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{"id":56,"date":"2014-12-04T00:09:14","date_gmt":"2014-12-03T22:09:14","guid":{"rendered":"http:\/\/protpi.ch\/blog\/?p=56"},"modified":"2021-03-15T17:30:00","modified_gmt":"2021-03-15T15:30:00","slug":"maldi-tof-ms-analysis-of-antibody-antigen-complexes-2","status":"publish","type":"post","link":"https:\/\/www.protpi.ch\/blog\/bioanalytics\/2014\/12\/maldi-tof-ms-analysis-of-antibody-antigen-complexes-2\/","title":{"rendered":"MALDI-TOF-MS analysis of antibody-antigen complexes"},"content":{"rendered":"<p style=\"text-align: right;\"><strong>Editor:<\/strong><br \/>\nProf. Dr. Christiane Zaborosch<\/p>\n<address style=\"text-align: right;\">Head of <a title=\"Center of Biochemistry\" href=\"http:\/\/icbc.zhaw.ch\/en\/lsfm\/institutes-centres\/institute-of-chemistry-and-biological-chemistry\/centers\/biochemistry.html\" target=\"_blank\" rel=\"noopener\">Center for Biochemistry<\/a><br \/>\nLecturer in Biochemistry<br \/>\nchristiane.zaborosch@zhaw.ch<\/address>\n<hr \/>\n<h2>\u00a0Introduction<\/h2>\n<p>In the development of new drugs, it is of interest in pharmaceutical research to determine the size of antibody-antigen complexes that have formed, since it is assumed that large complexes are cleared more quickly by the body, and thus the antigen is removed faster. Antibody-antigen complexes (mAb-Ag) can be analysed using MALDI-TOF-MS. However, non-covalent complexes are difficult to detect using MALDI-TOF-MS without sample preparation, as dissociation of the complex occurs during laser irradiation and co-crystallization with the matrix. In order to prevent dissociation during the MALDI process, chemical stabilization can be carried out by means of covalent cross-linking. For cross-linking of the antibodies with the antigen a homobifunctional, amine-reactive cross-linker BS(PEG)<sub>5<\/sub> was employed (spacer: 21.7 \u00c5). The crosslinker reacts with the free N-terminus and the \u03b5-amino group of lysine side chains of the proteins. The nucleophilic N atom of the protein&#8217;s primary amine breaks down the carbonyl group of the cross-linker and an amide bond is formed by nucleophilic substitution. To analyse the major cross-linked protein complexes (&gt;\u00a0150\u00a0kDa) with MALDI-TOF-MS, an HM1 high-mass ion conversion detector from CovalX was used.<\/p>\n<h2>Binding experiment with one mAb<\/h2>\n<p>To study the mAb-Ag complexes, a model system with IL-1\u03b2 (Ag) and two anti-human IL-1\u03b2 monoclonal antibodies XO01_\u03b32 and BS01 (mAb) was used. In the binding experiment XO01_\u03b32 or BS01 and IL-1\u03b2 were mixed at the ratio of 1:2. The complexes formed from Ag and mAb were crosslinked through the addition of BS(PEG)<sub>5<\/sub> in a 56-fold molar excess.<\/p>\n<figure id=\"attachment_57\" aria-describedby=\"caption-attachment-57\" style=\"width: 236px\" class=\"wp-caption alignnone\"><a href=\"http:\/\/protpi.ch\/blog\/wp-content\/uploads\/2014\/12\/MALDI_1.png\"><img loading=\"lazy\" decoding=\"async\" class=\"wp-image-57 size-medium\" src=\"http:\/\/protpi.ch\/blog\/wp-content\/uploads\/2014\/12\/MALDI_1-236x300.png\" alt=\"MALDI-Spectra\" width=\"236\" height=\"300\" srcset=\"https:\/\/www.protpi.ch\/blog\/wp-content\/uploads\/2014\/12\/MALDI_1-236x300.png 236w, https:\/\/www.protpi.ch\/blog\/wp-content\/uploads\/2014\/12\/MALDI_1-193x245.png 193w, https:\/\/www.protpi.ch\/blog\/wp-content\/uploads\/2014\/12\/MALDI_1.png 362w\" sizes=\"auto, (max-width: 236px) 100vw, 236px\" \/><\/a><figcaption id=\"caption-attachment-57\" class=\"wp-caption-text\">Figure 1: High-mass MALDI mass spectra of the binding experiment of IL-1\u03b2 and the mAb XO01_\u03b32. Ratio 1:2 mAb:Ag. A: before addition of the crosslinkers (control); B: after addition of BS(PEG)5, masses after removal of the crosslinker. Matrix: sinapic acid; Detector: HM1<\/figcaption><\/figure>\n<p>In the control spectrum (Fig. 1A) the peaks of the antigen IL-1\u03b2 occurred at 17.4 kDa and those of the mAb XO01_\u03b32 at 146.3\u00a0kDa. The peaks at 163.3\u00a0kDa and 180.3\u00a0kDa corresponded to complexes without crosslinking. In the spectrum after crosslinking (Fig. 1B), two very high intensity peaks were observed at 163.4\u00a0kDa and 180.2\u00a0kDa, which could be attributed to the [XO01_\u03b32*<strong>1<\/strong>IL-1\u03b2] and [XO01_\u03b32*<strong>2<\/strong>IL-1\u03b2] complexes. The mass of the crosslinker was subtracted from the calculated masses of the complexes. BS01 was also crosslinked with IL-1\u03b2 and analysed (data not shown).