins identified with |Δ| < 0.02

9. Identified E. herbicola proteins • DNA-binding protein HU-alpha • m/z 732.71, z 13+, E-value 7.5e-26, Δ-14.128 • Eight proteins identified with "large" |Δ|

10. Identified E. herbicola proteins • DNA-binding protein HU-alpha • m/z 732.71, z 13+, E-value 1.91e-58, Δ0.11 • Use "Sequence Gazer" to find mass shift

11. Identified E. herbicola proteins • DNA-binding protein HU-alpha • m/z 732.71, z 13+, E-value 7.5e-26, Δ-14.128 • Extract N- and C-terminus sequence supported by at least 3 b- or y-ions

12. E. herbicola protein sequences

13. E. herbicola sequences found in other species

14. Phylogenetic placement of E. herbicola Cladogram Phylogram phylogeny.fr – "One-Click"

15. Serratia proteamaculans CSR, RPS19 Citrobacter koseri RPL32 Enterobacter sakazakii RPS21 RPL30 Enterobacter sakazakii Sodalis glossinidius Photorhabdus luminescens* Erwinia tasmaniensis Enterobacter sp. 638 Genome Annotation Correction Some spectra match Glimmer predictions only!

16. Conclusions • Protein identification for unsequenced organisms. • Identification and localization for sequence mutations and post-translational modifications. • Extraction of confidently established sequence suitable for phylogenetic analysis. • Genome annotation correction. • New paradigm for phylogenetic analysis?

17. Acknowledgements • Dr. Catherine Fenselau • Colin Wynne, Joe Cannon • University of Maryland Biochemistry • Dr. Yan Wang • University of Maryland Proteomics Core • Dr. Art Delcher • University of Maryland CBCB • Funding: NIH/NCI

18. Shared "Biomarker" Proteins

19. Shared "Biomarker" Proteins