Protein characterization by mass spectrometry C7250 Part III Zbyněk Zdráhal RG Proteomics, CEITEC-MU Proteomics CF, CEITEC-MU NCBR FS MU zdrahal@sci.muni.cz Functional Genomics and Proteomics National Centre for Biomolecular Research Faculty of Science Masaryk University mu1 nfsnf Characterization of Protein Modifications MP900448552[1] Functional Genomics and Proteomics National Centre for Biomolecular Research Faculty of Science Masaryk University ...Post-translational modifications (PTMs) occur on nearly all proteins. Many domains within proteins are modified on multiple amino acid sidechains by diverse enzymes to create a myriad of possible protein species. How these combinations of PTMs lead to distinct biological outcomes is only beginning to be understood... A. P. Lothrop, M. P. Torres, S. M. Fuchs, FEBS Letters. 587 (2013) 1247–1257 PTM type number > 400 number of PTM sites ≈ 90 000 (detected experimentally) ≈ 230 000 (prediction) (SwissProt, per ≈ 530 000 proteins) G. A. Khoury et al., Sci. Rep. 1, 90; (2011); http://selene.princeton.edu/PTMCuration ...PTMs are known to act alone and in combination to regulate nearly all aspects of protein function... C7250 Why? Mutations of p53 or disruptions of p53 coordination, to a lesser extent, can disturb the normal physiological balance, if genome disarrangement reaches a critical value it leads to cancer Protein p53 https://encrypted-tbn0.gstatic.com/images?q=tbn:ANd9GcR1oGSRO3Nz6VVvIpqSbASgHtJqSW61KU3uFVk3KY87IW6 4x64_ p53 tetramer bound to DNA p53 exerts irreplaceable anti-neoplastic functions at homeostasis and thus is considered to be 'the guardian of the genome‘. B. Gu et al, Int. J. Biol. Sci. 2012, 8, 672-684. p53 is able to coordinate a regulatory network that supervises and responds to a variety of stress signals: ØDNA damage Øaberrant oncogenic activation Øtelomere erosion Øribosomal stress Øloss of cell-cell or cell-matrix adhesion Øhypoxia International Journal of Biological Sciences 08: 0672 image No. 01 Overview of p53 PTMs B. Gu et al, Int. J. Biol. Sci. 2012, 8, 672-684. Histone H3 most frequent PTMs C7250 * Type * Site localization * Site occupancy demands MS “screening” detailed characterization of individual modification Western blot detection of PTM type localization of selected single modification Specific staining of gels detection of PTM type without site localization (phospho, glyco proteins) Potential of MS in analysis of modifications Modification groups: Ø mutation (AA replacement) Ø Ø chemical Ø posttranslational Overview of PTMs and prediction SW tools : DeltaMass - https://www.abrf.org/delta-mass ExPASy - http://www.expasy.org/proteomics/post-translational_modification C7250 „Chemical modifications: q wanted modifications (carbamidomethylation Cys, N –terminal acetylation, quantification tags, etc.) q unwanted modifications (Met oxidation, deamidation N à D during sample preparation, drug adducts, etc.) j0195384 MCj02871590000[1] C7250 Zobrazit zdrojový obrázek Common Posttranslational Modifications C7250 Amines (K/N-terminus) Methylation +14.0269 Formylation +28.0104 Acetylation +42.0373 Lipoic acid +188.3147 Farnesylation +204.3556 Myristoylation +210.3598 Biotinylation +226.2994 Palmitoylation +238.4136 Stearoylation +266.4674 Geranylgeranylation +272.4741 Acids & amides (E/D/Q/N) Pyroglutamic acid (Q) -17.0306 Deamidation (Q/N) +0.9847 Carboxylation (E/D) +44.0098 Hydroxyl groups (S/T/Y) Phosphorylation +79.9799 Sulphation +80.0642 Carbohydrates (S/T/N) Pentoses +132.1161 Deoxyhexoses +146.1430 Hexosamines +161.1577 Hexoses +162.1424 N-acetylhexosamines +203.1950 Sialic acid +291.2579 Common Posttranslational Modifications Further details e.g.: http://themedicalbiochemistrypage.org/protein-modifications.php C7250 MS/MS fragmentation of peptides repetition MC900431582[1] N-terminus C-terminus + + + + b1 b2 b3 b4 b- ion series + + + + y1 y2 y3 y4 y- ion series C7250 N-terminus (c-series) C-terminus (z- series) CID vs ETD CID ETD b, y c, z C7250 Characterization of mutations Functional Genomics and Proteomics National Centre for Biomolecular Research Faculty of Science Masaryk University Blue tissue paper wild type 179 - 203 NWLTFNEPQTFTSFSYGTGVFAPGR 2824 E186Q Q 2823 peptide mass difference: - 1 Da 2800 2810 2820 2830 m/z 300 400 500 600 700 800 900 1000 a.i. 2799.73 2824. 65 Zm-p60.1 wild type 2799.85 2823. 82 Zm-p60.1 E186Q Confirmation of AA replacement at peptide level Protein: Zm-p60 (position 186) In-gel digestion MALDI-TOF MS 1 Da C7250 LC-MS/MS mutation D/E in position 210 confirmed ... DEEELQKENVKNTASLTGKITLSVTQSKPETGEVIGVFESIQPSDTDLGAKVPKDVKIQG ... Identification of AA replacement Protein detected in two variants in on 2-DE spot Initial information: Detection of two variants of tryptic peptide with mass difference: –14 Da ... DEEELQKENVKNTASLTGKITLSVTQSKPETGEVIGVFESIQPSDTDLGAKVPKDVKIQG ... MALDI-MS confirmation of tryptic peptide mass difference (2 proteases) localization of sequence region with the change MALDI-PSD ambiguous results C7250 933.5 951.7 970.6 +MS2(1004.0), 32.1min (#878) 0 100 200 300 920 930 940 950 960 970 m/z 927.2 945.0 956.4 +MS2(997.4), 32.6min (#887) 0 100 200 300 Ion b9 LSVTQSKPX D / E m/z MS/MS of peptide LSVTQSKPXTGEVIGVFES, MW 2006.0 (1992.0) D E 1+ 1+ -14 „Nemodif“ – detail of MS/MS spectrum „Modif“ - detail of MS/MS spectrum m/z 956 m/z 970 „Modif“ MS2 (997.4, 2+) „Nemodif“ MS2 (1004.4, 2+) Rt (min) EIC chromatogram EIC chromatogram m/z C7250 suggestion after methylation exchange of H for CH3 in each carboxyl group mass increase 14 Da/group VTQSKPX*TGE*VIG* y8 y8 nederiv m/z 787.2 pro D y8 methyl m/z 829.3 pro D D = 42 Da C7250 Confirmation of D in position 210 by methylation and LC-MS/MS m/z 750 760 770 780 790 800 810 820 830 840 850 0 50 100 150 200 250 300 350 400 450 500 550 [Abs. Int.] y8 787.7 y8 829.3 42 Da MS/MS spectrum of peptide VTQSKPXTGEVIG before and after methylation C7250 Characterization of modifications „chemical“ C7250 Functional Genomics and Proteomics National Centre for Biomolecular Research Faculty of Science Masaryk University unmodified peptide 2060 Peptide + X 2222 MALDI–MS spectrum of tryptic digests before a after modification (spectrum detail) 162 Da C7250 1256 1261 1266 1271 1276 m/z 100 200 300 400 500 600 700 800 900 1000 1100 1200 a.i. MALDI–MS spectrum – Met oxidation (spectrum detail) 16 Da Sample prep artefact (Sypro Ruby) C7250 1. gi|15803837 Mass: 13532 Score: 487 Queries matched: 5 50S ribosomal protein L14 [Escherichia coli O157:H7] Observed Mr(expt) Mr(calc) Delta Miss Score Peptide ……. 