<\/p>\n<h2>Multi-binding experiment with two mAbs<\/h2>\n<p>In the multi-binding experiment larger mAb-Ag complexes were generated and the exact masses determined using MALDI-TOF MS. The two mAbs which are directed against different epitopes were mixed with IL-1\u03b2 in a ratio of 1:2:1 (mAb1:Ag:mAb2) and then crosslinked with the crosslinker BS(PEG)<sub>5<\/sub> in a 56-fold molar excess.<\/p>\n<figure id=\"attachment_61\" aria-describedby=\"caption-attachment-61\" style=\"width: 300px\" class=\"wp-caption alignnone\"><a href=\"http:\/\/protpi.ch\/blog\/wp-content\/uploads\/2014\/12\/MALDI_2.png\"><img loading=\"lazy\" decoding=\"async\" class=\"size-medium wp-image-61\" src=\"http:\/\/protpi.ch\/blog\/wp-content\/uploads\/2014\/12\/MALDI_2-300x165.png\" alt=\"MALDI-Spectrum\" width=\"300\" height=\"165\" srcset=\"https:\/\/www.protpi.ch\/blog\/wp-content\/uploads\/2014\/12\/MALDI_2-300x165.png 300w, https:\/\/www.protpi.ch\/blog\/wp-content\/uploads\/2014\/12\/MALDI_2-400x220.png 400w, https:\/\/www.protpi.ch\/blog\/wp-content\/uploads\/2014\/12\/MALDI_2.png 449w\" sizes=\"auto, (max-width: 300px) 100vw, 300px\" \/><\/a><figcaption id=\"caption-attachment-61\" class=\"wp-caption-text\">Figure 2: High-mass MALDI mass spectrum of the multi-binding experiment of IL-1\u03b2 and the mAb XO01_\u03b32 and BS01 after crosslinking with BS(PEG)5 at a ratio of 1:2:1 mAb1:Ag:mAb2. Masses shown after removal of the crosslinker. Matrix: sinapic acid; Detector: HM1<\/figcaption><\/figure>\n<p>On the basis of the molecular masses, the peaks in the spectrum (Fig. 2) were attributed to the various mAb-Ag complexes (Tab. 1). Uncomplexed antigen was not detected; IL-1 \u03b2 was therefore fully bound into complexes.<\/p>\n<figure id=\"attachment_63\" aria-describedby=\"caption-attachment-63\" style=\"width: 300px\" class=\"wp-caption alignnone\"><a href=\"https:\/\/www.protpi.ch\/blog\/wp-content\/uploads\/2014\/12\/Table_Complexes.png\"><img loading=\"lazy\" decoding=\"async\" class=\"size-medium wp-image-63\" src=\"https:\/\/www.protpi.ch\/blog\/wp-content\/uploads\/2014\/12\/Table_Complexes-300x158.png\" alt=\"Table 1\" width=\"300\" height=\"158\" srcset=\"https:\/\/www.protpi.ch\/blog\/wp-content\/uploads\/2014\/12\/Table_Complexes-300x158.png 300w, https:\/\/www.protpi.ch\/blog\/wp-content\/uploads\/2014\/12\/Table_Complexes-400x211.png 400w, https:\/\/www.protpi.ch\/blog\/wp-content\/uploads\/2014\/12\/Table_Complexes.png 429w\" sizes=\"auto, (max-width: 300px) 100vw, 300px\" \/><\/a><figcaption id=\"caption-attachment-63\" class=\"wp-caption-text\">Table 1: Allocation of the mAb-Ag complexes formed<\/figcaption><\/figure>\n<h2>Conclusion<\/h2>\n<p>Using the crosslinker BS(PEG)<sub>5<\/sub> the two mAbs XO01_\u03b32 and BS01 were covalently crosslinked with the antigen IL-1\u03b2 and the resulting mAb-Ag-complexes were detected using MALDI-TOF-MS.<\/p>\n<p>[ratings]<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Editor: Prof. Dr. Christiane Zaborosch Head of Center for Biochemistry Lecturer in Biochemistry christiane.zaborosch@zhaw.ch \u00a0Introduction In the development of new drugs, it is of interest in pharmaceutical research to determine the size of antibody-antigen complexes that have formed, since it is assumed that large complexes are cleared more quickly by the body, and thus the [&hellip;]<\/p>\n","protected":false},"author":3,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[25],"tags":[11,12,6,9,27],"class_list":["post-56","post","type-post","status-publish","format-standard","hentry","category-bioanalytics","tag-antibody","tag-binding","tag-bioanalytics","tag-maldi","tag-mass-spectrometry"],"jetpack_featured_media_url":"","_links":{"self":[{"href":"https:\/\/www.protpi.ch\/blog\/wp-json\/wp\/v2\/posts\/56","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.protpi.ch\/blog\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.protpi.ch\/blog\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.protpi.ch\/blog\/wp-json\/wp\/v2\/users\/3"}],"replies":[{"embeddable":true,"href":"https:\/\/www.protpi.ch\/blog\/wp-json\/wp\/v2\/comments?post=56"}],"version-history":[{"count":12,"href":"https:\/\/www.protpi.ch\/blog\/wp-json\/wp\/v2\/posts\/56\/revisions"}],"predecessor-version":[{"id":549,"href":"https:\/\/www.protpi.ch\/blog\/wp-json\/wp\/v2\/posts\/56\/revisions\/549"}],"wp:attachment":[{"href":"https:\/\/www.protpi.ch\/blog\/wp-json\/wp\/v2\/media?parent=56"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.protpi.ch\/blog\/wp-json\/wp\/v2\/categories?post=56"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.protpi.ch\/blog\/wp-json\/wp\/v2\/tags?post=56"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}