proteoform profile mapping of the human serum complement component c9 reveals unexpected new features of n o and c glycosylation

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem) Article Proteoform profile mapping of the human serum Complement component C9 reveals unexpected new features of N-, O- and C-glycosylation Vojtech Franc, Yang Yang, and Albert J.R Heck Anal Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04527 • Publication Date (Web): 21 Feb 2017 Downloaded from http://pubs.acs.org on February 22, 2017 Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication They are posted online prior to technical editing, formatting for publication and author proofing The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the 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original U.S Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties Page of 30 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Analytical Chemistry Proteoform profile mapping of the human serum Complement component C9 reveals unexpected new features of N-, O- and C-glycosylation Vojtech Franc1,2, Yang Yang1,2,and Albert J.R Heck1,2* and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research Netherlands Proteomics Center, Padualaan 8, 3584 CH Utrecht, The Netherlands 10 11 12 13 14 15 16 Correspondence: Albert Heck, a.j.r.heck@uu.nl 17 ACS Paragon Plus Environment Analytical Chemistry 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 18 Abstract 19 The human complement C9 protein (~65 kDa) is a member of the complement pathway It 20 plays an essential role in the membrane attack complex (MAC), which forms a lethal pore on 21 the cellular surface of pathogenic bacteria Here, we charted in detail the structural micro- 22 heterogeneity of C9 purified from human blood serum, using an integrative workflow 23 combining high-resolution native mass spectrometry and (glyco)peptide-centric proteomics 24 The proteoform profile of C9 was acquired by high-resolution native mass spectrometry, 25 which revealed the co-occurrence of ~50 distinct MS signals Subsequent peptide-centric 26 analysis, through proteolytic digestion of C9 and LC-MS/MS measurements of the resulting 27 peptide mixtures, provided site-specific quantitative profiles of three different types of C9 28 glycosylation and validation of the native MS data Our study provides a detailed 29 specification, validation and quantification of 15 co-occurring C9 proteoforms, and the first 30 direct experimental evidence of O-linked glycans in the N-terminal region Additionally, next 31 to the two known glycosylation sites, a third novel, albeit low abundant N-glycosylation site 32 on C9 is identified, which surprisingly does not possess the canonical N-glycosylation 33 sequence N-X-S/T Our data also reveal a binding of up to two Ca2+ ions to C9 Mapping all 34 detected and validated sites of modifications on a structural model of C9, as present in the 35 MAC, hints at their putative roles in pore formation or receptor interactions The applied 36 methods herein represent a powerful tool for the unbiased in-depth analysis of plasma proteins 37 and may advance biomarker discovery, as aberrant glycosylation profiles may be indicative of 38 the pathophysiological state of the patients 39 40 Key words: complement component C9 / glycosylation / blood proteins / native mass 41 spectrometry / glycopeptide centric proteomics / N-glycosylation / O-glycosylation / C- 42 glycosylation 43 44 Abbreviations 45 ACN, acetonitrile; AMAC, ammonium acetate; DTT, dithiothreitol; EGF, epidermal growth 46 factor; EMR, extended mass range; ESI, electrospray ionization; EThcD, electron-transfer and 47 higher-energy collision dissociation; FDR, false discovery rate; HCD higher-energy 48 collisional dissociation; IAA, iodoacetamide; LC, liquid chromatography; LDLRA, low- ACS Paragon Plus Environment Page of 30 Page of 30 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Analytical Chemistry 49 density lipoprotein receptor class A repeat; MAC, membrane attack