Glycopeptide Identification with Byonic™ Software Marshall Bern www.proteinmetrics.com Protein Metrics – ASMS Asilomar, Oct. 2014 Byonic™ is an advanced proteomics search engine Byonic 7000 # No Mods 6000 # Common Mods (^q,^c,m,n,q) 4000 3000 2000 1000 1. Higher Sensitivity # Spectra 5000 11821 45511 14152 47603 ABRF iPRG Study on PTM finding, 2012 52781 14151 74564 23117 34284i 92653 87048i 40104i 23068 77777i 42424i 97053i 94158i 87133i 58409 11211 93128i 33564 58288v 71755v 0 2. Topdown Proteomics IgG1 Fc with glycan and M[+16]’s 2 Protein Metrics – ASMS Asilomar, Oct. 2014 Byonic™ is an advanced proteomics search engine 3. “Expert system” annotation 216 Da = Characteristic peak for phospho-Tyrosine 4. Multiple Identifications per MS2 scan Protein Metrics – ASMS Asilomar, Oct. 2014 3 Byonic™ is an advanced proteomics search engine 5. Glycopeptides! Protein Metrics – ASMS Asilomar, Oct. 2014 4 Outline for Rest of the Talk 1. How does Byonic work? 2. How to run Byonic 3. Interesting examples Protein Metrics – ASMS Asilomar, Oct. 2014 5 Byonic score is a sum of benefits and penalties Mouse brain synaptosome, run on Q-Exactive Data from Kati Medzihradszky, UCSF AT1B2_MOUSE peptide with man6 204 Da for HexNAc Y1 ion ~y12 means loss of full glycan y12 means glycan on Protein Metrics – ASMS Asilomar, Oct. 2014 6 Byonic annotates glycan (and peptide + glycan) peaks that match glycopeptide pieces for any reasonable glycan topology • Byonic will annotate an HCD peak at 657 Da for any glycan with HexNAc, Hex, NeuAc; 512 Da for any glycan with HexNAc, Hex, and Fuc; etc. • Byonic will annotate Pep+HexNAc for any glycopeptide with HexNAc Mouse brain Contactin-1 glycopeptide HexNAc(5)Hex(4)Fuc(2) 512 Da = Diagnostic peak for antennal fucose Protein Metrics – ASMS Asilomar, Oct. 2014 7 Is it possible to localize O-glycans with CID ? HexNAc(2) Hex(2) NeuAc(2) = 1312.455 Da rEPO made in HEK cells Run on Orbitrap Velos HCD NCE = 35% Data from Khoo, Academia Sinica, Taiwan • ~y11 + 2 HexNAc rules out two HexNAc-Hex-NeuAc’s • Byonic placed the O-glycan correctly (AASAA not AISPP) on 178 of 185 observations of the peptide ~y11 + HexNAc Pep_1+ ~y11 + HexNAc Hex ~y12 + HexNAc Protein Metrics – ASMS Asilomar, Oct. 2014 Pep – 18 ~y11 + 2 HexNAc 8 Outline for Rest of the Talk 1. How does Byonic work? 2. How to run Byonic 3. Interesting examples Protein Metrics – ASMS Asilomar, Oct. 2014 9 Modification Fine Control At most 3 common modifications per peptide At most 1 rare modification per peptide Allow up to three M[+16]’s but only one W[+32] Protein Metrics – ASMS Asilomar, Oct. 2014 10 Glycan modifications follow the same rules Pre-defined database of ~300 N-glycan compositions Rare1 at most one per peptide A peptide can have one man5, one man6, and one man7, along with a rare modification Protein Metrics – ASMS Asilomar, Oct. 2014 11 Glycan database size and modification fine control can have a huge impact on search size! HIV gp120 sequence: …GK.LICTTAVPWNASWSNK.SLEDIWDNMTWMQWER.EIDNYTNT… • 300 N-glycans, all rare1, total rare max 1, no missed cleavages LICT…NK peptide gives ~600 glycoforms • 300 N-glycans, common1, total common max 3, no missed cleavages LICT…NK peptide gives 300 × 300 = 90,000 glycoforms • 300 N-glycans, common1, total common max 3, one missed cleavage LICT…ER peptide gives 300 × 300 × 300 = 2.7 x 107 glycoforms Protein Metrics – ASMS Asilomar, Oct. 2014 12 Glycan database size and modification fine control can have a huge impact on search size! Human Ig Alpha-1 Sequence: …HVK.HYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPR.LSH… • 10 