Comprehensive Proteoform Characterization of Plasma Complement Component C8αβγ by Hybrid Mass Spectrometry Approaches
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The human complement hetero-trimeric C8αβγ (C8) protein assembly (~ 150 kDa) is an important component of the membrane attack complex (MAC). C8 initiates membrane penetration and coordinates MAC pore formation. Here, we charted in detail the structural micro-heterogeneity within C8, purified from human plasma, combining high-resolution native mass spectrometry and (glyco)peptide-centric proteomics. The intact C8 proteoform profile revealed at least ~ 20 co-occurring MS signals. Additionally, we employed ion exchange chromatography to separate purified C8 into four distinct fractions. Their native MS analysis revealed even more detailed structural micro-heterogeneity on C8. Subsequent peptide-centric analysis, by proteolytic digestion of C8 and LC-MS/MS, provided site-specific quantitative profiles of different types of C8 glycosylation. Combining all this data provides a detailed specification of co-occurring C8 proteoforms, including experimental evidence on N-glycosylation, C-mannosylation, and O-glycosylation. In addition to the known N-glycosylation sites, two more N-glycosylation sites were detected on C8. Additionally, we elucidated the stoichiometry of all C-mannosylation sites in all the thrombospondin-like (TSP) domains of C8α and C8β. Lastly, our data contain the first experimental evidence of O-linked glycans located on C8γ. Albeit low abundant, these O-glycans are the first PTMs ever detected on this subunit. By placing the observed PTMs in structural models of free C8 and C8 embedded in the MAC, it may be speculated that some of the newly identified modifications may play a role in the MAC formation.
KeywordsComplement component C8 Membrane attack complex Glycosylation Plasma proteins Native mass spectrometry Ion-exchange chromatography Glycopeptide-centric proteomics N-glycosylation O-glycosylation C-glycosylation
Epidermal growth factor
Extended mass range
Electron-transfer and higher-energy collision dissociation
False discovery rate
Higher-energy collisional dissociation
Low-density lipoprotein receptor class A repeat
Membrane attack complex
Tandem mass spectrometry
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Extracted ion chromatogram
In humans, the complement system forms the first line of defense against microbial infections. Three major complement cascades (the classical, the alternative, and the lectin pathway) can initiate the terminal pathway. This process includes the formation of the membrane attack complex (MAC), consisting of the complement component C5, 6, 7, 8, and 9, an important innate immune effector that forms cytotoxic pores in the bacterial cell membrane. Although MAC assembly and its action have been extensively functionally and structurally investigated for many years, the molecular mechanism behind these processes and the role of protein post-translational modifications (PTMs) therein remain largely elusive. Recent progress in structural biology has provided several cryoEM maps on the MAC and new insights into the molecular architecture of this fascination assembly, albeit that PTMs are mostly not visible in such structural models .
Materials and Methods
Chemicals and Materials
Complement component C8 (UniProt Code: P07357 (α), P07358 (β), P07360 (γ)) purified from pooled human blood plasma (several healthy donors) was acquired from Complement Technology, Inc. (Texas, USA). The sample was purified according to a reported standard protocol  (the certificate of analysis is attached in the Supporting information – S1). Dithiothreitol (DTT), iodoacetamide (IAA) and ammonium acetate (AMAC) were purchased from Sigma-Aldrich (Steinheim, Germany). Formic acid (FA) was from Merck (Darmstadt, Germany). Acetonitrile (ACN) was purchased from Biosolve (Valkenswaard, The Netherlands). POROS Oligo R3 50-μm particles were obtained from PerSeptive Biosystems (Framingham, MA, USA) and packed into GELoader pipette tips (Eppendorf, Hamburg, Germany). Sequencing grade trypsin was obtained from Promega (Madison, WI). Asp-N, PNGase F, and Sialidase were obtained from Roche (Indianapolis, USA).
Sample Preparation for Native MS
Unprocessed protein solution in a phosphate buffer at pH 7.2, containing ~ 30–40 μg of C8, was buffer exchanged into 150 mM aqueous AMAC (pH 7.5) by ultrafiltration (vivaspin500, Sartorius Stedim Biotech, Germany) using a 10 kDa cutoff filter. The resulting protein concentration was measured by UV absorbance at 280 nm and adjusted to 2–3 μM prior to native MS analysis. The enzyme Sialidase was used to remove sialic acid residues from C8. PNGase F was used to cleave the N-glycans of C8 . All samples were buffer exchanged into 150 mM AMAC (pH 7.2) prior to native MS measurements.