complex; MS, mass 50 spectrometry; MS/MS, tandem mass spectrometry; PTM, post-translational modification; 51 SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TFA, trifluoroacetic 52 acid; TSP, thrombospondin; XIC, extracted ion chromatogram 53 54 Introduction 55 Post-translational modifications (PTMs) of proteins regulate their activity, localization, 56 turnover, interactions and many other important physiological processes1, Of all possible 57 PTMs, protein glycosylation is one of the most abundant yet structurally diverse PTM, 58 making it analytically and biochemically challenging to monitor3 This is because, the 59 enzymes involved in the glycosylation machinery can produce diverse glycosylation patterns 60 on proteins and heterogeneous populations of glycans at every occupied glycosylation site4-6 61 In addition, these modifications are also present in non-stoichiometric amounts with multiple 62 varieties of chemical moieties involved Thus, new methods are needed for their detailed 63 analysis Recent progress in high-resolution native electrospray ionization (ESI) mass 64 spectrometry (MS) can provide novel means to facilitate the in-depth analysis of all co- 65 appearing modifications, at least when they are distinguishable in mass7-9 In combination 66 with peptide-centric proteomics, this approach is very useful for high detail analysis of PTMs 67 on proteins from different biological sources and can provide direct assessment of the 68 biosimilarity among similar therapeutic proteins10 Through integrating native MS data and 69 peptide-centric proteomics, one can obtain information about composition, stoichiometry, 70 site-specificity and relative abundance of the modifications at each site Moreover, a direct 71 cross-evaluation of both data sets (native MS and peptide-centric MS) validates completeness 72 of each approach and provides reliability for the quantitative profiling of protein proteoforms 73 So far in the analysis of protein proteoforms most MS methods typically use denaturing 74 conditions prior to the ESI process11-14 These conditions inevitably disrupt protein tertiary 75 and quaternary structures Although native MS is often regarded somewhat less sensitive, it 76 provides the advantage that the resulting mass spectra are less congested, as the ion signals are 77 distributed over substantially less number of charge states, and over a wider m/z window 78 Collectively, such hybrid mass spectrometry strategies have the potential to become beneficial 79 for the study of biologically important (glyco)proteins, whereby knowledge about their 80 precise modifications is crucial in understanding their activity and function ACS Paragon Plus Environment Analytical Chemistry 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 81 Most proteins in human blood plasma are decorated by a plethora of PTMs, particularly 82 involving glycosylation, and the complement component protein C9 is not an exception15 83 Human C9 is primarily produced in the liver and plays a key role in the formation of the 84 membrane attack complex (MAC), together with the other complement proteins C5, 6, and 85 While several cryoEM maps have recently become available for the MAC.16, 17, no detailed 86 structure is available for its C9 component Still amino acid alignments have identified several 87 domains in C9 based on its homology to other proteins These include the N-terminal type 88 thrombospondin (TSP) domain, a low-density lipoprotein receptor class A repeat (LDLRA), a 89 number of potential transmembrane regions and the C-terminal epidermal growth factor 90 (EGF)-like domain (Figure 1)18 The majority of the detailed characterization studies of the 91 PTMs occurring on C9 dates back to the previous century, when techniques used to perform 92 such analysis were very cumbersome In these early studies, C9 was reported to be N- 93 glycosylated19-21, however, no current evidence exists regarding the composition and 94 heterogeneity of these N-linked glycans Moreover, although suspected, no