LC MS/MS-CID showing hexosylation at Thr64 of the peptide Gt₆₄KDFLPIELQSLEVSK of MYPU_3230.

<p>The assigned b and y ions are shown in blue and red, respectively. Glycosylation of Q and S glycosites is absent in this spectrum as illustrated. The PEAKS peptide score (-10lgP) for this spectrum was 87. The charge state of the parental ion was <i>z</i> = 3.</p

LC MS/MS-CID showing hexosylation at Asn335 of the peptide STLEYTINNSQELQn₃₃₅ILKQTYEEFTK of MYPU_3200.

<p>The assigned b and y ions are shown in blue and red, respectively. Glycosylation of N, Q, T, S, and Y glycosites is absent in this spectrum as illustrated. The PEAKS peptide score (-10lgP) for this spectrum was 60. The charge state of the parental ion was <i>z</i> = 3.</p

Hexosylation of the peptide STLEYTINNSQELQNILKQTYEEFTK of MYPU_3200.

<p>Orbitrap MS showing the doubly and triply charged ions. The monoisotopic mass of the doubly charged species at 1648.8077 is consistent with the hexosylated peptide at <i>z</i> = 2 with a mass accuracy of 0.0012 Da. The 54.0175 shift for <i>z</i> = 3 between non-glycosylated and hexose forms equates to a mass shift of 162.0525 Da with a mass accuracy of 0.0003 Da. Monoisotopic values for the calculated theoretical and experimental masses of the peptide are given in bold. The images presented were obtained from an LC peak of MS scans and are expanded to show the charge states of each form.</p

Hexosylation of the peptide GTKDFLPIELQSLEVSK of MYPU_3230.

<p>Orbitrap MS showing the doubly and triply charged ions. The 81.0262 shift for <i>z</i> = 2 between the non-glycosylated and glycosylated peptides equates to a mass shift of 162.0524 Da, which corresponds to the addition of hexose (162.0528 Da) with a mass accuracy of 0.0004 Da. The 54.0169 shift for <i>z</i> = 3 between non-glycosylated and glycosylated forms equates to a mass shift of 162.0507 Da, which corresponds to hexosylation with a mass accuracy of 0.0021 Da. Monoisotopic values for the calculated theoretical and experimental masses of the peptide are given in bold. The images presented were obtained from an LC peak of MS scans and are expanded to show the charge states of each form.</p

The effect of substrate on glycoconjugate synthesis by <i>M</i>. <i>pulmonis</i> and <i>M</i>. <i>pneumoniae</i>.

<p>Panels A and B show the relative abundance of glucose or xylose, respectively, linked to protein as determined by GC. The values represent averages of the areas under the curves from 3 replicates of gas chromatograms that were converted to μg of sugar per mg of protein. The colors of the bars representing the different substrates used to supplement the medium are shown on the right. Plus or minus standard error bars are shown and the asterisks indicate a significant difference between a sample and control.</p

Glycosites identified in this study.

<p>^a Accession numbers for MYPU_3200, MYPU_3230, MYPU_3460, and MARTH_403 are CAC13493, CAC13496, CAC13519, and YP_001999972, respectively.</p><p>Glycosites identified in this study.</p

LC MS/MS-CID showing hexosylation at Gln49 of the peptide ITDLLSq₄₉KEVTETQK of MYPU_3460.

<p>The assigned b and y ions are shown in blue and red, respectively. Glycosylation of Q, S, and T glycosites is absent in this spectrum as illustrated. The PEAKS peptide score (-10lgP) for this spectrum was 74. The charge state of the parental ion was <i>z</i> = 3.</p

Hexosylation of the peptide ITDLLSQKEVTETQK of MYPU_3460.

<p>Orbitrap MS showing the doubly and triply charged ions. The 81.027 shift for <i>z</i> = 2 between the non-glycosylated and glycosylated peptides equates to a mass shift of 162.054 Da, which corresponds to the addition of hexose (162.0528 Da) with a mass accuracy of 0.0012 Da. The 54.0177 shift for <i>z</i> = 3 between non-glycosylated and glycosylated forms equates to a mass shift of 162.0531 Da, which corresponds to hexosylation with a mass accuracy of 0.0003 Da. The calculated theoretical and experimental values for <i>m/z</i> are given in bold. The images presented were obtained from an LC peak of MS scans and are expanded to show the charge states of each form.</p

Naturally Occurring Structural Isomers in Serum IgA1 <i>O</i>-Glycosylation

IgA is the most abundantly produced antibody and plays an important role in the mucosal immune system. Human IgA is represented by two isotypes, IgA1 and IgA2. The major structural difference between these two subclasses is the presence of nine potential sites of <i>O</i>-glycosylation in the hinge region between the first and second constant region domains of the heavy chain. Thr²²⁵, Thr²²⁸, Ser²³⁰, Ser²³² and Thr²³⁶ have been identified as the predominant sites of <i>O</i>-glycan attachment. The range and distribution of <i>O</i>-glycan chains at each site within the context of adjacent sites in this clustered region create a complex heterogeneity of surface epitopes that is incompletely defined. We previously described the analysis of IgA1 <i>O</i>-glycan heterogeneity by use of high resolution LC–MS and electron capture dissociation tandem MS to unambiguously localize all amino acid attachment sites in IgA1 (Ale) myeloma protein. Here, we report the identification and elucidation of IgA1 <i>O</i>-glycopeptide structural isomers that occur based on amino acid position of the attached glycans (positional isomers) and the structure of the <i>O</i>-glycan chains at individual sites (glycan isomers). These isomers are present in a model IgA1 (Mce1) myeloma protein and occur naturally in normal human serum IgA1. Variable <i>O</i>-glycan chains attached to Ser²³⁰, Thr²³³ or Thr²³⁶ produce the predominant positional isomers, including <i>O</i>-glycans composed of a single GalNAc residue. These findings represent the first definitive identification of structural isomeric IgA1 <i>O</i>-glycoforms, define the single-site heterogeneity for all <i>O</i>-glycan sites in a single sample, and have implications for defining epitopes based on clustered <i>O</i>-glycan variability

Serum galactose-deficient-IgA1 and IgG autoantibodies correlate in patients with IgA nephropathy

<div><p>IgA nephropathy is an autoimmune disease characterized by IgA1-containing glomerular immune deposits. We previously proposed a multi-hit pathogenesis model in which patients with IgA nephropathy have elevated levels of circulatory IgA1 with some <i>O</i>-glycans deficient in galactose (Gd-IgA1, autoantigen). Gd-IgA1 is recognized by anti-glycan IgG and/or IgA autoantibodies, resulting in formation of pathogenic immune complexes. Some of these immune complexes deposit in the kidney, activate mesangial cells, and incite glomerular injury leading to clinical presentation of IgA nephropathy. Several studies have demonstrated that elevated circulatory levels of either Gd-IgA1 or the corresponding autoantibodies predict progressive loss of renal clearance function. In this study we assessed a possible association between serum levels of Gd-IgA1 and IgG or IgA autoantibodies specific for Gd-IgA1 in serum samples from 135 patients with biopsy-proven IgA nephropathy, 76 patients with other renal diseases, and 106 healthy controls. Our analyses revealed a correlation between the concentrations of the autoantigen and the corresponding IgG autoantibodies in sera of patients with IgA nephropathy, but not of disease or healthy controls. Moreover, our data suggest that IgG is the predominant isotype of Gd-IgA1-specific autoantibodies in IgA nephropathy. This work highlights the importance of both initial hits in the pathogenesis of IgA nephropathy.</p></div