939.45 1876.89 1876.88 0.02 0 (125) MIQEQTMLNVADNSGAR 947.44 1892.87 1892.87 -0.01 0 159 MIQEQTMLNVADNSGAR + Oxidation (M) 947.45 1892.88 1892.87 0.01 0 (147) MIQEQTMLNVADNSGAR + Oxidation (M) 955.45 1908.89 1908.87 0.03 0 (118) MIQEQTMLNVADNSGAR + 2 Oxidation (M) ……. Protein identification – MS/MS data (Mascot) (Petra P. Vz. 3, 080821) D m/z 8 (16) ……. 16 (32) C7250 EIC: m/z 939.5 (2+) m/z 947.4 (2+) m/z 955.4 (2+) LC-MS/MS 1x Ox 1x Ox 2x Ox C7250 ? MS/MS spectrum of unmodified peptide - MIQEQTMLNVADNSGAR C7250 MIQEQTMLNVADNSGAR Shifts in m/z of selected fragments for individual M(ox) peptide forms y10 y10 – LNVADNSGAR b9 – M1IQEQTM7LN b9 y11 y11 – M7LNVADNSGAR no 1 Ox 1 Ox 2 Ox M1 M7 both y10 0 0 0 0 y11 0 0 16 16 b9 0 16 16 32 C7250 M7 - ox unmodified M1 - ox M1, M7- ox y10 – LNVADNSGAR y11 – M7LNVADNSGAR y10 1016,5 y10 1016,5 y10 1016,5 y10 1016,5 y11 1147,6 y11 1163,6 y11 1163,6 y11 1147,6 b9 1089,5 b9 1105,5 b9 1105,5 b9 1121,5 b9 – M1IQEQTM7LN + shift of the whole b series (b3 - M1IQ) +16 +16 +32 +16 +16 0 C7250 LC-MS/MS spectrum (detail) Confirmation of modification of Cys 200.3 246.1 268.1 285.4 300.0 315.3 349.3 383.3 410.1 428.2 448.2 478.5 522.2 0 1000 2000 3000 Intens. 200 250 300 350 400 450 500 m/z y2 y3 y4 C …VC*AR 200.4 246.4 285.5 314.9 384.0 410.5 441.4 455.9 468.1 493.3 0.00 0.25 0.50 0.75 1.00 1.25 4 x10 Intens. 200 250 300 350 400 450 500 m/z y3* y2 C + modification modification C7250 Fryčák P. et al., J. Mass Spectrom., 40 (10), 1309-1318 (2005) 5340 Ac Ac ? 5340 5382 Fragment with one E Ac Fragment with two E Ac Non-acetylated fragment Histone H3, detection of acetylation (in-vitro) MALDI-MS digestu (spectrum detail) 0 250 500 750 1000 1250 1500 5250 5300 5350 5400 5450 5500 5550 5600 5650 5700 5750 m/z C7250 T G G K* 1246.4 c 11 1303.4 c 12 1360.4 c 13 1530.4 c 14 42 Da Histone H3, localization acetylace K(14) MALDI-ISD MS (spectrum detail) ARTKQTARKSTGGKAPR c14 c13 C7250 Functional Genomics and Proteomics National Centre for Biomolecular Research Faculty of Science Masaryk University Characterization of modifications „posttranslational “ Phosphorylation Phosphorylation sites db: http://www.phosphosite.org/homeAction.do http://phospho.elm.eu.org Whereas phosphorylation of serine, threonine or tyrosine results in the formation of a phosphoester linkage, phosphorylation of histidine residues occurs on nitrogen atoms, producing a phosphoramidate bond. Phosphohistidines have a large standard free energy of hydrolysis making them the most unstable of any known phosphoamino acid. Klumpp et al, Eur. J. Biochem. 