Native MS Analysis
Samples were analyzed on a modified Exactive Plus Orbitrap instrument with extended mass range (EMR) (Thermo Fisher Scientific, Bremen) using a standard m/z range of 500–10,000, as described in detail previously . The voltage offsets on the transport multi-poles and ion lenses were manually tuned to achieve optimal transmission of protein ions at elevated m/z. Nitrogen was used in the higher-energy collisional dissociation (HCD) cell at a gas pressure of 6–8 × 10−10 bar. MS parameters used: spray voltage 1.2–1.3 V, source fragmentation 30 V, source temperature 250 °C, collision energy 30 V, and resolution (at m/z 200) 30,000. The instrument was mass calibrated as described previously, using a solution of CsI .
Native MS Data Analysis
The accurate masses of the observed C8 proteoforms were calculated manually averaging over all detected charge states of C8. For PTM composition analysis, data were processed manually and glycan structures were deduced based on known biosynthetic pathways. Average masses were used for the PTM assignments, including hexose (e.g., Glucose, Glc; mannose, Man; Galactose, Gal; 162.1424 Da), N-acetylhexosamine (e.g., GlcNAc or GalNAc; 203.1950 Da) and N-acetylneuraminic acid (NeuAc, 291.2579 Da). All used symbols and text nomenclature are according to recommendations of the Consortium for Functional Glycomics.
Dual-Column Ion-Exchange Chromatography Separation of Purified C8
An Agilent 1290 Infinity HPLC system (Agilent Technologies, Waldbronn. Germany) consisting of a vacuum degasser, binary pump, refrigerated autosampler with 500-μL injector loop, thermostated two column compartment, auto collection fraction module and multi-wavelength detector, was used in this study. The dual-column set-up, comprising a tandem WAX-CAT (PolyWAX LP, 200 × 2.1 mm i.d., 5 μm, 1000 Å; PolyCAT A, 50 × 2.1 mm i.d., 5 μm, 1000 Å) two-stage set-up. All columns were obtained from PolyLC Inc. (Columbia, USA) . The column compartment was cooled to 17 °C while the other bays were chilled to 4 °C minimize sample degradation. Mobile phase Buffer A consisted of 100 mM AMAC in water, and mobile phase Buffer B consisted of 2.5 M AMAC in water. A small amount (final concentration of 3 mM) of NaN3 was added to minimize microbial outgrowth to each solution, which was filtered using a 0.22 μm disposable membrane cartridge (Millipore) before use. Injections were typically 250 μg total protein per run. Elution was achieved using multi-step gradient, consisting of five transitions with increasing proportions of Buffer B: (step 1; equilibration) 0%B, 0–6 min; (step 2; salt gradient) 0–60%B, 6–42 min; (step 3; high salt rinse) 60–100%B, 42–60 min; (step 4; high salt wash) 100%B, 60–61 min; (step 5; restoration) 100–0%B. The flow rate was set to 800 μL min−1. The chromatograms were monitored at 280 nm and peak based fractions collected using an automated fraction collector.
In-Solution Digestion for Peptide-Centric Glycoproteomics
Intact human C8 protein in PBS buffer (10 mM sodium phosphate, 145 mM NaCl, pH 7.3) at a concentration of 1 mg/ml was reduced with 5 mM DTT at 56 °C for 30 min and alkylated with 15 mM IAA at room temperature for 30 min in the dark. The excess of IAA was quenched by using 5 mM DTT. C8 was digested overnight with trypsin at an enzyme-to-protein-ratio of 1:100 (w/w) at 37 °C. Another C8 sample was digested for 4 h by using Asp-N at an enzyme to-protein-ratio of 1:75 (w/w) at 37 °C and the resulted peptide mixtures were further treated with trypsin (1:100; w/w) overnight at 37 °C. All proteolytic digests containing modified glycopeptides were desalted by GELoader tips filled with POROS Oligo R3 50 μm particles , dried and dissolved in 40 uL of 0.1% FA prior liquid chromatography (LC)-MS and MS/MS analysis.
