Use of Cysteine Aminoethylation To Identify the Hypervariable

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Use of Cysteine Aminoethylation to Identify the Hypervariable Peptides of an Antibody Nick DeGraan-Weber, and James Patrick Reilly Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02732 • Publication Date (Web): 13 Dec 2017 Downloaded from http://pubs.acs.org on December 13, 2017

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Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Analytical Chemistry

Use of Cysteine Aminoethylation to Identify the Hypervariable Peptides of an Antibody Authors: Nick DeGraan-Weber[a] and James P. Reilly[a],* [a]

Department of Chemistry, Indiana University, 800 East Kirkwood Avenue, Bloomington, Indiana 47405 Email: [email protected]

Address reprint requests to James P. Reilly, Department of Chemistry, Indiana University, 800 East Kirkwood Avenue, Bloomington, Indiana 47405, [email protected]

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 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

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ABSTRACT: Aminoethylation of cysteines can provide enzymatically cleavable sites. The ability to obtain peptides containing antibody complementarity determining regions (CDRs) with aminoethylated cysteines was investigated. Because cysteines are often located N-terminal to CDRs, digestion with LysN enables acquisition of peptides with CDRs. Lys-N peptides containing an aminoethylated cysteine at the N-terminus were also amidinated. Subsequent collisional activation yields a unique loss of 118 Da that originates from this modified residue, providing a signature ion for cysteine-containing peptides. The relative cleavage efficiencies for Lys-N and trypsin are also compared.


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INTRODUCTION Monoclonal antibodies are widely used in a variety of protein drug therapies.1,2 Many antibodies have similar structures; they contain two light and two heavy chains that are connected through disulfide bonds to create a 150 kDa protein.2-4 The resulting structure is Y-shaped, the variable region of which binds with antigens. The six short strings of amino acids that are most involved in this interaction are called complementarity determining regions (CDRs).2-4 Figure 1 shows part of the variable regions of ten IgG monoclonal antibodies. The amino acids in bold are the CDRs that range between five and nineteen residues. The three light chain CDRs (LCDR) are referred to as LCDR1, LCDR2, and LCDR3 (from N- to C-terminal), and those in the heavy chain (HCDR) are referred to as HCDR1, HCDR2, and HCDR3. As is evident in the figure, amino acids outside of the CDRs tend to be similar in all ten antibodies. Of particular relevance to the present work, the locations of the cysteines before LCDR1, LCDR3, HCDR1, and HCDR3 (indicated in red) are consistent. These cysteines are in intra-chain disulfide bonds that stabilize the structure and binding activity.2 Tandem mass spectrometry is now commonly employed in the analysis of biomolecules.5-7 In bottom-up proteomics proteins are first digested into peptides that are subsequently characterized by collision-induced dissociation (CID) mass spectrometry.6 This activation method generates b- and y-type ions that facilitate peptide identification.5,8,9 An alternative approach that has been applied to biomolecules such as antibodies involves top-down analysis using electron capture or electron transfer dissociation.10,11 Analysis of antibodies by tandem mass spectrometry is particularly difficult when the sample contains a mixture of many antibodies, as is the case in an immune response.12-15 In the human serum, 70-75% of antibodies are of the IgG class.16 Since much of the sequence tends to be similar from one antibody to another, (Figure 1), the CDRs are the primary distinguishing features. However, identifying