269, 1067-1071 (2002) Protein Phosphorylation is of Fundamental Importance in Biological Regulation cca 10-30% of all proteins are phosphorylated * S, T, Y 1800 : 200 : 1 ??? * H ??? C7250 Phospho.ELM version 9.0 (September 2010) contains 8,718 substrate proteins from different species covering 3,370 tyrosine, 31,754 serine and 7,449 threonine instances. ANd9GcS7DgpmMhdFVpn6PlY8T92DyDkPS8RmVbgExRVf7Hx161sytJTUMw 9 aminoacid, which might be phosphorylated serine (Ser) > threonine (Thr) > tyrosine (Tyr) histidine (His) aspartic acid (Asp), glutamic acid (Glu) lysine (Lys), arginine (Arg), cysteine (Cys) ANd9GcSGPXPo1GVda5zic7cVH_KH58S54DXtkkd61xmwD7JEB4QKn5XzAw C7250 Phosphorylation Phosphoproteome - troublemaker * most of signaling proteins are low abundant * proteins are usually in several phospho variants * proteins can be easily dephosphorylated during sample preparation by phosphatases present in sample * signal suppression in MS - preferential ionization of unmodified peptides (only small portion out of total peptide content is phosphorylated; lower ionization efficiency of phosphopeptides) C7250 Sample preparation * phosphostatus stabilization - phosphatase inhibitors (as soon as possible) - denaturation (e.g. FASP - lysis in SDT buffer) * phosphopeptide(protein) enrichment - TiO2 (or other metal oxides, MOAC – „metal oxide affinity chromatography“) - IMAC („immobilized metal affinity chromatography“) - SCX resp. SAX or HILIC („ion exchange or hydrophilic interaction chromatography“) - immunoprecipitation by specific antibody I.L. Batalha, Trends in Biotechnology 30 (2), 100-110 (2012) MS analysis other types of MS/MS fragmentation CID ETD (ECD) electron transfer (capture) dissociation HCD higher-energy collision dissociation EThcD electron-transfer/higher-energy collision dissociation Frese at al., J. Proteome Res., 12, 1520−1525 (2013) neutral loss scan (different variants) precursor scan C7250 phosphoproteins (Pro-Q Diamond , blue) proteins (SYPRO Ruby, red). (https://www.lifetechnologies.com/order/catalog/product/P33300) Specific staining of phosphoproteins, 2D GE alternatives Metabolic tagging by 32P radioactivity measurement immunoblotting phosphatase treatment phosphoproteins display a basic shift in their pI after the dephosphorylation. comparison 2D gels C7250 Visualization of total protein and phosphoproteins in a 2D gel. http://www.lifetechnologies.com/content/dam/LifeTech/Documents/spectra/images/33300.jpg Mobility Shift Detection of Phosphorylated Proteins SDS-PAGE using an Phos-tagTM complex with two manganese(II) ions Mn2+–Phos-tagTMTM c(Mn2+) mM E. Kinoshita et al., Molecular & Cellular Proteomics, 5, 749-757 (2006) C7250 specific binding eluce Immobilized metal affinity chromatography (IMAC) charging (Cu2+, Fe3+, Ga3+) C7250 IMAC enrichment of -Casein phosphopeptides (1 pmol of tryptic digest) Ziptip MC Technical note - Millipore C7250 normal peptides phosphopeptides Maldi-MS w/o enrichment LOGO Casein (1 ug) after tryptic digestion C7250 Maldi-MS enrichment IMAC (Poros), Fe3+ LOGO C7250 peptides phosphopeptides Casein (1 ug) after tryptic digestion Maldi-MS TiO2 enrichment LOGO