Presentation on theme: "Proteomics and Glycoproteomics (Bio-)Informatics of Protein Isoforms Nathan Edwards Department of Biochemistry and Molecular & Cellular Biology Georgetown."— Presentation transcript:
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 Phyloproteomics using top-down mass-spec. Characterization of glycoprotein microheterogeneity by mass-spectrometry 2
Mass Spectrometer 3 Ionizer Sample + _ Mass Analyzer Detector MALDI Electro-Spray Ionization (ESI) Time-Of-Flight (TOF) Quadrapole Ion-Trap Electron Multiplier (EM)
Tandem Mass Spectrometry (MS/MS) 8 Precursor selection
Tandem Mass Spectrometry (MS/MS) 9 Precursor selection + collision induced dissociation (CID) MS/MS
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 10
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 11
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 (N n ): 161,286 (49.7%) Tumor samples (N t ): 163,068 (50.3%) 4270 proteins identified in total 2-unique generalized protein parsimony 23
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 26
27 CID Protein Fragmentation Spectrum from Y. rohdei
28 CID Protein Fragmentation Spectrum from Y. rohdei Match to Y. pestis 50S Ribosomal Protein L32
38 E. herbicola sequences found in other species
39 Phylogenetic placement of E. herbicola Phylogram Cladogram phylogeny.fr – "One-Click"
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 40
Altered N-Glycosylation in Cancer 41 N X S/T COO- NH3+ Fut-VIII (α1-6 Fuc) Comunale, 2010 GnT-V (β1-6 GlcNAc) Wang, 2007 ST-VI Gal1 (α 2-6 NeuAc) Hedlund, 2008 Fut-VI (α1-3 Fuc) Higai,2008 Glycosyltransferase Expression or Glycan Analyses GalNAc Sialic Acid Gal GlcNAc Man K. Chandler
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 42
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 44
Haptoglobin (HPT_HUMAN) NLFLNHSE*NATAK MVSHHNLTTGATLINE VVLHPNYSQVDIGLIK Haptoglobin Standard 45 N-glycosylation motif (NX/ST) * Site of GluC cleavage Pompach et al. Journal of Proteome Research 11.3 (2012): 1728–1740.
Tuning the filters… Oxonium ions: Number & intensity Match tolerance "Intact-peptide" fragments: Number & intensity Match tolerance Glycan composition: ICScore Constrain search space Match tolerance Glycan database: Constrain search space Match tolerance Precursor ion: Non-monoisotopic selection Sodium adducts Charge state Peptide search space: Semi-specific peptides Non-specific peptides Peptide MW range Variable modifications 46
Tuning the filters… We estimate the number of false-positives… …so that the user can tune the search parameters 47
Application of Exoglycosidases to locate Fucose At ITIH4 site N517 48 LPTQNITFQTE K. Chandler
Your consent to our cookies if you continue to use this website.