All peptides (typically 300 fmol of C8 peptides) were separated and analyzed using an Agilent 1290 Infinity HPLC system (Agilent Technologies, Waldbronn. Germany) coupled on-line to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Reversed-phase separation was accomplished using a 100 μm inner diameter 2 cm trap column (in-house packed with ReproSil-Pur C18-AQ, 3 μm) (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) coupled to a 50 μm inner diameter 50 cm analytical column (in-house packed with Poroshell 120 EC-C18, 2.7 μm) (Agilent Technologies, Amstelveen, The Netherlands). Mobile-phase solvent A consisted of 0.1% FA in water, and mobile-phase solvent B consisted of 0.1% FA in ACN. The flow rate was set to 300 nL/min. A 45 min gradient was used as follows: 0–10 min, 100% solvent A; 10.1–35 min 10% solvent B; 35–38 min 45% solvent B; 38–40 min 100% solvent B; 40–45 min 100% solvent A. Nanospray was achieved using a coated fused silica emitter (New Objective, Cambridge, MA) (outer diameter, 360 μm; inner diameter, 20 μm; tip inner diameter, 10 μm) biased to 2 kV. The mass spectrometer was operated in positive ion mode and the spectra were acquired in the data-dependent acquisition mode. For the MS scans, the mass range was set from 300 to 2000 m/z at a resolution of 60,000 and the AGC target was set to 4 × 105. For the MS/MS measurements, HCD and electron-transfer and higher-energy collision dissociation (EThcD) were used in the two LC-MS/MS runs for every sample. First, HCD was performed with two independent scan events. One scan event with a normalized collision energy of 15% and the other with 35%. The second LC-MS/MS run was performed using EThcD. A supplementary activation energy of 20% was used for EThcD. For the MS/MS scans the mass range was set from 100 to 2000 m/z and the resolution was set to 30,000; the AGC target was set to 5 × 105; the precursor isolation width was 1.6 Da and the maximum injection time was set to 300 ms.
LC-MS/MS Data Analysis
Raw data were interpreted by using the Byonic software suite (Protein Metrics Inc.)  and further validation of the key MS/MS spectra was manually checked. The following parameters were used for data searches: precursor ion mass tolerance, 10 ppm; product ion mass tolerance, 20 ppm; fixed modification, Cys carbamidomethyl; variable modification: Met oxidation, Trp Mannosylation, and both N- and O-glycosylation from mammalian glycan databases. A non-enzyme specificity search was chosen for all samples. The database used contained the C8 protein amino acid sequence (Uniprot Code: P02748). Profiling and relative quantification of PTM modified C8 peptides were achieved by use of the extracted ion chromatograms (XICs) from two independently processed C8 samples. The peptide mixtures were prepared with different combinations of proteolytic enzymes as described above (1. Trypsin; 2. AspN + Trypsin). Both samples were analyzed in two independent LC-MS/MS runs using EThcD and HCD respectively. Each peptide that contains PTM sites was normalized individually so that the sum of all its proteoform areas was set at 100%. The average peptide ratios from all measurements were taken as a final estimation of the abundance. The XICs were obtained using the software Thermo Proteome Discoverer 22.214.171.1248. The glycan structures of each glycoform were manually annotated. Hereby reported glycan structures are depicted without the linkage type of glycan units since the acquired MS/MS patterns do not provide such information.
Integrating Native MS and Peptide-Centric Proteomic Data
Reliability and completeness of the obtained proteoform profiles of C8 were assessed by an integrative approach combining the native MS data with the glycopeptide centric proteomics data. Details of this approach have been described in detail previously . Briefly, in silico data construction of the “intact protein spectra” was performed based on the masses and relative abundances of all site-specific PTMs derived from the glycopeptide centric analysis. Subsequently, the constructed spectrum was compared to the experimental native MS spectra of C8. The similarity between the two independent data sets (Native MS spectra and constructed spectra based on glycopeptide centric data) was expressed by a Pearson correlation factor. All R scripts used for the spectra simulation are available at github (https://github.com/Yang0014/glycoNativeMS).