C7250 peptides phosphopeptides Casein (1 ug) after tryptic digestion 1070 1120 1170 m/z 500 1000 1500 2000 2500 a.i. 80 Da MALDI–MS spectrum of peptide w/o and with phosphorylation HRTSS*VPEY LOGO C7250 1 x Phos 2 x Phos Casein digest TiO2 MALDI-MS Confirmation of phosphorylation by alkaline phosphatase C7250 ESI–MS (IT) spectrum of peptide w/o and with phosphorylation positive mode 165.0 226.8 285.1 334.7 380.1 408.2 447.7 495.0 538.3 569.3 640.4 668.3 873.5 916.9 1076.4 200 400 600 800 1000 1200 m/z 0.0 0.5 1.0 1.5 2.0 2.5 7 x10 Intens. m/z 538.3, 2+ m/z 578.3, 2+ 40 Da LOGO C7250 Characterization of Histone H4 phosphorylation by Aurora B kinase (K. Šedová) Experimental procedure • protein phosphorylation in vitro • tryptic digestion • TiO2 phosphoenrichment • LC-MS/MS analysis – neutral loss scan (ETD) in ion trap design of MS analysis precursor selection based on intesity MS/MS CID MS fragmentation of selected precursors by CID selection of new precursors detection of neutral loss in CID MS/MS spectra corresponding to loss of H3PO4 (D m/z 49, 32.6) fragmentation of precursors displaying loss of H3PO4 by soft fragmentation technique (ETD no neutral loss detection MS/MS ETD yes C7250 5 10 15 20 25 30 35 Time [min] 0.0 0.2 0.4 0.6 0.8 1.0 6 x10 Intens. 1 5 10 15 20 25 30 35 Time [min] 0.0 0.5 1.0 1.5 2.0 2.5 7 x10 Intens. LC-MS chromatogram EIC chromatogram of selected ion - m/z 559.0 TIC EIC 2. +MS, 21.9-22.1min #915-#923 0.0 0.2 0.4 0.6 0.8 1.0 1.2 5 x10 Intens. 557.0 557.5 558.0 558.5 559.0 559.5 560.0 560.5 561.0 561.5 m/z m/z 559.0 559.3 559.6 560.0 3+ 0.3 C7250 m/z 559.0 MS/MS spectrum, CID fragment of m/z 526.3, 3+ corresponds to neutral loss (32.7) potential phosphopeptide m/z 559.0 MS/MS spectrum, ETD C7250 Biotools Score Score (Biotools) (Mascot) CID RKTVTAMDVVYALK 520 23 RKTVTAMDVVYALK 518 22 ETD RKTVTAMDVVYALK 6149 75 RKTVTAMDVVYALK 774 47 m/z Charge RT (min) Expect Mr 559.0 3+ 22.063 1674.0 Identification of phosphopeptide by database searching MS/MS Ion Search (MASCOT) MASCOT •gi|223582 Mass: 11230 Score: 74 Queries matched: 2 histone H4 Observed Mr(expt) Mr(calc) Delta Miss Score Expect Rank Peptide 558.98 1673.93 1673.86 0.076 2 (23) 7.8 1 K.RKTVTAMDVVYALK.R + Phospho (ST) CID 558.98 1673.93 1673.86 0.076 2 75 5e-05 1 K.RKTVTAMDVVYALK.R + Phospho (ST) ETD Modifications: Optional: Phospho (ST) Search Parameter: Charge=2+ and 3+, MS Tol.:0.500000 Da, MSMS Tol.:0.500000 Da, Trypsin Mascot 2.2.03, NCBInr NCBInr_20081101.fasta C7250 m/z 559.0 MS/MS spectrum, CID RKTVTAMDVVYALK based on CID MS/MS data localization of phosphorylation is not possible C7250 RKTVTAMDVVYALK m/z 559.0 MS/MS spectrum, ETD C7250 based on ETD MS/MS data phosphorylation is reliably assigned to T(3) T(3) x T(5) T3 + phospho (101 + 80) T5 (101) T3 (101) T5 + phospho (101 + 80) 403.3 c3 502.3 c4 C7250 C7250 Li et al., Front. Plant Sci., 6, 430 (2015) a46 Ubiquitination Ubiquitination is an enzymatic, protein post-translational modification (PTM) process in which the carboxylic acid of the terminal glycine from the di-glycine motif in the