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Proteomics and Glycoproteomics (Bio-)Informatics of Protein Isoforms

Proteomics and Glycoproteomics (Bio-)Informatics of Protein Isoforms. Nathan Edwards Department of Biochemistry and Molecular & Cellular Biology Georgetown University Medical Center. Outline. Tandem mass-spectrometry of peptides Detection of alternative splicing protein isoforms

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Proteomics and Glycoproteomics (Bio-)Informatics of Protein Isoforms

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  1. Proteomics and Glycoproteomics(Bio-)Informatics of Protein Isoforms Nathan Edwards Department of Biochemistry and Molecular & Cellular Biology Georgetown University Medical Center

  2. Outline • Tandem mass-spectrometry of peptides • Detection of alternative splicing protein isoforms • Phyloproteomics using top-down mass-spec. • Characterization of glycoprotein microheterogeneity by mass-spectrometry

  3. Sample + _ Detector Ionizer Mass Analyzer Mass Spectrometer • ElectronMultiplier(EM) • Time-Of-Flight (TOF) • Quadrapole • Ion-Trap • MALDI • Electro-SprayIonization (ESI)

  4. Mass Spectrum

  5. Mass is fundamental

  6. Enzymatic Digest and Fractionation Sample Preparation for MS/MS

  7. Single Stage MS MS

  8. Tandem Mass Spectrometry(MS/MS) Precursor selection

  9. Tandem Mass Spectrometry(MS/MS) Precursor selection + collision induced dissociation (CID) MS/MS

  10. Why Tandem Mass Spectrometry? • MS/MS spectra provide evidence for the amino-acid sequence of functional proteins. • Key concepts: • Spectrum acquisition is unbiased • Direct observation of amino-acid sequence • Sensitive to small sequence variations

  11. Unannotated Splice Isoform • Human Jurkat leukemia cell-line • Lipid-raft extraction protocol, targeting T cells • von Haller, et al. MCP 2003. • LIME1 gene: • LCK interacting transmembrane adaptor 1 • LCK gene: • Leukocyte-specific protein tyrosine kinase • Proto-oncogene • Chromosomal aberration involving LCK in leukemias. • Multiple significant peptide identifications

  12. Unannotated Splice Isoform

  13. Unannotated Splice Isoform

  14. Translation start-site correction • Halobacterium sp. NRC-1 • Extreme halophilic Archaeon, insoluble membrane and soluble cytoplasmic proteins • Goo, et al. MCP 2003. • GdhA1 gene: • Glutamate dehydrogenase A1 • Multiple significant peptide identifications • Observed start is consistent with Glimmer 3.0 prediction(s)

  15. Halobacterium sp. NRC-1ORF: GdhA1 • K-score E-value vs PepArML @ 10% FDR • Many peptides inconsistent with annotated translation start site of NP_279651

  16. What if there is no "smoking gun" peptide…

  17. What if there is no "smoking gun" peptide…

  18. What if there is no "smoking gun" peptide…

  19. HER2/Neu Mouse Model of Breast Cancer • Paulovich, et al. JPR, 2007 • Study of normal and tumor mammary tissue by LC-MS/MS • 1.4 million MS/MS spectra • Peptide-spectrum assignments • Normal samples (Nn): 161,286 (49.7%) • Tumor samples (Nt): 163,068 (50.3%) • 4270 proteins identified in total • 2-unique generalized protein parsimony

  20. Nascent polypeptide-associated complex subunit alpha 7.3 x 10-8

  21. Pyruvate kinase isozymes M1/M2 2.5 x 10-5

  22. Phyloproteomics • Fragment intact proteins (top-down MS) • Match the spectra to protein sequences • Place the organism phylogenetically • Works even for unknown microorganisms without any available sequences

  23. CID Protein Fragmentation Spectrum from Y. rohdei

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

  25. Exact match sequence…

  26. Phylogeny: Protein vs DNA Protein Sequence 16S-rRNA Sequence

  27. What about mixtures?

  28. 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|

  29. 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

  30. E. herbicola protein sequences

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

  32. Glycoprotein Microheterogeneity • Glycosylation is important, but our analytic tools are rather rudimentary • Detach glycans (PNGase-F) and analyze glycans • Detach glycans (PNGase-F) and analyze peptides • Get glycan structures, but no association with protein or protein site, or • Get glycosylation sites, but no association with glycan structures. • We analyze glycopeptides directly… • Challenges all facets of glycoproteomics

  33. Altered N-Glycosylation in Cancer Glycosyltransferase Expression or Glycan Analyses GalNAc Sialic Acid Gal GlcNAc Man Fut-VIII (α1-6 Fuc) Comunale, 2010 Fut-VI (α1-3 Fuc) Higai,2008 GnT-V (β1-6 GlcNAc) Wang, 2007 ST-VI Gal1 (α 2-6 NeuAc) Hedlund, 2008 NH3+ N X S/T COO- K. Chandler

  34. The informatics challenge • Identify glycopeptides in large-scale tandem mass-spectrometry datasets • Many glycopeptide enriched fractions • Many tandem mass-spectra / fraction • Good, but not great, instrumentation • QStar Elite – CID, good MS1/MS2 resolution • Strive for hypothesis-generating analysis • Site-specific glycopeptide characterization • Glycoform occupancy in differentiated samples

  35. CID Glycopeptide Spectrum

  36. Observations • Oxonium ions (204, 366) help distinguish glycopeptides from peptides… • …but do little to identify the glycopeptide • Few peptide b/y-ions to identify peptides… • …but intact peptide fragments are common • If the peptide can be guessed, then… • …the glycan's mass can be determined

  37. Haptoglobin Standard • N-glycosylation motif (NX/ST) * Site of GluC cleavage Pompach et al. Journal of Proteome Research 11.3 (2012): 1728–1740. Haptoglobin (HPT_HUMAN) NLFLNHSE*NATAK VVLHPNYSQVDIGLIK MVSHHNLTTGATLINE

  38. Tuning the filters… • We estimate the number of false-positives……so that the user can tune the search parameters

  39. Application of Exoglycosidasesto locate Fucose • At ITIH4 site N517 LPTQNITFQTE LPTQNITFQTE LPTQNITFQTE LPTQNITFQTE K. Chandler

  40. NVVFVIDK ITIH4 Glycopeptide K. Chandler

  41. Similar Glycopeptides Spectra( mass Δ ~ +162 Da) ? +162 Da MVSHHNLTTGATLINE

  42. Fragmented Glycopeptides( mass Δ ~ +162 Da) ? +162 Da MVSHHNLTTGATLINE MVSHHNLTTGATLINE

  43. Propagating Annotations VVL+A1G1 MVS+A2G2 MVS+A2G2 VVL+A2G2 MVS+A2G2 MVS+A1G1 MVS+A1G1 G. Berry

  44. Summary Mass-spectrometry coupled with protein chemistry and good informatics can look beyond the obvious to the unexpected... …and there is plenty to find!

  45. Acknowledgements • Edwards lab • Kevin Chandler • Gwenn Berry • Fenselau lab (UMD) • Colin Wynne • Avantika Dhabaria • Goldman lab (GU) • Kevin Chandler • Petr Pompach • NSF Graduate Fellowship (Chandler) • Funding: NCI

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