I-TASSER Structural Modeling of C8
Detailed descriptions of I-TASSER can be found in Refs [24, 25]. Briefly, the structural monomer of poly-C9 (EMDB code: 3134)  was selected as a template for the structural remodeling of the soluble C8β (UniProt code: P07358) to its putative membrane form. Using Monte Carlo simulations, the template and regions modeled with ab initio methods are assembled into a large number of full length conformations. By clustering the conformations, cluster centroids are identified, and the final models are built by additional refinements of the cluster centroids. The models were eventually processed using PyMOL Molecular Graphic System.
Native MS of the C8 Complement Assembly
The observed heterogeneity among the most abundant species in the C8 proteoform profile is represented by mass differences of 162 Da corresponding to Hex moieties. C8α and β are structurally and genetically related protein subunits, both belonging to the MAC protein family . In addition to other structural similarities, both the α and β subunits contain two highly conserved TSP domains, harboring C-mannosylations [16, 32]. Previous C8 studies suggested four fully occupied C-mannosylation sites at each subunit. However, our native MS experiments on C8 strongly suggest structural heterogeneity caused by partial occupancy of the C-mannosylation sites. Our final calculation of the overall PTM composition of the most abundant C8 proteoform includes two N-glycans and seven mannoses (143,075.63 + 2 × 2206.01 + 7 × 162.14 = 148,622.63 Da) which in the end corresponds to an acceptable small mass error 6.52 ppm with respect to the observed C8 mass; 148,623.60 Da. These findings were further supported by our peptide-centric proteomic analyses, which are described below.
Non-denaturing Ion Exchange Chromatography of C8 Enables the More Detailed Study of Individual Components
Peptide-Centric LC-MS/MS Analysis of C8
To further validate our findings and calculations based on the native MS measurements, we next performed proteolytic digestion of C8 using different proteases and the resulting peptide mixtures were analyzed by LC-MS/MS. Data interpretation provided information about the site location, glycan type, composition and abundance of N-glycosylated, O-glycosylated and C-mannosylated proteoforms. Starting our description with C8α, MS/MS of the tryptic peptide with amino acid sequence 425GGSSGWSGGLAQNR 438 clearly confirmed the composition of the high abundant N-glycans at N437 with the major glycoform corresponding to the N-glycan composition of HexNAc4Hex5NeuAc2. The MS/MS spectra revealed that the major N-glycan composition on this site is. The native MS measurements also revealed a novel low abundant N-glycosylation site on C8α, which we unfortunately did not detect in our LC-MS/MS data, probably due to its low abundance. Additionally, we detected peptides originating from the TSP domains; 31AATPAAVTCQLSNWSEWTDCFPCQDKK57 and 538ADGSWSCWSSWSVCR552. These peptides contain the sequence motif WXXW, which is known to be frequently C-mannosylated . Our EThcD and HCD MS/MS spectra unequivocally confirmed that in C8α, W44, and W542 are fully occupied by C-mannosylation, whereas W47, W545, and W548 are only partially occupied.
A similar set of modifications was found on C8β. From the three potential N-glycosylation sites following the canonical N-X-S/T motif, two were found to be occupied. Interpretation of the LC-MS/MS spectra of the glycopeptides with the sequences 86YAYLLQPSQFHGEPCNFSDKEVEDCVTNRPCR117 and 233EYESYSDFERNVTEK247 revealed high mannose N-glycans with low occupancy on N101 and biantennary complex N-glycans on N243. Attachment of C-mannoses was confirmed by fragmentation of the peptides 55SVDVTLMPIDCELSSWSSWTTCDPCQK81 and 541NTPIDGKWNCWSNWSSCSGR560. In C8β W70 and W551 are fully occupied and W73, W548, W554 are partially modified. Furthermore, we identified and quantified two distinct positional isomers of the peptide 425NTPIDGKWNCWSNWSSCSGR425 modified by two Man either on W548 and W551 or W551 and W554 (Supplementary Fig. 2). Finally, C8γ was found to be partially O-glycosylated close to its N-terminus. EThcD and HCD MS/MS spectra of the O-glycopeptides with amino acid sequence 28RPASPISTIQPK39 exposed the structural composition of the attached O-glycans. These were HexNAc1, HexNAc1Hex1 and HexNAc1Hex1NeuAc1 attached to T35. All MS/MS spectra supporting these modifications can be found in the Supplementary data – S4.