activated ubiquitin forms an amide bond to the epsilon amine of the lysine in the modified protein. Protein ubiquitination regulates many cellular processes including transcription, endocytosis, cell cycle control, signal transduction, stress response, DNA repair as well as proteasomal-mediated degradation C7250 S. Liu, Z.J. Chen , Cell Research (2011) 21:6–21 The complex Ub code contains numerous variants of homotypic and heterotypic (mixed or branched) chains. Based on the eight possible linkages (M1, K6, K11, K27, K29, K33, K48, and K63) between two Ub moieties, at least 92 different Ub chain types exist. Stolz A. et al., Trends in Cell Biology, 28 (1), 1-3 (2018) Akutsu M. et al., J. Cell Sci., 129, 875-880 (2016) Ubiquitination ubiquitin – protein 8.5 kDa (76 AA) MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLV LRLRGG K heterogeneity of modified forms C7250 * localization of modified AA sites * determination of polyubiquitin crosslinks The most studied polyubiquitin chains - lysine48-linked - target proteins for destruction N.J. Denis, Proteomics 2007, 7, 868–874 Strategy of ubiquitinated site analysis C7250 > Score Mr(calc) Delta Sequence 45.2 2630.4003 -0.0059 KTVTAMDVVYALKRQGRTLYGF 45.2 2630.4003 -0.0059 KTVTAMDVVYALKRQGRTLYGFGG 0.3 2630.3792 0.0152 KSAPAPKKGSKKAVTKAQKKD All matches to this query Mascot 79 –100 658.6059 2630.3944 2630.4003 -2.23 0 45 0.0013 1 R.KTVTAMDVVYALKRQGRTLYGF.G + UBI_dT (K) 1 SGRGKGGKGL GKGGAKRHRK VLRDNIQGIT KPAIRRLARR GGVKRISGLI 51 YEETRGVLKV FLENVIRDAV TYTEHAKRKT VTAMDVVYAL KRQGRTLYGF 101 GG Histone H4 (trypsin) False-positive identification or K(91) is really ubiquitinated ??? C7250 BBAP monoubiquitylates histone H4 at lysine 91 and selectively modulates the DNA damage response." Yan Q., Dutt S., Xu R., Graves K., Juszczynski P., Manis J.P., Shipp M.A. Mol. Cell 36:110-120 (2009) C7250 K(1) is not ubiquitinated based on hte MS/MS data is not possible to decide K(91)ubi or C-terminal GG False-positive identification or K(91) is really ubiquitinated ??? D.Bustos, MCP, 2012 11: 1529-1540 Characterization of ubiquitinations using immunoprecipitation Scheme of experiment C7250 Ubiquitination Semiquantitative assessment of site occupancy site occupancy of individual Ubi sites sample vs control site occupancy within polyubiquitin chains sample vs control C7250 de Groot R. E.A. et al., Sci. Signal., 7 (317), ra26 (2014) Characterization of ubiquitinations using immunoprecipitation II UbiSite antibody the antibody recognizes 13 C-terminal aminoacids of ubiquitin method principle experimental scheme Nat. Struct. Mol. Biol., 25 (July), 631–640, (2018) two human cell lines Hep2 Jurkat proteasomal inhibitors treatment BOR - bortezomib AP15 - b-AP15 C7250 Numbers of ubiquitination sites and ubiquitinated proteins identified in the two cell lines (n = 3 independent biological replicates). Numbers on the top of bars indicate identified ubiquitination sites; numbers within bars indicate identified proteins. Overlap of ubiquitination sites and ubiquitinated proteins between Hep2 and Jurkat cells. Numbers