direct proof has 95 been reported for the presence of O-linked glycans C9 is additionally modified by a rarer type 96 of glycosylation; C-mannosylation22 With such a diverse repertoire of modifications, C9 97 presents not only a challenging analytical target, but also imparts a potential variability in its 98 physiological functioning Exemplary findings to support this statement come from reports 99 wherein the extracellular Ser phosphorylation of C9 by ecto-protein kinases in cancer cells 100 K562 was proposed to serve as a protective mechanism against complement in tumor cells23 101 Moreover, C9 with fucosylated N-glycans has been suggested as a biomarker for squamous 102 cell lung cancer, as patients tend to show overproduction of these proteoforms24 A detailed 103 map of the full proteoform profile of serum-derived Complement component C9 is, therefore, 104 of high interest 105 Here, we report an unbiased and in-depth analysis of the complement component C9 protein 106 isolated from pooled human blood serum (of at least three donors) using modern, hybrid MS 107 technologies Our data provides a detailed view of the modifications co-occurring on C9 We 108 validate all identified PTMs from high quality tandem mass spectrometry (MS/MS) spectra 109 using a peptide-centric approach In addition to the earlier reported C9 modifications, our data 110 revealed the attachment of mucin type of O-glycosylation in the N-terminal part of C9, 111 providing the first experimental evidence of this modification on C9 Except information 112 about PTMs, our native MS measurements suggest binding of up to two Ca2+ ions on C9 113 Since the N-terminal region of C9 seems to play a crucial role in the C9 polymerization ACS Paragon Plus Environment Page of 30 Page of 30 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Analytical Chemistry 114 process and thus also in the assembly of MAC25, the role of the here identified O- 115 glycosylation site may act as a target for further functional investigations Moreover, we 116 identified a novel low abundant N-glycosylation site on N215 that is highly conserved 117 throughout mammals and does not adhere to the typical N-glycosylation sequon N-X-S/T (X 118 can be any amino acid except P) 119 120 Materials and Methods 121 Chemicals and materials 122 Complement component C9 (Uniprot Code: P02748) purified from pooled human blood 123 plasma (more than three healthy donors) was acquired from Complement Technology, Inc 124 (Texas, USA) The sample was purified according to a standard protocol26 (the certificate of 125 analysis is attached in the Supporting information – S5) Dithiothreitol (DTT), iodoacetamide 126 (IAA) and ammonium acetate (AMAC) were purchased from Sigma-Aldrich (Steinheim, 127 Germany) Formic acid (FA) was from Merck (Darmstadt, Germany) Acetonitrile (ACN) was 128 purchased from Biosolve (Valkenswaard, The Netherlands) POROS Oligo R3 50 µm 129 particles were obtained from PerSeptive Biosystems (Framingham, MA, USA) and packed 130 into GELoader pipette tips (Eppendorf, Hamburg, Germany) Sequencing grade trypsin was 131 obtained from Promega (Madison, WI) Glu-C, Asp-N, PNGase F and Sialidase were obtained 132 from Roche (Indianapolis, USA) 133 134 Sample preparation for native MS 135 Unprocessed protein solution in a phosphate buffer at pH 7.2, containing ~ 30-40 µg of C9, 136 was buffer exchanged into 150 mM aqueous AMAC (pH 7.5) by ultrafiltration (vivaspin500, 137 Sartorius Stedim Biotech, Germany) using a 10 kDa cut-off filter The resulting protein 138 concentration was measured by UV absorbance at 280 nm and adjusted to 2-3 µM prior to 139 native MS analysis The enzyme Sialidase was used to remove sialic acid residues from C9 140 PNGase F was used to cleave the N-glycans of C926 All samples were buffer exchanged to 141 150 mM AMAC (pH 7.2) prior to native MS measurements 142 ACS Paragon Plus Environment Analytical Chemistry 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 143 Native MS analysis 144 Samples were analyzed on a modified