O-glycans, common5, Total common max = 5 HYT…PR peptide gives (12 choose 5) × 510 = 7,434,375,000 glycoforms • 100 different sums of O-glycan masses, rare1 HYT…PR peptide gives 1200 glycoforms (but no site localization) • Can search anything in between – e.g., try to resolve the O-glycan on only the first and last S/T Protein Metrics – ASMS Asilomar, Oct. 2014 13 Glycan databases • Use any text editor to write/edit • Use literature, prior knowledge, glycomics experiments, etc. • Setting the database to common1 is the same as setting each glycan in it to common1. • Big impact on performance! • Byonic keywords: Hex, HexNAc, NeuAc, NeuGc, Fuc, dHex, GlcNAc, GalNAc, Man, Glc, Gal, Pent, Xyl, GlcA, IdoA, Sodium, Na, Sulfo, Methyl, Acetyl, Phospho, DiNAcBac, Pseudaminic, Legionaminic, Etn, pEtn, … Protein Metrics – ASMS Asilomar, Oct. 2014 14 One more option, meant for “one-protein” samples Glycopeptide assignments will be shown regardless of Byonic score or FDR Protein Metrics – ASMS Asilomar, Oct. 2014 15 One more option, meant for “one-protein” samples Uncheck this box Glycopeptide assignments will be shown regardless of Byonic score or FDR Protein Metrics – ASMS Asilomar, Oct. 2014 16 Practical tips for Glycoproteomics • “Right size” the protein database • Include all the proteins in the sample, but not too many more. • “Right size” the glycan database by iterative search • Start from a small glycan database and work up. • Start large and work down by removing glycans with little support. • Be careful with “Total common max” – start small (like 1 or 2) and work up only if you need to • Manually check all glycopeptide assignments prone to error • Glycans containing 2 or more Fuc’s or 1 Fuc and 1 NeuGc • Glycopeptides with close-together oxidations and glycosylations • Glycopeptides with 2 or more potential glycosylation sites Protein Metrics – ASMS Asilomar, Oct. 2014 17 Outline for Rest of the Talk 1. How does Byonic work? 2. How to run Byonic 3. Interesting examples Protein Metrics – ASMS Asilomar, Oct. 2014 18 Bacterial Glycosylation Campylobacter jejuni, run on Orbitrap Velos with HCD fragmentation, HCD NCE = 45% Data from Nick Scott, UBC Nothaft et al, Mol Cell Proteomics 11 (11), 2012 HexNAc(5)Hex(1) diNAcBacillosamine(1) Protein Metrics – ASMS Asilomar, Oct. 2014 Y1_1+ Glycopep_2+ Glycan contains diNAcBac Byonic looks for Y1 = Peptide + diNAcBac (228 Da) 19 O-glycosylation (and N-terminal alkylation!) on MHC class I peptides Human B cell line, run on Orbitrap Elite with EThcD fragmentation, HCD NCE = 32% Data from Albert Heck, Utrecht Mommen et al, PNAS 111 (12), 2014 from RNA-binding protein 27 (cytoplasm and nucleus!) Pep_1+ [+28]IPRPPIT[+365]QSSL [+28]IPRPPIT[+656]QSSL Pep_1+ Note: [+14/+28]RPPIT[+203]QSSL (called O-GlcNAc) found in leukemia samples [Hunt lab, ASMS 2014] Protein Metrics – ASMS Asilomar, Oct. 2014 20 O-glycosylation (without N-terminal alkylation) on MHC class I peptides Human B cell line, run on Orbitrap Elite with EThcD fragmentation, HCD NCE = 32% Data from Albert Heck, Utrecht Mommen et al, PNAS 111 (12), 2014 IPRPPIT[+365]QSSL c10 Pep_1+ from RNA-binding protein 27 (cytoplasm and nucleus!) Protein Metrics – ASMS Asilomar, Oct. 2014 26 Summary • • Byonic offers: • Glycopeptide search in a full-service proteomics search engine • Support for all types of fragmentation and mass analysis • Special features for one-protein samples Byonic is keeping up with MS hardware. • If you can fragment it, we can identify it! Protein Metrics – ASMS Asilomar, Oct. 2014 www.proteinmetrics.com 22
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