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these regions can be challenging.14,15 Database matching may be implemented if the sequence is known, but sequence similarities can result in false positives.17 Alternatively if the sequence is not known, de novo sequencing is needed to elucidate the CDR amino acids.14,15 Aminoethylation of cysteine with 2-bromoethylamine has previously been investigated.18,19 This process results in a structure with a side chain length similar to that of lysine, however, the aminoethylated cysteine still contains a sulfur atom. Trypsin has been used to digest aminoethylated proteins and cleavages C-terminal to the modified cysteines were observed.20-21 Digestion of aminoethylated Bence-Jones proteins was shown to cleave between variable and constant regions.23-28 We have recently found that the enzyme, Lys-N, can recognize aminoethylated cysteines, and subsequent amidination and low-energy fragmentation results in a signature loss of 118 Da that originates from this residue. Likewise, identification of cysteine-containing peptides from antibodies and proteins was previously reported by Cotham et. al. and Parker et. al., in which a chromophore label on cysteines was selectively photodissociated.29,30 This method facilitated identification of HCDR3. Other methods for cysteine mapping include ICAT, in which thiol groups are labeled prior to digestion. ICAT labeled peptides can then be extracted and isolated.31 In the present work, the ability to selectively cleave at cysteine residues and identify peptides that contain CDRs is investigated with a known prototype antibody, rituximab. The antibody is reduced and aminoethylated prior to enzymatic digestion with trypsin or Lys-N. To generate peptides, proteolytic cleavage sites need to be located on both sides of each CDR. Since cysteines are located Nterminal to four of the six CDRs, aminoethylation provides a cleavage site for both Lys-N and trypsin. Lysines and arginines are often found C-terminal, but only sometimes N-terminal, to CDRs. When Lys-N is employed, the resulting CDR peptides will contain the aminoethylated cysteine. However, tryptic peptides will not include this residue. In the present experiments, CDR-containing peptides are collisionally fragmented in an ion-trap/orbitrap mass spectrometer with an electrospray ionization (ESI)


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source. Aminoethylated cysteine-containing peptides are also amidinated and fragmented by CID. As a method that helps to identify cysteine-containing peptides in general, this approach facilitates the detection of CDR peptides in particular. Given the sequence similarity across IgG antibodies (Figure 1), it also has broad applicability.


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EXPERIMENTAL SECTION Materials and Chemicals. 2-bromoethylamine hydrobromide, trizma hydrochloride, and trypsin were obtained from Sigma (St. Louis, MO, USA). Ammonium bicarbonate was purchased Mallinckrodt Chemicals (Phillipsburg, NJ, USA) and concentrated sodium hydroxide was from VWR Analytical (Radnor, PA, USA). Dithiothreitol was obtained from Bio-Rad (Hercules, CA, USA) and rituximab was provided by Genentech (South San Francisco, CA, USA). Antibody Aminoethylation, Digestion, and Amidination. Rituximab was first reduced with 5 mM dithiothreitol at 37°C for one hour. The reduced antibody was aminoethylated in a 400 mM trizma solution with the reagent ethylene imine (1500:1 reagent to protein) prepared from 2 M 2bromoethylamine and 5 M sodium hydroxide.32 The reaction proceeded in a sonicator for two hours. A 3 kDa spin filter then removed reagents and buffers with the addition of 100 mM ammonium bicarbonate. 2 μg of Lys-N or 1 μg of trypsin was added to this aminoethylated antibody in 90% ammonium bicarbonate and 10% acetonitrile and digested overnight at 37°C. Lys-N peptides were mixed in equal volumes with the amidinating reagent, S-methyl thioacetimidate (SMTA) (43.4 g/L SMTA in 250 mM trizma solution). The reaction mixture remained at room temperature for one hour. Mass Spectrometry. Protein digests were separated on a Thermo Scientific EASY-nLC 1200 liquid chromatograph with a reversed-phase Thermo Scientific Acclaim PepMap RSLC C18 column (Waltham, MA, USA). The eluent was electrosprayed into a Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer. Peptide ions were fragmented by CID in a data-dependent acquisition with a 3 m/z isolation width and 35 eV collision energy. Spectra were analyzed with a database matching program developed in-house using the known sequence of rituximab.