Exactive Plus Orbitrap instrument with extended mass 145 range (EMR) (Thermo Fisher Scientific, Bremen) using a standard m/z range of 500-10,000, 146 as described in detail previously27 The voltage offsets on the transport multi-poles and ion 147 lenses were manually tuned to achieve optimal transmission of protein ions at elevated m/z 148 Nitrogen was used in the higher-energy collisional dissociation (HCD) cell at a gas pressure 149 of 6-8 × 10-10 bar MS parameters used: spray voltage 1.2-1.3 V, source fragmentation 30 V, 150 source temperature 250 °C, collision energy 30 V, and resolution (at m/z 200) 30,000 The 151 instrument was mass calibrated as described previously, using a solution of CsI27 152 153 Native MS data analysis 154 The accurate masses of the observed C9 proteoforms were calculated manually averaging 155 over all detected charge states of C9 For PTM composition analysis, data were processed 156 manually and glycan structures were deduced based on known biosynthetic pathways 157 Average masses were used for the PTM assignments, including hexose/mannose/galactose 158 (Hex/Man/Gal, 159 (HexNAc/GlcNAc/GalNAc, 203.1950 Da), and N-acetylneuraminic acid (NeuAc, 291.2579 160 Da) All used symbols and text nomenclature are according recommendations of the 161 Consortium for Functional Glycomics 162.1424 Da), N-acetylhexosamine/N-acetylglucosamine 162 163 In-solution Digestion for Peptide-centric glycoproteomics 164 Intact human C9 protein in PBS buffer (10 mM sodium phosphate, 145 mM NaCl, pH 7.3) at 165 a concentration of mg/ml was reduced with mM DTT at 56 °C for 30 and alkylated 166 with 15 mM IAA at room temperature for 30 in the dark The excess of IAA was 167 quenched by using mM DTT C9 was digested overnight with trypsin at an enzyme-to- 168 protein-ratio of 1:100 (w/w) at 37 °C Another C9 sample was digested for hours by using 169 Asp-N at an enzyme to-protein-ratio of 1:75 (w/w) at 37 °C and the resulted peptide mixtures 170 were further treated with trypsin (1:100; w/w) overnight at 37 °C All proteolytic digests 171 containing modified glycopeptides were desalted by GELoader tips filled with POROS Oligo ACS Paragon Plus Environment Page of 30 Page of 30 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Analytical Chemistry 172 R3 50 µm particles28, dried and dissolved in 40 uL of 0.1% FA prior liquid chromatography 173 (LC)-MS and MS/MS analysis 174 175 LC-MS and MS/MS analysis 176 All peptides (typically 300 fmol of C9 peptides) were separated and analyzed using an Agilent 177 1290 Infinity HPLC system (Agilent Technologies, Waldbronn Germany) coupled on-line to 178 an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) 179 Reversed-phase separation was accomplished using a 100 µm inner diameter cm trap 180 column (in-housed packed with ReproSil-Pur C18-AQ, µm) (Dr Maisch GmbH, 181 Ammerbuch-Entringen, Germany) coupled to a 50 µm inner diameter 50 cm analytical 182 column (in-house packed with Poroshell 120 EC-C18, 2.7 µm) (Agilent Technologies, 183 Amstelveen, The Netherlands) Mobile-phase solvent A consisted of 0.1% FA in water, and 184 mobile-phase solvent B consisted of 0.1% FA in ACN The flow rate was set to 300 nL/min 185 A 45 gradient was used as follows: 0-10 min, 100% solvent A; 10.1-35 10% solvent 186 B; 35-38 45% solvent B; 38-40 100% solvent B; 40-45 100% solvent A 187 Nanospray was achieved using a coated fused silica emitter (New Objective, Cambridge, MA) 188 (outer diameter, 360 µm; inner diameter, 20 µm; tip inner diameter, 10 µm) biased to kV 189 The mass spectrometer was operated in positive ion mode and the spectra were acquired in the 190 data dependent acquisition mode For the MS scans the mass range was set from 300 to 2,000 191 m/z at a resolution of 60,000 and the AGC target was set to 4×105 For the MS/MS 192 measurements HCD and electron-transfer and higher-energy collision dissociation (EThcD) 193 were used HCD was performed with normalized collision energy of 15% and 35% 194 respectively A supplementary activation energy of 