Supporting Information Parsimonious Charge Deconvolution for Native Mass Spectrometry

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1 Supporting Information Parsimonious Charge Deconvolution for Native Mass Spectrometry Marshall Bern* 1, Tomislav Caval 2, Yong J. Kil 1, Wilfred Tang 1, Christopher Becker 1, Eric Carlson 1, Doron Kletter 1, K. Ilker Sen 1, Nicolas Galy 2, Dominique Hagemans 2, Vojtech Franc 2, Albert J.R. Heck* 2 1 Protein Metrics Inc., San Carlos, CA Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Science4Life, Utrecht University, and Netherlands Proteomics Centre, Padualaan 8, 3584 CH, Utrecht Contents Table S1. Amino acid sequences of the analyzed proteins Table S2. Therapeutic antibodies Figure S1. Simultaneous deconvolution of monomer and dimer Figure S2. Properdin qualitative and quantitative proteoform profile Figure S3. Improved properdin qualitative and quantitative proteoform profile Figure S4. Annotated glycopeptide fragmentation spectra of properdin peptides with triantennary glycans Figure S5. Annotated peptide fragmentation of Daclizumab revealing the GG clipping Figure S6. Annotated peptide fragmentation spectra of Daclizumab showing part of the signal peptide Table S3. Observed glycoforms of the Cetuximab Fd fragment S-1

2 Table S1. Amino acid sequences and expected average masses of the analyzed proteins Daclizumab light chain 23,215 Da average isotope mass (with reduced Cys) DIQMTQSPSTLSASVGDRVTITCSASSSISYMHWYQQKPGKAPKLLIYTTSNLASGVPARFSGSGSGTEF TLTISSLQPDDFATYYCHQRSTYPLTFGQGTKVEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC Daclizumab heavy chain with signal peptide 48,717 Da (pyro-glu N-terminus, reduced Cys, and no signal peptide nor C-terminal Lys) MGWSWIFLFLLSGTAGVHSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYRMHWVRQAPGQGLEWIGYI NPSTGYTEYNQKFKDKATITADESTNTAYMELSSLRSEDTAVYYCARGGGVFDYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Infliximab light chain 23,439 Da (with reduced Cys) DILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGSPRLLIKYASESMSGIPSRFSGSGSGTD FTLSINTVESEDIADYYCQQSHSWPFTFGSGTNLEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC Infliximab_heavy_chain 49,388 Da (with reduced Cys and without C-terminal Lys) EVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAEIRSKSINSATHYAESVKGRF TISRDDSKSAVYLQMTDLRTEDTGVYYCSRNYYGSTYDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK Cetuximab light chain 23,427 Da (with reduced Cys) DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC Cetuximab heavy chain 49,226 Da (pyro-glu N-terminus, reduced Cys, and no C-terminal Lys) QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK S-2

3 Properdin (Factor P) 48,494 Da with reduced Cys, 48,450 Da with disulfide-bonded Cys DPVLCFTQYEESSGKCKGLLGGGVSVEDCCLNTAFAYQKRSGGLCQPCRSPRWSLWSTWAPCSVTCSEGS QLRYRRCVGWNGQCSGKVAPGTLEWQLQACEDQQCCPEMGGWSGWGPWEPCSVTCSKGTRTRRRACNHPA PKCGGHCPGQAQESEACDTQQVCPTHGAWATWGPWTPCSASCHGGPHEPKETRSRKCSAPEPSQKPPGKP CPGLAYEQRRCTGLPPCPVAGGWGPWGPVSPCPVTCGLGQTMEQRTCNHPVPQHGGPFCAGDATRTHICN TAVPCPVDGEWDSWGEWSPCIRRNMKSISCQEIPGQQSRGRTCRGRKFDGHRCAGQQQDIRHCYSIQHCP LKGSWSEWSTWGLCMPPCGPNPTRARQRLCTPLLPKYPPTVSMVEGQGEKNVTFWGRPLPRCEELQGQKL VVEEKRPCLHVPACKDPEEEEL S-3

4 Table S2. Overview of investigated therapeutic antibodies S-4

5 (a) (b) S-5

6 (c) (d) Figure S1. Simultaneous deconvolution of monomer and dimer. The m/z spectrum of native properdin (a) shows three separated peak series: unfolded monomer at m/z , folded monomer at , and dimer at Protein Metrics Intact gave an accurate deconvolution (b), but Thermo Protein Deconvolution 4.0 gave large artifact peaks (c) with minimum number of charge states set to 3, and lost the dimer (d) with minimum number of charge states set to 5. (Protein Metrics Intact does not ask nor even allow the user to set the minimum number of charge states.) The deconvolutions shown in Figure 2 of the main text show zooms on the peaks in (b) and (d). S-6

7 Figure S2. Properdin qualitative and quantitative proteoform profile. Yang et al. analyzed properdin using the z=14 + charge state as shown at the top. This charge state is also shown in Figure 2a of the main text. Protein Metrics Intact enabled a reanalysis of the same properdin data with a deconvolved mass spectrum rather than the m/z spectrum of a single charge state. The code 14, 4, a indicates 14 C-linked mannoses in thrombospondin repeats, 4 GlcFuc s, and the singly sialylated N-glycan a. (This combination has the same mass as 13, 3.5, a, where 3.5 means 3 GlcFuc s and one Fuc, but Fuc without Glc appears to be rare, so we assigned the peak to 14, 4, a as in Yang et al.) Blue and purple ellipses indicate signals most likely caused by salt adducts. S-7

8 Figure S3. Improved properdin qualitative and quantitative proteoform profile. Based on the annotation of many salt adducts in our earlier analysis of properdin (Figure S2), we improved de-salting, giving us a cleaner deconvolved mass spectrum revealing several new low-abundant proteoforms, which we assign to tri-antennary N-glycans. Example structures with the correct oligosaccharide compositions are shown in cartoon form. It is interesting that the tri-antennary N-linked glycans seem to occur only on the proteoforms with 15 C- mannosylations. S-8

9 Glycan c Glycan d S-9

10 Glycan e HexNAc(5)Hex(6)Fuc(1)NeuAc(2 ) Figure S4. Annotated glycopeptide fragmentation spectra of properdin peptides with triantennary glycans. The interpretation of the small peaks in Figure S3 as proteoforms with triantennary N-glycans is supported by these MS/MS spectra from digested properdin. S-10

11 (a) (b) Figure S5. Annotated peptide fragmentation of Daclizumab revealing the GG clipping. Figure 4 of the main text shows peaks indicating hydrolysis at GG in the heavy chain CDR3. This interpretation is supported by HCD (a) and EThcD (b) MS/MS spectra from digested Daclizumab showing peptides with termini at the clip site. Spectrum (a) is from a sample digested with GluC and Trypsin, and (b) from a sample digested with AspN and Trypsin. S-11

12 (a) (b) Figure S6. Annotated peptide fragmentation spectra of Daclizumab showing part of the signal peptide. HCD (a) and EThcD (b) MS/MS spectra from digested samples also support the interpretation of the 340 Da mass delta as VHS (the C-terminal amino acid residues of the signal peptide) on the N-terminus of the heavy chain. S-12

13 Table S3. Observed glycoforms of the Cetuximab Fd fragment. The calculated masses in the fourth column assume pyro-glu N-terminus and two intact intrachain disulfide bonds. The glycan compositions show mostly fucosylated biantennary N-glycans with Gal-α-Gal termini. Of the masses above with assigned glycoforms, 22.8% by abundance include antennal fucose, misassigned in previous literature due to arithmetic errors. S-13