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RESULTS AND DISCUSSION Lys-N Digestion of Aminoethylated Rituximab. Lys-N peptides were LC separated, electrosprayed, and collisionally activated. The CID mass spectrum for the doubly charged Lys-N peptide C(+43)QQWTSNPPTFGGGT that contains LCDR3 is in Figure 2. The aminoethylated cysteine is indicated by +43 Da. Intense y8 and b7 ions are formed from cleavage N-terminal to proline.33 Other low intensity y- and b-type ions also appear. Other CDR peptides produced by Lys-N display similar fragmentation patterns. The peptide containing HCDR1, KASGYTFTSYNMHWV, results from cleavage at a lysine adjacent to an aminoethylated cysteine. Its mass spectrum is shown in Figure S1. With a lysine at the N-terminus and a histidine near the C-terminus, a mixture of b- and y-type ions is observed from fragmenting the doubly and triply charged peptides. The other CDR peptides, KQTPGRGLEWIGAIYPGNGDTSYNQ (HCDR2) in Figure S2, KPWIYATSNLASGVPVRF (LCDR2) in S3, and ARSTYYGGDWYF (HCDR3) in S4, also generate mixtures of b- and y-type ions. KPWIYATSNLASGVPVRF[S] is formed by non-specific cleavage at the Cterminus, while [C]ARSTYYGGDWYF[N] is from non-specific cleavages at both termini. Lys-N has been previously reported to be less specific than trypsin.34 In summary, Lys-N digestion of aminoethylated rituximab resulted in five of the six CDR peptides being detected and identified, although surprisingly, only one contained an aminoethylated cysteine at the N-terminus. Amidination of Lys-N Peptides. We have found that amidination modifies aminoethylated cysteines and improves the basicity of primary amines for analysis in mass spectrometry.35,36 Subsequent collisional activation of amidinated aminoethylated peptides with sequestered charges produces an intense signature loss of 118 Da corresponding to loss of HS-CH2-CH2-NH-CNH-CH3 from the labeled cysteine.


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Peptides obtained from Lys-N digestion of aminoethylated rituximab were amidinated with SMTA, LC separated, electrosprayed, and analyzed by CID. The LCDR3 peptide (+41)C(+84)QQWTSNPPTFGGGT was identified in the +2 charge state. (Here, the +41 indicates an amidinated N-terminal amine, while the +84 denotes an amidinated aminoethylated cysteine). The CID mass spectrum for this peptide in Figure 3 includes an abundant y8 ion from cleavage at proline and a y14 ion whose formation is facilitated by the amidination label at the N-terminus.33,35 Low intensity [MH118]+, [M+2H-118]2+, and b-118 ions are also observed. Since there are very few basic residues in this peptide, most likely there is a mobile proton that leads to alternative fragmentation pathways. However, an MS3 experiment involving collisional activation of the singly-charged fragment, b7-NH3, yielded an abundant [MH-118]+ and several b-118 ions, as shown in Figure S5. This demonstrates that even when a peptide such as CQQWTSNPPTFGGGT does not produce an intense 118 Da signature loss (Figure 3), one of its fragments may just do this (Figure S5). The +3 and +4 charged (+41)C(+84)RASSSVSYIHWFQQ were fragmented by CID and the resulting mass spectra are displayed in Figure 4. This peptide also contains LCDR1 and was not observed without amidination. Collisional activation of the +3 charged peptide yields abundant [M+2H-118-NH3]2+ and [M+2H-118]2+ ions, as shown in Figure 4a. The formation of the low intensity fragment, y142+, is facilitated by the amidination label at the N-terminus.36 The CID mass spectrum for the +4 charged peptide appears in Figure 4b. Abundant b133+ and b143+ ions, as well as low intensity [M+3H-118]3+ and b118 ions are produced. Comparison of the spectra in Figures 4a and 4b demonstrates that the loss of 118 Da is slower than fragmentations that are charge-induced. The CID mass spectra for the HCDR1 peptide, (+41)K(+41)ASGYTFTSYNMHWV, with +2 and +3 charges are in Figure 5a and b. This peptide is amidinated at both the N-terminal amine and the lysine side chain. Fragmentation of the doubly charged peptide produces abundant b132+ and b142+ ions. These fragments are likely protonated at an amidination site and the histidine side chain. Fragmentation of