20% was used for EThcD For the MS/MS 195 scans the mass range was set from 100 to 2,000 m/z and the resolution was set to 30,000; the 196 AGC target was set to 5×105; the precursor isolation width was 1.6 Da and the maximum 197 injection time was set to 300 ms 198 199 LC/MS and MS/MS data analysis 200 Raw data were interpreted by using the Byonic software suite (Protein Metrics Inc.)29 and 201 further validation of the key MS/MS spectra was performed manually The following 202 parameters were used for data searches: precursor ion mass tolerance, 10 ppm; product ion ACS Paragon Plus Environment Analytical Chemistry 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 203 mass tolerance, 20 ppm; fixed modification, Cys carbamidomethyl; variable modification: 204 Met oxidation, Trp Mannosylation, and both N- and O-glycosylation from mammalian glycan 205 databases A non-enzyme specificity search was chosen for all samples The database used 206 contained the C9 protein amino acid sequence (Uniprot Code: P02748) Profiling and relative 207 quantification of PTM modified C9 peptides were achieved by use of the extracted ion 208 chromatograms (XICs) from two independently processed C9 samples The peptide mixtures 209 were prepared with different combinations of proteolytic enzymes as described above (1 210 Trypsin; AspN + Trypsin) For peak area calculations, the first three isotopes were taken 211 from each manually validated peptide proteoform Integrated peak areas were normalized for 212 all PTM sites individually and the average peptide ratios from the two samples were taken as 213 a final estimation of the abundance The XICs were obtained using the software Skyline30 214 The glycan structures of each glycoform were manually annotated Hereby reported glycan 215 structures are depicted without the linkage type of glycan units, since the acquired MS/MS 216 patterns not provide such information 217 218 Combining native MS and peptide-centric proteomic data 219 Reliability and completeness of the obtained proteoform profiles of C9 were assessed by an 220 integrative approach combining the native MS data with the glycopeptide centric proteomics 221 data Details of this approach have been described in detail previously10 Briefly, in silico data 222 construction of the “intact protein spectra” was performed based on the masses and relative 223 abundances of all site-specific PTMs derived from the glycopeptide centric analysis 224 Subsequently, the constructed spectrum was compared to the experimental native MS spectra 225 of C9 The similarity between the two independent data sets (Native MS spectra and 226 constructed spectra based on glycopeptide centric data) was expressed by a Pearson 227 correlation factor All R scripts used for the spectra simulation are available at github 228 (https://github.com/Yang0014/glycoNativeMS) All C9 proteoforms predicted from the 229 peptide-centric data were further filtered by taking 0.5 % cut-off in relative intensity of the 230 peaks in the experimental native spectrum and mass deviations were manually checked 231 232 Results 233 Native MS analysis provides hints about novel unexpected PTMs and Ca2+ binding to C9 ACS Paragon Plus Environment Page of 30 Page of 30 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Analytical Chemistry 234 We started our investigation by first acquiring high-resolution native ESI-MS spectra of the 235 human complement component C9 (Figure 2a) The recorded native MS spectrum of C9 236 shows at least five different charge states, ranging from [M + 13H]13+ to [M + 17H]17+ Each 237 charge state contains various ion series that correspond to different masses and thus different 238 proteoforms of C9 Based on their distinguishable masses, taking a 1% cut-off in relative 239 intensity of the peaks, we can distinguish at least ~50 co-occurring MS signals Since we 240 suspected these could correspond to different proteoforms of C9, we set out to further 241 examine and validate our findings 242 To simplify the visualization of the C9 