1. Volume 18, Issue 1, Pages 237-247 (January 2017)
Proteomic Analysis of Pemphigus Autoantibodies Indicates a Larger, More Diverse, and More Dynamic Repertoire than Determined by B Cell Genetics 
Jing Chen, Qi Zheng, Christoph M. Hammers, Christoph T. Ellebrecht, Eric M. Mukherjee, Hsin-Yao Tang, Chenyan Lin, Huijie Yuan, Meng Pan, Jana Langenhan, Lars Komorowski, Don L. Siegel, Aimee S. Payne, John R. Stanley 
Cell Reports 
Volume 18, Issue 1, Pages (January 2017)
DOI: /j.celrep
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2. Cell Reports 2017 18, 237-247DOI: (10.1016/j.celrep.2016.12.013)
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3. Figure 1
Proteomic Platform to Identify Circulating Pemphigus Anti-Dsg Abs
(A) IgG heavy chains from Dsg-binding abs and from abs that do not bind to Dsg are analyzed by LC-MS/MS. Resultant spectra are searched against a custom database of all VH sequences from the same patient to identify ab peptides. Informative peptides match H-CDR3 amino acid sequences that define clonotypes of abs.
(B) ELISA assays of PV or PF sera before and after affinity chromatography show that anti-Dsg ab is depleted by AP. PBMC, peripheral blood mononuclear cell; AP, affinity purification.
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4. Figure 2 Most of the Abs in Circulation Were Not Identified by APD
Each MS-identified ab is shown as a circle (peptide was detected in both bound and unbound fractions) or triangle (peptide was detected only in either bound or unbound fraction). A colored circle or triangle indicates that a particular ab is clonally related to one B cell clonotype identified by APD. In each patient, different APD clonoypes are indicated by different colors. APD clonotypes did not match any abs identified by MS in the PV1a, PV3, or PV16 samples.
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5. Figure 3
Recombinant scFv with VH Amino Acid Sequences Identified by MS from PV3a Serum Bind Dsg3
(A) Two scFv (GB4P4-7 and GB8P3-8) with different VH amino acid sequences as identified in MS, synthesized and isolated as described in Experimental Procedures, bind Dsg3 by ELISA.
(B) The same two scFvs bind to the cell surface of keratinocytes in monkey esophagus where Dsg3 locates. Scale bar, 10 μm.
(C) PV3a serum, but not normal human serum, inhibits binding of these scFvs to Dsg3 on ELISA.
See also Table S4.
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6. Figure 4
Characterization of Circulating Ab Clonotypes in PV and PF patients
(A and B) VH gene usage of clonotypes in PV patients (A) and PF patients (B).
(C) Intensity (as a measure of the amount of ab) of each of the 56 clonotypes found in PV3a. Arrows indicate the four “dominant” clones, which together produce more than 50% of total abs. Data are from four bound replicates. Error bar, SEM.
(D) Most clonotypes do not have variable H-CDR3 amino acid sequences.
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7. Figure 5
Anti-Dsg Ab and B Cell Genetic Clonotypes over Time in Three Patients
(A) Circulating ab clonotypes at each time point. “Ab repertoire” represents MS-identified abs at each time point and corresponds to the number of IgG clonotypes in Table 1; “B cell/ab repertoire” refers to both the ab detected at a particular time point by MS plus genetic ab sequences found at that time point identified by a pemphigus ab found at another time point by MS (i.e., NGS genetic sequences identified as pemphigus abs by matches to MS sequences of the affinity-purified pemphigus ab from a different time point). Many anti-Dsg B cell and circulating ab clones persist for many years.
(B) Intensity of circulating ab clones at different time points. Each column is one clonotype. Data indicate that the repertoire of circulating ab clonotypic expression is variable over time and that although some ab clonotypes may persist, their ab production varies over time.
See also Figures S2–S4 and Tables S5 and S6.
Cell Reports  , DOI: ( /j.celrep )
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