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the triply charged peptide produces abundant b82+, b92+, b102+, b112+, and b122+, that contain only the two basic amidination sites. Other CDR peptide spectra include (+41)K(+41)QTPGRGLEWIGAIYPGNGDTSYNQ with +3 charges in Figure S6, the semi-Lys-N peptide (+41)K(+41)PWIYATSNLASGVPVRF with +3 charges in S7, and peptide (+41)ARSTYYGGDWYF with +2 charges in S8. All CID mass spectra contain abundant b-type ions that are made intense by the basic N-termini amidino group. Fragmentation of both (+41)K(+41)PWIYATSNLASGVPVRF and (+41)ARSTYYGGDWYF yields abundant yN-1 ions whose formation is facilitated by the N-terminal amidination label.36 b1 ions, that are not normally observed from standard peptides, are also formed due the amidination label at the N-terminus.36 In some CID mass spectra, these ions are absent because their masses are below the mass cutoff. However for the spectrum in Figure S7, a b1 ion is identified because this fragment contains two amidination labels at the N-terminal lysine. These results indicate that although six peptides containing each of the CDRs are identified, having aminoethylated cysteines N-terminal to CDRs was not as helpful as expected. Only LCDR1 and LCDR3 were identified with an N-terminal cysteine, while HCDR1 and HCDR3 contained an N-terminal lysine or exhibited a non-specific cleavage. Since there is some sequence variability across monoclonal antibodies (Figure 1), it is possible that another antibody might yield Lys-N cleavages at aminoethylated cysteines located C-terminal to CDRs. In any case, the signature loss of 118 Da from an amidinated aminoethylated cysteine is a useful diagnostic ion for helping to identify peptides with N-terminal cysteines. Tryptic Digestion of Aminoethylated Rituximab. Peptides from the tryptic digestion of aminoethylated rituximab were LC separated and electrosprayed into an ion trap mass spectrometer. CID mass spectra for the doubly and triply charged HCDR1 peptide, ASGYTFTSYNMHWVK, appear in


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Figures 5c and d. With a C-terminal basic group, mainly y-type ions were detected from the fragmentation of both charge states. However, the doubly charged peptide yielded more abundant singly charged y3, y4, y5, y6, y7, y8, y9, y10, and y11 ions that facilitate sequencing. HCDR1 was also observed with an N-terminal lysine in the Lys-N digestion of rituximab, as previously shown in Figure S1. As one would expect, these CID spectra yielded considerably more b-type ions. HCDR2 was identified from the fragmentation of the +2 and +3 charged GLEWIGAIYPGNGDTSYNQK as seen in Figure S9. Both spectra contained many y-type ions and some btype fragments. The triply charged peptide produced abundant multiply charged y-type ions, such as y112+, y122+, and y152+. The formation of intense y11 and y112+ ions is facilitated by the N-terminal amine of proline in the presence of mobile protons.33 One peptide contained LCDR1, LCDR2, and two missed cleavage sites, probably resulting from prolines being C-terminal to two lysines.37 The CID mass spectra for the +3, +4, and +5 charged peptide, ASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVR, are in Figure S10. Interestingly, fragmentation of the +3 charged peptide formed primarily y-type ions, while +4 and +5 charge states formed considerably more b-type ions. This is most likely due to the presence of histidine and multiple lysines in the middle of the sequence and only one arginine residue at the C-terminus. Cleavage at one of these lysines occurred to some extent resulting in the LCDR2 peptide, PWIYATSNLASGVPVR. The CID mass spectrum for this doubly charged peptide in Figure S11a has many singly charged y-type and b-type ions. The intense y3 and b13 ions are formed by cleavage N-terminal to proline.33 The triply charged peptide displayed in Figure S11b contains a number of doubly charged b-type ions. The N-terminal half of ASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVR contains LCDR1 and its CID spectra are in Figure S12. ASSSVSYIHWFQQKPGSSPK was fragmented in the doubly charge state (Figure S12a) and yielded many singly charged b- and y-type ions. Fragmentation of the +3 and +4 charged peptide ions (Figures S12b and c) produced primarily y-type ions with multiple charges.