proteoform profile, we focused on the most intense 243 charge state (15+) The average mass of the protein backbone of C9 is 60,954.02 Da In this 244 mass calculation we used the mass of the C9 backbone sequence lacking the N-terminal signal 245 peptide, corrected by the mass shift induced by the twelve disulfide bonds present in C9 (-24 246 x 1.0079 Da) Compared to, for instance, chicken ovalbumin31 and CHO derived 247 erythropotein10, which we previously analyzed by high-resolution native mass spectrometry, 248 the native mass spectra of C9 are remarkably less heterogeneous Especially since according 249 to previously published data, C9 has been shown to be C-mannosylated22 at the TPS domain 250 and N-glycosylated19-21 at two sites in the MACPF domain 251 Looking on the inset on Figure 2a, the mass difference of 656 Da between the abundant peaks 252 with 253 HexNAc1Hex1NeuAc1 The same mass difference can be observed between the abundant 254 peaks with m/z of 4,415.89 and 4,459.61 This may either correspond to variability in the 255 number of antennas on the N-glycans or the additional attachment of mucin type O-glycans 256 To address this, we next treated the protein with various enzymes that cleave off parts of the 257 glycan moieties The removal of N-glycans or sialic acid residues resulted in specific mass 258 shifts, allowing us to calculate and partly predict the PTM composition of C9 For cleavage of 259 N-glycosylations we used PNGaseF (Figure 2b, c, d) and sialidase for the specific removal of 260 sialic acids (Figure 2e) We subsequently subjected these treated C9 samples to native MS 261 analysis The incubation of C9 with PNGase F for hours at room temperature resulted in a 262 removal of one of the two N-glycan chains (Figure 2c) The mass difference of 2,206 Da 263 between the most abundant intact C9 proteoform and the N-deglycosylated C9 indicated the 264 attachment of a N-glycan with the composition of HexNAc4Hex5NeuAc2 A prolonged 265 treatment (48 hours; 37 °C) of C9 with PNGase F resulted into a second major mass shift of 266 2,206 Da corresponding to a loss of the second N-glycan (Figure 2d) A closer look at the m/z of 4,435.28 and 4,479.02 corresponds to ACS Paragon Plus Environment the glycan composition Analytical Chemistry 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 459 the fact that such analyses happen under denaturing conditions Our data indicate that C9 can 460 possibly bind up to two Ca2+ ions The exact site of Ca2+ binding in C9 has not been identified 461 yet, nevertheless one of the possible candidate sites is located at cysteine-rich LDLRA domain 462 of C943 Another possible Ca2+ binding site is at the C9 N-terminal part, which also harbors O- 463 glycosylation sites The 12 N-terminal amino acids of C9 exhibit a strong negative charge and 464 the 16 N-terminal amino acids contain a consensus sequence for Ca2+ binding proteins25 As 465 mentioned above, the N-terminal region of C9, located at the outside of the MAC (Figure 4d, 466 e), has been shown to play a significant role in C9 polymerization, hinting at a possible 467 regulating role of the here identified O-glycosylation and Ca2+ binding in the assembly of the 468 MAC or interactions with some other molecules or receptors 469 In conclusion, here we provide an unbiased detailed specification of three different types of 470 glycosylation on C9 isolated from human blood serum and quantification and validation of 15 471 C9 proteoforms In total, we achieved more than 90% correlation between the native MS data 472 and peptide centric data Mapping all sites of modifications on a structural model of C9, as 473 present in the MAC, hints at their putative roles in pore formation or receptor interactions 474 More general, the here applied combination of MS methods represents a powerful tool for the 475 in-depth analysis of plasma proteins and may advance thus biomarker discovery 476 477 Acknowledgements 478 We acknowledge the support from the Netherlands Organization for Scientific Research 479 (NWO) funding the large-scale proteomics facility Proteins@Work (project 184.032.201) 480 embedded in the Netherlands Proteomics Centre Y.Y and A.J.R.H are supported by the EU 481 funded ITN project ManiFold, grant 317371 This project has received additional funding 482 from the European Union’s Horizon 2020 research and innovation programme f under grant 483 agreement 668036 (RELENT) and 686547 (MSMed) V.F A.J.R.H acknowledge further 484 support by the NWO TOP-Punt Grant 718.015.003 485 486 Supporting Information Available: Supplementary Figures S1, S2, S3 and S4, the 487 certificate of analysis of the purified C9 sample (S5) and Supplementary Tables S1 and S2 488 This material is available free of charge via the internet at http://pubs.acs.org ACS Paragon Plus Environment Page 16 of 30 Page 17 of 30 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 489 Analytical Chemistry References 490 Seo, J.; Lee, K J Biochem Mol Biol 2004, 37, 35-44 491 Spiro, R G Glycobiology 2002, 12, 43R–56R 492 Hart, G W Curr Opin Cell Biol 1992, 4, 1017-1023 493 Rudd, P M.; Dwek, R 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Paragon Plus Environment Analytical Chemistry 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 521 19 Biesecker, G.; Müller-Eberhard, H J J Immunol 1980, 124, 1291–1296 522 20 Biesecker, G.; Gerard, C.; Hugli, T E J Biol Chem 1982, 257, 2584-2590 523 21 Kontermann, R.; Rauterberg, E W Mol Immunol 1989, 26, 1125-1132 524 22 Hofsteenge, J.; Blommers, M.; Hess, D.; Fumarek, A.; Miroshnichenko, O J Biol Chem 1999, 525 274, 32786-32794 526 23 Paas, Y.; Bohana-Kashtan, O.; Fishelson, Z Immunopharmacology 1999, 42, 175–185 527 24 Narayanasamy, A.; Ahn, J M.; Sung, H J.; Kong, D H.; Ha, K S.; Lee, S Y.; Cho, J Y J 528 Proteomics 2011, 74, 2948-2958 529 25 Taylor, K M.; Trimby, A R.; Campbell, A K Immunology 1997, 91, 20-27 530 26 Rosati, S.; Yang, Y.; Barendregt, A.; Heck, A J R Nat Protoc 2014, 9, 967–976 531 27 Rose, R J.; Damoc, E; Denisov, E.; Makarov, A.; Heck, A J R Nat Methods 2012 9, 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T E J Biol Chem 1985, 206, 14802-14809 547 36 Vance, A.; Wu W.; Ribaudo, R K.; Segal, D M.; Kearse, K P J Biol Chem 1997, 272, 23117- 548 23122 549 37 Miletich, J P.; Broze, G J J J Biol Chem 1990, 265, 11397-11404 550 38 Yasuda, D.; Imura, Y.; Ishii, S.; Shimizu, T.; Nakamura, M FASEB J 2015, 29, 2412-2422 551 39 Zielinska, F.; Gnad, J.; Wisniewski, M.; Mann, M Cell 2010, 141, 897-907 552 40 Sun, S.; Zhang, H Anal chem 2015, 87, 11948-11951 553 41 Lizak, C.; Gerber, S.; Numao, S.; Aebi, M.; Locher, K P Nature 2011, 474, 350-355 554 42 Hounsell, E F.; Davies, M J.; Renouf, D V Glycoconj J 1996, 13, 19–26 555 43 Thielens, N M.; Lohner, K.; Esser, A F J Biol Chem 1988, 15, 6665-6670 ACS Paragon Plus Environment Page 18 of 30 Page 19 of 30 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 556 Analytical Chemistry 44 Yang, J.; Yan, R.; Roy, A.; Xu, D.; Poisson, J.; Zhang, Y Nat Methods 2015, 12, 7-8 ACS Paragon Plus Environment Analytical Chemistry 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 557 FIGURE LEGENDS 558 Figure 559 Schematic of domain composition and primary structure of C9 The scheme includes 560 previously reported sites of C-mannosylation22 and N-glycosylation on C919-21 The glycan 561 nomenclature used is indicated at the bottom 562 563 Figure 564 Full native ESI-MS spectrum of the intact C9 sprayed from aqueous ammonium acetate (a) 565 The charge states are indicated Zoom in on the 15+ charged state in the inset reveals 566 approximately 50 distinct ion signals (b) Zoom in on the 15+ charged state, centered around 567 m/z 4,500, in the native mass spectra of intact C9 In (c) alike spectrum of C9 treated with 568 PNGase F for hours reveals partial N-deglycosylation In (d) C9 was treated with PNGase F 569 for 48 hours In (e) C9 was first enzymatically desialylated, and in (f) C9 was partially 570 denatured, prior to native MS analysis The differences in mass between C9 proteoforms in 571 the unprocessed and