indicate the number of identified ubiquitination sites (left) or proteins (right). Nat. Struct. Mol. Biol., 25 (July), 631–640, (2018) Characterization of ubiquitinations using immunoprecipitation II UbiSite antibody C7250 Ubiquitin-like proteins a77 Glycosylation one of the most common post-translational modifications of proteins in eukaryotic cells. involved in a wide range of biological functions such as receptor binding, cell signaling, immune recognition, inflammation, and pathogenicity. basic types of glycans: * N-linked * O-linked * GPI anchors - C-linked - glykace http://www.uniprot.org/manual/carbohyd Variation in the degrees of saturation at available glycosylation sites results in heterogeneity in the mass and charge of glycoproteins Signal Supression C7250 nlink olink N - linked O - linked C7250 glycans are attached to the protein backbone via an amide bond to an asparagine during protein synthesis N-linked glycosylations N-X-S(T) X not to stand for P subtypes: * High-mannose * Hybrid * Complex C7250 N-linked:High-mannose subtype b-D-Mannose b-D-N-Acetylglucosamine C7250 N-linked: Hybrid subtype a-N-Acetylneuraminic acid (Sialic Acid) b-D-Galactose C7250 N-linked: Complex subtype a-L-Fucose C7250 glycans are linked via the hydroxyl group of serine or threonine O-linked glycosylations examples: b-D-N-Acetylgalactosamine C7250 GPI (glycosylphosphatidylinositol) anchors anchors are linked via C-terminus, membrane bound proteins C7250 Characterization of glycoproteins * specific detection of glycosylated proteins * protein identification * site localization * determination of glycan structure C7250 Specific detection of glycosylated proteins Pro-Q Emerald 300 - glyco only Sypro Ruby - all alternative detection techniques: colorimetric detection fluorescence detection specific enrichment: affinity chromatography (lectins, m-Aminophenylboronic Acid) C7250 chemical: Hydrazinolysis Hydrazine hydrolysis has been found to be effective in the complete release of unreduced O- and N-linked oligosaccharides. Alkaline b-Elimination - only O-linked (some exceptions) Trifluoromethanesulfonic Acid - glycan destruction enzymatic: PNGase F N-linked, all removed, if not PNGase A N-linked, all removed, if not Endoglycosidase H N-linked, cleaves after first Endoglycosidase F1, F2, F3 N-linked, cleave specifically with respect to glycane structure O-Glycosidase O-linked. all removed b-galactosidase cleaves before ... Deglycosylation C7250 Site assigment, glycan structure elucidation * glycosylation „only“ at S or T (O-linked) NXS(T) (N-linked) prediction of potential glyco site * combination of MS and MS/MS techniques * * separation of glycoproteins or glycopeptides * deglycosylation strategies * glycan derivatisation C7250 MALDI-MS spectrum of glycosylated and non-glycosylated protein size of glycan part 30000 40000 50000 m/z 50 100 150 200 250 300 350 a.i. D = 2.8 kDa heterogeneity C7250 glycosylated protein protein after deglycosylation Endo Hf 1D GE of protein before and after deglycosylation confirmation of glycosylation