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VEAEDAATYYC(+43)QQWTSNPPTFGGGTK contains LCDR3 and an aminoethylated cysteine. However, cleavage on the C-terminal side of the aminoethylated cysteine was not observed, despite the fact that trypsin can cleave at this modified residue.38,39 Note that as discussed above, Lys-N was able to cleave next to this aminoethylated cysteine. Fragmentation of the doubly charged peptide as displayed in Figure S13a formed many y-type ions. The abundant y9 ion is the result of cleavage on the N-terminal side of proline.33 Many singly and doubly charged y-type ions were observed in the CID spectrum for the triply charged peptide in Figure S13b. The b172+ ion is complementary to the y9 ion. It is evident that the proteolytic cleavage efficiency at aminoethylated cysteines varies with the enzyme used. In order to compare the cleavage efficiencies of Lys-N and trypsin, the number of lysines, arginines, and cysteines along with the occurrence ratios for these residues in rituximab are displayed in Table 1. Also in this table are the experimentally observed cleavage sites for the peptides that were identified from Lys-N and tryptic digestions. Lys-N cleaved at eight cysteines and 39 lysines. The ratio of these numbers is 0.205; since this is smaller than the ratio of cysteines to lysines in rituximab, 0.254 this indicates that Lys-N prefers to cleave at lysine residues relative to aminoethylated cysteines. By comparison, trypsin was found to cleave at 37 lysine and arginine residues, but only two cysteines. The ratio of these numbers, 0.054, is much smaller than the 0.254 ratio of these residues in rituximab. This suggests that trypsin cleaves more slowly at cysteines than at lysines and arginines, consistent with previous work.38,39 Likewise, one can infer from these data that trypsin cleaves more slowly at aminoethylated cysteines than Lys-N. It should be noted that other factors including the accessibility of residues and the inability to detect very small peptides may affect this comparison of enzymatic activity of trypsin and Lys-N. Although this is a limited sample, some trends are apparent and further studies of the relative activity of these two enzymes are warranted. CONCLUSIONS


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Digestion of an aminoethylated antibody with Lys-N and trypsin yielded peptides that contained all six complementarity determining regions. Fragmentation of Lys-N peptides generated b-type ions, while fragmentation of tryptic peptides produced primarily y-type ions. Lys-N was able to cleave next to aminoethylated cysteines more readily than trypsin, since no tryptic CDR peptides resulted from cleavage at this modified residue. The digestion comparison in Table 1 should be applied to a larger sample to verify this conclusion. It was previously reported that trypsin cleaves less efficiently at aminoethylated cysteines than lysines and arginines and this is consistent with the present findings.38,39 Some Lys-N CDR peptides result from non-specific cleavage, and this may have a positive or negative impact on these types of experiments. Additionally, a Lys-N peptide with an amidinated aminoethylated cysteine and sequestered protons tends to form a unique 118 Da loss from this modified residue that can facilitate identification of these peptides. Another step of collisional activation on ions that have lost 118 Da can be performed to provide more information. This approach was able to identify CDR peptides by database matching of a known antibody. De novo sequencing of CDR peptides from an unknown antibody may be possible utilizing photodissociation to obtain complete coverage of amino acid sequences.40 Isotopically labeling the aminoethylation or amidination tag can also provide quantitative information. The enzyme IdeS can be implemented to remove the constant region of the antibody. In this case, most of the cysteine -containing peptides identified using these methods would actually include CDRs. ASSOCIATED CONTENT Supporting Information. Includes supporting figures. This material is available free of charge via the internet at http://pubs.acs.org. ACKNOWLEDGEMENTS This work was supported by the National Institutes of Health grant R01GM103725.