Integration of Native MS and Peptide-Centric Data
Next, we made a correlative comparison between the native MS spectrum of the intact fully assembled C8 with an in silico constructed MS spectrum based on all the quantitative information we gathered from the LC-MS/MS peptide centric data (Fig. 4c). All C8 species predicted from the peptide-centric data were filtered by taking 1% cutoff in relative intensity of the peaks in the experimental native spectrum and mass deviations were manually checked. This validation process resulted in a list containing 14 distinct C8 variants and covered most of the detected signals of the C8 proteoforms (Supplementary Table 2). This comparison reveals a high degree of consistency between our native MS and peptide-centric MS approach (R = 0.95), indicating that we have nearly annotated all proteoforms detected in the native MS spectrum. The unmatched low abundant ion signals mostly correspond to adducts bearing Na+ and/or K+ ions, which are frequently observed in the ESI ionization process. Some of the peaks in the constructed spectrum show a different intensity compare to the experimental native MS spectrum. This is caused by the fact that labile PTMs are easily lost during the peptide-centric LC-MS/MS analysis [35, 36, 37] and ionization at the peptide level is more biased when compared to the native intact protein measurements. Although we could explain most of the ion signals observed in the native MS spectra originating from the fully assembled C8, this analysis did not cover some of the lower abundant modifications that we only detected on the individual sub-complexes and proteins following the fractionation of C8 sub-complexes and subunits by IEX. Therefore, these low abundant species are not included in the final list of the validated C8 proteoforms. These include the C8 proteoforms containing the novel albeit low abundant N-glycosylation sites on C8α and C8β.
C8 is a protein assembly consisting of three distinct subunits. C8 possesses an atypical structure and unusual physicochemical properties relative to other related complement proteins and even to most plasma proteins in general. Due to a complicated assembly process and secretion pathway of C8, it is not fully clear, which structural variants of C8 are present in circulation and what is their biological relevance. The proposed biosynthetic mechanism suggests that precursors of C8αγ and β are likely associated intracellularly, and the pre-mature C8 complex is converted to its final form by terminal glycosylation reactions within the Golgi complex . Mature C8 is then thought to be secreted intact. Alternatively, precursors of C8αγ and β could be processed independently or associated with their mature counterparts. Since less β subunit is synthesized in the liver compared to the α and γ subunits, independent secretion of C8β is not expected under normal conditions. However, independent secretion of αγ hetero-dimers and the β subunit may still be possible, especially in cases of hereditary C8 deficiency, wherein the αγ dimer and/or β subunit are found in their non-bound forms [38, 39]. Our native MS analysis of the Complement component C8 purified from pooled plasma hints at the co-existence of free C8αγ and fully assembled C8 in the sample solution. Up to now, no reports exist on the presence of free C8αγ dimers in normal plasma. In our analysis, we work with relatively high concentrations of C8 (~ 1 mg/mL), which is approximately 20× higher than the physiological concentration in normal human plasma. Therefore, we cannot exclude whether the C8αγ dimer is actually present in human plasma or whether it is an artifact of the purification process, or some product of dynamic assembly and disassembly between the C8 subunits. For that reason, although we find it an interesting finding, we focused our analysis on the proteoforms from the fully assembled C8 complex.