C7250 MALDI-MS spectrum of deglycosylated protein confirmation of glycosylation Endoglycosidase Hf 30000 40000 50000 m/z 20 40 60 80 100 120 140 160 180 a.i. deglycosylated 30000 40000 50000 m/z 20 40 60 80 100 120 140 160 180 a.i. 30000 40000 50000 m/z 20 40 60 80 100 120 140 160 180 a.i. C7250 35000 40000 45000 50000 m/z 50 100 150 200 250 300 350 400 450 500 a.i. Daver = 1.2 kDa MALDI-MS of intact protein C7250 P5_all_zz MALDI-MS of tryptic digests * differential peptide * identical protein C7250 2900 3100 3300 3500 3700 3900 m/z 100 150 200 250 300 350 400 450 500 a.i. Detail of spectra of protein digest before and after deglycosylation PNGase A peptide 3966 disappeared new peptide 2797, overlapping peptide 2795 * N-glycosylation * Daver = 1169 Da C7250 2770 2790 2810 m/z 100 150 200 250 300 350 400 450 500 a.i. C7250 Detail of spectra of protein digest before and after deglycosylation tryptic peptide - 2796 Da …PHIFDYSGS… , N is transformed to D during deglycosylation by PNGase A original sequence is …PHIFNYSGS… (mass 2795 Da) Peptide was also confirmed by LC-MS/MS analysis (original one was not found in sample before deglycosylation) Glycan mass 1170 Da corresponds to already reported glycan xylose+fucose+3*mannose+2*N-acetylglukosamin So glycan structure was not confirmed by MS/MS. Summary C7250 …missing parts have potential N-glycosylation sites... • MLRNVCPVLILLIIGATAQDPTDVGEAFANVEWSVAELKRVLVMGVPRDCGELFLSGQNHSGVYNIYPYKDSLLPVSAYCDMETDGGGWTVFQRRGQFG NPVYYFYKKWADYAHGFGDPAKEYWLGNNVLHALTSDKAMSLRIEKNHSLETLTAEYSVFKVASEEEYFKINVGGYIGSK GSDAFSIANGSMFTASDQDHDTYTNNCAVEFKG AWYTSCHGSNLNGLNLNGEHPSYADGIEWSAR GGSTGLYYYSYPNVEMKVRDAHFISRVADGRAS from lecture of Petr Man C7250 2nd_site_MS hexose increments ... glycopeptide... MS/MS from lecture of Petr Man C7250 2nd_site_MS2 MS/MS from 1202.2 - – glycopeptide, type of glycan identified from lecture of Petr Man H – Hexose N – HexNAc C7250 2nd_site_MS3 MS/MS from 971.7 – peptide with one HexNAc – site of glycosylation identified from lecture of Petr Man b2 N(HexNAc)H y13 SLETLTAEYSVFK H – His N – Asn C7250 • MLRNVCPVLILLIIGATAQDPTDVGEAFANVEWSVAELKRVLVMGVPRDCGELFLSGQNHSGVYNIYPYKDSLLPVSAYCDMETDGGGWTVFQRRGQFG NPVYYFYKKWADYAHGFGDPAKEYWLGNNVLHALTSDKAMSLRIEKNHSLETLTAEYSVFKVASEEEYFKINVGGYIGSK GSDAFSIANGSMFTASDQDHDTYTNNCAVEFKG AWYTSCHGSNLNGLNLNGEHPSYADGIEWSAR GGSTGLYYYSYPNVEMKVRDAHFISRVADGRAS from lecture of Petr Man C7250 MALDI-MS spectrum of ribonuclease B 162 Da 163 Da glycan C7250 MS MS2 MS3 MS4 MSn of ribonuclease B glycans (AP-MALDI-IT MS) C7250 Combination of deglycosylation enzymes C7250 Sequential deglycosylation by different enzymes (MALDI-MS) C7250 C7250 Glycan profiling and structural analysis of glycans NSCLC - Bronchoalveolar Carcinoma Bronchoalveolar Adenocarcinoma Large Cell Carcinoma 0 1 2 3 4 4 x10 2000 2500 3000 3500 4000 m/z 0 2 4 6 4 x10 0 0.5 1.0 1.5 4 x10 3x 3x 4x 4x 6x 5x 5x 3x 6x 4x 5x 3x 5x 3x 4x 3x 4x 5x 3x GlcNAc; Fuc Gal; Man; MALDI-TOF-MS spectra of N-glycans after desialylation Lattová E. et al., J. Proteome Res., 15 (8), 2777-2786 (2016) a67 And this is the end