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Tables Table 1: Comparison of rituximab residues with Lys-N and tryptic cleavages. Sample Lys Arg Cys Ratio Ratio (C/K) (C/K+R) Rituximab 49 14 16 0.327 0.254 Lys-N Cleavages 39 8 0.205 Trypsin Cleavages 28 9 2 0.054


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Figures Li ght Chai n QMTQSPSSLSASVGDRVTI TCKASQNI D- - - - KYLNWYQQKPGKAPKLLI YNTNNLQTGVPSRFSGSGSGTDFTFTI VSTQSPAI MSASPGEKVTMTCSASSSRS- - - - - YMQWYQQKPGTSPKRWI YDTSKLASGVPARFSGSGSGTSYSL TI QMTQSPSSLSASVGDRVTI TCSASQDI S- - - - NYLNWYQQKPGKAPKVLI YFTSSLHSGVPSRFSGSGSGTDFTL TI VLTQSPDFQSVTPKEKVTI TCRASQSI G- - - - SSLHWYQQKPDQSPKLLI KYASQSFSGVPSRFSGSGSGTDFTL TI LLTQSPVI LSVSPGERVSFSCRASQSI G- - - - TNI HWYQQRTNGSPRLLI KYASESI SGI PSRFSGSGSGTDFTL SI VLSQSPAI LSASPGEKVTMTCRASSSVS- - - - - YMHWYQQKPGSSPKPWI YAPSNLASGVPARFSGSGSGTSYSL TI QLTQSPSSLSASVGDRVTI TCRASQSVDYDGDSYMNWYQQKPGKAPKLLI YAASYLESGVPSRFSGSGSGTDFTL TI VLSQSPAI LSASPGEKVTMTCRASSSVS- - - - - YI HWFQQKPGSSPKPWI YATSNLASGVPARFSGSGSGTSYSL TI QMTQSPASLSVSVGETVTI TCRASENI Y- - - - SNLAWYQQKQGKSPQLLVYAATNLADGVPSRFSGSGSGTQYSL KI QMTQSPSSLSASVGDRVTI TCRASQDVN- - - - TAVAWYQQKPGKAPKLLI YSASFLYSGVPSRFSGSRSGTDFTL TI

SSLQPEDI ATYYCLQHI SRPRTFGQGTKVEI KRTVAAPSVFI SSMEAEDAATYYCHQRSS- - YTFGGGTKLEI KRTVAAPSVFI SSLQPEDFATYYCQQYSTVPWTFGQGTKVEI KRTVAAPSVFI NSLEAEDAAAYYCHQSSSLPFTFGPGTKVDI KRTVAAPSVFI NSVESEDI ADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFI SRVEAEDAATYYCQQWSFNPPTFGAGTKLELKRADAAPTVFI SSLQPEDFATYYCQQSHEDPYTFGQGTKVEI KRTVAAPSVFI SRVEAEDAATYYCQQWTSNPPTFGGGTKLELKRADAAPSVFI NSLQSEDFGSYYCQHFWGTPYTFGGGTRLEI KRADAAPTVFI SSLQPEDFATYYCQQHYTTPPTFGQGTKVEI KRTVAAPSVFI

FPPSDEQL KSGTA FPPSDEQL KSGTA FPPSDEQL KSGTA FPPSDEQL KSGTA FPPSDEQL KSGTA FPPSDEQL KSGTA FPPSDEQL KSGTA FPPSDEQL KSGTA FPPSDEQL KSGTA FPPSDEQL KSGTA

Al emt uzumab Basi l i xi mab Bevaci zumab Canaki numab Cet uxi mab I br i t umomab Omal i zumab Ri t uxi mab Sat umomab Tr ast uzumab