As with many other human plasma proteins, the C8 complex is synthetized in the liver. As a consequence, C8 is expected to contain mostly complex biantennary N-glycans with varying degrees of sialylation. The primary structures of C8α and β contain two and three potential N-glycosylation sites, respectively. Two of them (N437 on α and N243 on β) were known to harbor complex N-glycans, but with a lower level of sialylation compared to the other complement components. Additionally, C8β was also found to attach bisected glycan structures . In comparison to these previous reports, we did not observe any abundant bisected structures and all detected N-glycopeptides attaching biantennary N-glycans showed relatively homogenous structures containing mostly sialylated antennas. These minor discrepancies may be explained by a variability between analyzed samples and/or more advanced technological possibilities in the current study. Beside the two known N-glycosylation sites, the native MS measurements of the IEX fractions revealed some low abundant glycoforms of C8β (fraction 3) and C8αγ (fraction 4) both containing additional N-glycans. Although the β chain was predicted to harbor an N-glycan on N101 based on the sequence, no experimental evidence had been reported previously. Our data show that N101 can be indeed glycosylated; however, its occupancy is low. Interestingly, we detected only the presence of high mannose N-glycans, suggesting a low accessibility of this potential N-glycosylation site for the glycosylation machinery in the Golgi. The second low abundant N-glycan detected on C8αγ is likely located on C8α, since the γ subunit does not possess any potential N-glycosylation site. C8α contains a potential N-glycosylation site (N43) in the first TSP domain that overlaps with a sequon for C-mannosylation. Previous reports dealing with the C-mannosylation of C8 protein speculate that C-mannosylation precedes the addition of N-glycans on this site . Although the mass differences observed in the native MS spectra of the αγ dimer clearly revealed the presence of unreported complex biantennary N-glycans, we did not detect any N-glycopeptide confirming the position of this N-glycan probably due to its very low abundance. Nevertheless, if N43 was modified by this N-glycan, we could speculate that the proposed function of the Man at W44 precluding the attachment of N-glycans may be actually correct. The question remains if that could be possible, since W44 is fully C-mannosylated and therefore the N-glycan would have to co-exist next to this Man. Another option is that the detected N-glycan is located on another potential, non-canonical, N-glycosylation site.
C-mannosylation is a significant feature of the MAC. It has been hypothesized to play an important role in adhesion of the terminal complement proteins to each other and in the assembly of the MAC [40, 41]. Regarding to C-mannosylation of C8 in particular, we identified that occupancy of the potential C-mannosylation sequons in TSP domains of α and β subunit is the major source of micro-heterogeneity in C8. This is in contradiction with older data, which suggested only completely modified or non-modified W on C8 . Interestingly, we also identified positional isomers on C-terminal TSP of C8β. Our peptide-centric data on C8β show that the second W in the WXXWXXW motif is always modified while the second Man is added either on the first or the third W without apparent preference. We did not observe this on analogical C-man sites on the C-terminal TSP of C8α, where the first and the second W are clearly preferred to be modified compare to the third W in the motif. This may support hypothesis that the signal for C-mannosylation could be formed apart from the WXXW motif by the three-dimensional structure of a protein . Since mechanistic insights about C-mannosylation have not been clarified, our results provide another important piece of information contributing to the discussion about this quite rare modification.
The last modification detected in our work was found on C8γ. There are several possible functions of C8γ described; however, a more precise biological role of C8γ in the complement system remains rather elusive. Here, we provide the first experimental evidence for O-glycans located on T35. Although detected O-glycosylated peptides showed very low abundance which is typically caused by weak ionization response of O-glycopeptides in general , the native MS profile of C8 clearly revealed proteoforms containing O-glycans with more than 2% of relative peak intensity. It could be that this modification is present only in parts of the population requiring analysis of C8 from individuals to determine a possible functional significance.
More generically, our analysis was performed on C8 purified from pooled plasma originating from several healthy donors. It may therefore also be of interest to investigate whether the occupancies we here report of all PTMs are equal of different in each individual. Such studies come within reach if more efficient purification methods would become available, and the analysis of individual proteoform profiles can be done more automatically. Our full analysis reported here would still take a week per individual.
The applied complementary mass-spectrometry based methods herein represent powerful tools for the unbiased in-depth analysis of C8 but also other plasma glycoproteins. In the near future we can foresee that we should be able to analyze glycoproteoform profile of individual proteins, isolated from individual personalized body fluids, such as blood, serum and urine. Such proteoform profiles may represent a new level of analysis in biomarker discovery.
We acknowledge the support from the Netherlands Organization for Scientific Research (NWO) funding the large-scale proteomics facility Proteins@Work (project 184.032.201) embedded in the Netherlands Proteomics Centre. A.J.R.H. acknowledges further support by the NWO TOP-Punt Grant 718.015.003. This project has received additional funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement 668036 (RELENT) and 686547 (MSMed). J.Z. acknowledges support from the Chinese Scholarship Council (CSC).
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