treated samples allows the deduction of the PTM composition of these 572 most abundant C9 proteoforms The mass of the most abundant ion in the unprocessed 573 sample, at m/z of 4,435.28, is 66,516.20 Da, from which a calculated composition of PTMs 574 can be derived Hex12HexNAc9NeuAc6 unambiguously The 15 positive charges come from 13 575 H+ and Ca2+ ion, due to the presence of a bound Ca2+ ion, as revealed in (f) 576 577 Figure 578 Low energy HCD MS/MS (a) and EThcD MS/MS (b) spectra of the peptide harboring the 579 novel, non-canonical N-glycosylation site at N215, derived by tryptic digestion of C9 Both 580 spectra were acquired for the same precursor with m/z of 1,124.13 Da Sequential 581 fragmentation of the N-glycan moiety in the spectrum (a) allowed deduction of its glycan 582 composition while the EThcD spectrum (b) provided confirmation of the peptide sequence 583 and position of the N-glycan “P” = peptide backbone of the glycopeptide 584 585 Figure 586 Summary of assignments of PTMs on C9 (a) Native MS spectrum of C9, zoomed in on 587 charge state [M+13H+Ca2+]15+ The overall PTM composition of the most abundant ACS Paragon Plus Environment Page 20 of 30 Page 21 of 30 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Analytical Chemistry 588 proteoform with m/z of 4,435.28 was deduced as described in Figure Mass differences 589 among the peaks correspond to various glycan units or glycans (b) Relative abundances of 590 peptide proteoforms were estimated from their corresponding peptide ion currents (XICs) 591 Each PTM modified peptide was normalized individually so that the sum of all proteoforms 592 was set to 100% For clarity, only parts of the peptide sequence carrying PTMs are shown 593 below the graph (c) A comparison of the intact C9 native MS spectrum with the in silico 594 constructed spectrum based on the peptide-centric proteomics data The correlation is very 595 high (R=0.92) (d) Structural model of poly-C916 and mono-C9, whereby the sites of the 596 modifications are indicated The poly-C9 model was chosen as template for the mono-C9 597 using I-Tasser44 and the model was processed by means of the PyMOL Molecular Graphic 598 System, Version 1.8 Schrödinger, LLC (e) Overview of the C9 sequence with all identified 599 PTM sites, wherein the newly discovered O-glycosylation sites at the N-terminus and the 600 novel N-glycosylation site at residue 236 are highlighted in orange and purple, respectively ACS Paragon Plus Environment Analytical Chemistry 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 601 602 Figure ACS Paragon Plus Environment Page 22 of 30 Page 23 of 30 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Analytical Chemistry 603 604 Figure ACS Paragon Plus Environment Analytical Chemistry 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 605 606 Figure ACS Paragon Plus Environment Page 24 of 30 Page 25 of 30 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Analytical Chemistry 607 608 Figure 609 610 ACS Paragon Plus Environment Analytical Chemistry 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 611 612 For table of Contents Only 613 ACS Paragon Plus Environment Page 26 of 30 Page 27 of 30 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Analytical Chemistry Figure Schematic of domain composition and primary structure of C9 76x38mm (300 x 300 DPI) ACS Paragon Plus Environment Analytical Chemistry 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Figure Full native ESI-MS spectrum of the intact C9 84x108mm (300 x 300 DPI) ACS Paragon Plus Environment Page 28 of 30 Page 29 of 30 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Analytical Chemistry Figure Low energy HCD MS/MS (a) and EThcD MS/MS (b) spectra of the peptide harboring the novel, non-canonical N-glycosylation site at N215 83x75mm (300 x 300 DPI) ACS Paragon Plus Environment Analytical Chemistry 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Figure Summary of assignments of PTMs on C 105x68mm (300 x 300 DPI) ACS Paragon Plus Environment Page 30 of 30