DI QI DI EI DI QI DI QI DI DI

Al emt uzumab Basi l i xi mab Bevaci zumab Canaki numab Cet uxi mab I br i t umomab Omal i zumab Ri t uxi mab Sat umomab Tr ast uzumab

Heavy Chai n QVQLQESGPGLVRPSQTLSLTCTVSGFTFTD- FYMNWVRQPPGRGLEWI GFI RDKAKGYTTEYNPSVKGRVTMLVDTSKNQFSLRLSSVTAADTAVYYCAREG- HTAA- - - PFDYWGQGSL VTVSSASTKGPSV - - QLQQSGTVLARPGASVKMSCKASGYSFTR- YWMHWI KQRPGQGLEWI GAI YPGNSD- - TSYNQKFEGKAKL TAVTSASTAYMELSSL THEDSAVYYCSR- - - DYGY- - - YFDFWGQGTTLTVSSASTKGPSV EVQLVESGGGLVQPGGSLRLSCAASGYTFTN- YGMNWVRQAPGKGLEWVGWI NTYTGE- - PTYAADFKRRFTFSLDTSKSTAYLQMNSL RAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTL VTVSSASTKGPSV QVQLVESGGGVVQPGRSLRLSCAASGFTFSV- YGMNWVRQAPGKGLEWVAI I WYDGDN- - QYYADSVKGRFTI SRDNSKNTLYLQMNGL RAEDTAVYYCAR- DLRTG- - - - PFDYWGQGTL VTVSSASTKGPSV QVQLKQSGPGLVQPSQSLSI TCTVSGFSLTN- YGVHWVRQSPGKGLEWL GVI WSGGN- - - TDYNTPFTSRLSI NKDNSKSQVFFKMNSL QSNDTAI YYCARALTYYDY- - - EFAYWGQGTL VTVSAASTKGPSV QAYLQQSGAELVRPGASVKMSCKASGYTFTS- YNMHWVKQTPRQGLEWI GAI YPGNGD- - TSYNQKFKGKATL TVDKSSSTAYMQLSSL TSEDSAVYFCARVVYYSNS- YWYFDVWGTGTTVTVSA- - - - - PSV EVQLVESGGGLVQPGGSLRLSCAVSGYSI TSGYSWNWI RQAPGKGLEWVASI - TYDGS- - TNYADSVKGRFTI SRDDSKNTFYLQMNSL RAEDTAVYYCARGSHYFG- - HWHFAVWGQGTL VTVSS- - - - GPSV QVQLQQPGAELVKPGASVKMSCKASGYTFTS- YNMHWVKQTPGRGLEWI GAI YPGNGD- - TSYNQKFKGKATL TADKSSSTAYMQLSSL TSEDSAVYYCARSTYYGGD- - WYFNVWGAGTTVTVSAASTKGPSV QVQLQQSDAELVKPGASVKI SCKASGYTFTD- HAI HWAKQKPEQGLEWI GYI SPGNDD- - I KYNEKFKGKATL TADKSSSTAYMQLNSL TSEDSAVYFCKRS- - - - - - - - - YYGHWGQGTTLTVSSASTKGPSV EVQLVESGGGLVQPGGSLRLSCAASGFNI KD- TYI HWVRQAPGKGLEWVARI YPTNGY- - TRYADSVKGRFTI SADTSKNTAYLQMNSL RAEDTAVYYCSR- - - WGGDGFYAMDYWGQGTL VTVSSASTKGPSV

Figure 1: Comparison of the partial variable regions for light and heavy chains from different monoclonal antibodies. Amino acids that are in bold are CDRs.

100

y8

812.4

C(+43)QQ W T S N P PT F G G GT2+

b7

% y4‡ 0 200

y4

y 5‡ b3 y5 400

2+

y8‡ b14 b7* b4* b4 y7 b5 b6 y9 y10 b8 y11 600

800

1000

b10 y

b11 12

1200

b12 b13

1400

1600

m/z

Figure 2: CID mass spectra of doubly charged C(+43)QQWTSNPPTFGGGT[K] (812.4 m/z) containing LCDR3. * indicates ammonia loss and ‡ denotes water loss.

