1. Top-down characterization of proteins in bacteria with unsequenced genomes Nathan Edwards Georgetown University Medical Center

2. 2 Microorganism Identification Homeland-security/defense applications Long history of fingerprinting approaches Clinical applications in strain identification: Selection of treatment and/or antibiotics New applications in microbiome analysis: Bacterial colonies in gut,.... Chronic wound infections Compete with genomic approaches? PCR, Next-gen sequencing Primary sales-pitch is speed.

3. Microorganism Identifications Match spectra with proteome (or genome) sequence for (species) identity Provides robust match with respect to instrumentation and sample prep Many bacteria will never be sequenced or "finished"... Pathogen simulants, for example...but many have – about 2500 to date. 3

4. Microorganism Identifications Match spectra with proteome (or genome) sequence for (species) identity Provides robust match with respect to instrumentation and sample prep Many bacteria will never be sequenced or "finished"... Pathogen simulants, for example...but many have – about 2500 to date. Can we use the available sequence to identify proteins from unknown, unsequenced bacteria? Yes, for some proteins in some organisms! 4

5. 5 Intact protein LC-MS/MS 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 CAD product ion scan 15,000 @ 400 m/z

6. 6 CID Protein Fragmentation Spectrum from Y. rohdei

7. 7 Enterobacteriaceae Protein Sequences Exhaustive set of all Enterobacteriaceae family protein sequences from Swiss-Prot, TrEMBL, RefSeq, Genbank, and [CMR]...plus Glimmer3 predictions on RefSeq Enterobacteriaceae genomes 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.

8. 8 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.

9. Other tools Explored using standard search engines: Decharge and format as charge +1 spectrum X!Tandem scoring plugin (ProSight, delta M) OMSSA, Mascot, etc… MS-Tools: MS-Deconv, MS-TopDown, MS-Align, MS-Align+, MS-Align-E! 9

10. 10 CID Protein Fragmentation Spectrum from Y. rohdei Match to Y. pestis 50S Ribosomal Protein L32

11. Exact match sequence… 11

12. Phylogeny: Protein vs DNA 12 Protein Sequence16S-rRNA Sequence

13. What about mixtures? 13

14. 14 Shared Small Ribosomal Proteins

15. 15 Shared Small Ribosomal Proteins

16. 16 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

17. 17 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| Identified E. herbicola proteins

18. 18 DNA-binding protein HU-alpha m/z 732.71, z 13+, E-value 1.91e-58 Use "Sequence Gazer" to find mass shift ΔM mode can "tolerate" one shift for free! Identified E. herbicola proteins

19. ProSightPC: ΔM mode 19 Protein Sequence Experimental Precursor ΔMΔM b- and y-ions Also: PIITA - Tsai et al. 2009

20. ProSightPC: ΔM mode 20 Protein Sequence Experimental Precursor ΔMΔM b- and y-ions ΔMΔM b'- and y'-ions Also: PIITA - Tsai et al. 2009 Match a single "blind" mass-shift for free!

21. ProSightPC: ΔM mode 21 Protein Sequence Experimental Precursor ΔMΔM b-, b'-, y- and y'-ions ΔMΔM Also: PIITA - Tsai et al. 2009 Match a single "blind" mass-shift for free!

22. 22 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 Identified E. herbicola proteins

23. 23 E. herbicola protein sequences

24. 24 E. herbicola sequences found in other species

25. 25 Phylogenetic placement of E. herbicola Phylogram Cladogram phylogeny.fr – "One-Click"

26. Genome annotation errors UniProt: E. coli Cell division protein ZapB 22 (371) E. coli strains 26 MQFRRGMTMSLEVFEKLEAKVQQAIDTITL… 3 (204) 17 (166) 0 (2)

27. Genome annotation errors UniProt: E. coli Cell division protein ZapB 22 (371) E. coli strains Need ±1500 Da precursor tolerance… 27 MQFRRGMTMSLEVFEKLEAKVQQAIDTITL… 3 (204) 17 (166) 0 (2)

28. 28 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?

29. 29 Acknowledgements Dr. Catherine Fenselau Avantika Dhabaria, Joe Cannon*, Colin Wynne* University of Maryland Biochemistry Dr. Yan Wang University of Maryland Proteomics Core Dr. Art Delcher University of Maryland CBCB Funding: NIH/NCI