100

y8

853.4

%

0 200

(+41)C(+84) QQ W T SN PPTFGGGT2+

[M+2H-118]2+

b3-118* 400

2+ b5-118* b13 b7* b7-118 b y10 7 y11 b4-118*

600

800

1000

y14 *]+ y14* [MH-118 [MH-118]+

y12 1200

1400

1600

m/z

Figure 3: CID mass spectra of doubly charged (+41)C(+84)QQWTSNPPTFGGGT[K] (853.4 m/z) containing LCDR3. * indicates ammonia loss.


18

Page 19 of 20 1 2 3 4 5 6 7 8 9 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


100

(a)

[M+2H-118*]2+

641.9

(+41)C(+84) RASSSVSYIHWFQQ3+ [M+2H-118]2+

% y142+ 0 200 100

400

(b)

600

800

1000

1200

b143+

481.7

(+41)C(+84)RASSSVSYIHWF Q Q4+

b133+ [M+3H-118]3+ [b13-118]2+ b144+

y2*y2

Y 0 200

[b14-118]2+

[M+3H**]3+

400

600

800

1000

m/z

Figure 4: CID mass spectra of (a) +3 (641.9 m/z) and (b) +4 charged (481.7 m/z) (+41)C(+84)RASSSVSYIHWFQQ[K] containing LCDR1. * indicates ammonia loss.

100

(a)

b14*2+ [M+2H]2+ b13*2+ (+41)K(+41) A SGYT F TS Y N M H WV2+

937.4

[M+2H*‡]2+

%

a13*2+ *2+ y102+ b12 y4 y5

y3 0 200 100

(b)

400

625.3

y2

b1 0 200

100

(c)

600

b12*

a14*2+ * b9*y8 b10*y9 b11

800

1000

1200

400

600

1600

1800

800

y3 b ‡ 5 b5

y10 b12‡ b12y11 b11 y11‡ b13 b14 b11 y10‡ b13‡ b14‡

y7 y y 2+ 6 y 2+ y4 b6‡ 11y 2+ 132+ y5 12 y14 * y7 b b 6

400

600

‡

7

800

1000

y9

y8

A S G Y T F T S Y N M H W V K2+

0 200 100

1400

** 2+

b82+ [M+2H ] 3+ b9‡2+ 2+ b10*2+ (+41)K(+41) ASGYT F T S Y N M H W V b9 y5 b8*2+ * b112+ y82+ b5 b72+ b13*2+ 2+ 2+ b6* b10 y b122+ b13 2+ b7‡2+ 3 y14 *2+ b6 b62+ b14 2+ b14

896.4

a4

b13* y y13 14

1000

1200

1400

1600

1800

y132+

(d) 597.9 A S G Y T F T S YNMHWVK3+

a4 b4

0 200

400

y82+ ‡3+ b5 y13 600

y10

y11 2+

m/z

2+ y13 y122+

‡2+

y142+ y7

800

1000

y8

y9 1200

Figure 5: CID mass spectra of (a) doubly (937.4 m/z) and (b) triply charged (625.3 m/z) (+41)K(+41)ASGYTFTSYNMHWV[K] containing HCDR1. (c) and (d) contain doubly (896.4 m/z) and triply charged (597.9 m/z) [K]ASGYTFTSYNMHWVK. * indicates ammonia loss and ‡ denotes water loss.


19

mAb Page Analytical Chemistry 20 ofCDR 20 Lys-N + Cys

Cys label

R

CID 1 2 M-118 Paragon Plus -NH3Environm 3 % 4 5 m/z

Use of Cysteine Aminoethylation To Identify the Hypervariable

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