Retention Time Prediction for Glycopeptides in Reversed-Phase Chromatography for Glycoproteomic Applications.

The sequence-specific retention calculator algorithm (SSRCalc) [ Krokhin , O. V. Anal. Chem. 2006 , 78 , 7785 ] was adapted for the prediction of retention times of N-glycopeptides separated by reversed-phase high performance liquid chromatography (RPLC). The retention time shifts (dHI = HIglyco - HIdeglyco, where HI is the hydrophobicity index, measured in percent acetonitrile units) used for modeling were measured for 602 glycopeptides versus 123 of their deglycosylated analogues. Our method used a tryptic digest of 12 purified glycoproteins, glycopeptide enrichment, deglycosylation with PNGaseF, and RPLC-MS/MS analysis of combined (deglycosylated and intact) peptide mixtures. On average, glycosylation yields a 0.79% acetonitrile unit decrease in retention, compared with the hydrophobicity indices of their deglycosylated analogues. These values, however, are drastically different for asialo (-1.37% acetonitrile units), monosialylated (-0.47% acetonitrile units), disialylated (+0.61% acetonitrile units), and trisialylated (+1.94% acetonitrile units) glycans. Peptide retention time shifts upon glycosylation (dHI) vary depending on the number of monosaccharide units, the presence or absence of sialic acid, peptide hydrophobicity, and the number of position-dependent features. These features are mostly driven by competing effects of acidic residues (aspartic acid and sialic acid) on ion-pair formation and by nearest-neighbor effects of hydrophilic glycans. The accuracy of the modified prediction model for glycopeptides approaches that of the prediction for nonmodified species (R2 = 0.97 vs 0.98). However, retention time prediction based on the experimental retention values of deglycosylated analogues (HIglyco = HIdeglyco + dHI, R2 = 0.995) is much more accurate, thus providing a solid support for glycopeptide identification in complex samples based on mass and retention time.

Poor Patient and Graft Outcome After Induction Treatment by Antithymocyte Globulin in Recipients of a Kidney Graft After Nonrenal Organ Transplantation.

BACKGROUND: End-stage renal failure occurs in a substantial number of patients having received a nonrenal transplantation (NRT), for whom a kidney transplantation is needed. The medical strategy regarding the use of immunosuppression (IS) for a kidney graft in patients after an NRT is not well established. The prekidney grafts long-term IS advocates for a mild induction, such as using anti-IL-2R antibodies, whereas addition of new incompatibilities and anti-HLA preimmunization may suggest using stronger IS such as induction by polyclonal antithymocyte globulins (ATG). METHODS: We performed Cox multivariate and propensity score analysis of our validated transplant database to study the impact of the type of induction therapy on kidney graft survival of recipients of a kidney graft after NRT. RESULTS: We report here that kidney transplantation after NRT treated with an ATG induction has a poorer outcome (kidney and recipient survival) than that with an anti-IL-2R induction. After accounting for potential baseline differences with a multivariate Cox model, or by adjusting on a propensity score, we found that despite patients having received ATG cumulate more risk factors, ATG appears independently involved. As animal-derived biotherapeutics induce antiglycan antibodies and particularly anti-N-glycolylneuraminic acid (Neu5Gc) IgGs which may activate endothelial cells in patients and grafts, we also investigated the magnitude and the nature of the anti-Neu5Gc elicited by the induction and showed that induction was associated with a shift in anti-Neu5Gc IgG repertoire. Possible reasons and mechanisms of a deleterious ATG usage in these patients are discussed. CONCLUSIONS: Our study suggests that ATG induction after a kidney transplantation in recipients already under maintenance IS for a NRT should be used cautiously.

Absolute Quantitation of Glycoforms of Two Human IgG Subclasses Using Synthetic Fc Peptides and Glycopeptides.

Immunoglobulins, such as immunoglobulin G (IgG), are of prime importance in the immune system. Polyclonal human IgG comprises four subclasses, of which IgG1 and IgG2 are the most abundant in healthy individuals. In an effort to develop an absolute MALDI-ToF-MS quantitative method for these subclasses and their Fc N-glycoforms, (glyco)peptides were synthesized using a solid-phase approach and used as internal standards. Tryptic digest glycopeptides from monoclonal IgG1 and IgG2 samples were first quantified using EEQYN(GlcNAc)STYR and EEQFN(GlcNAc)STFR standards, respectively. For IgG1, a similar glycopeptide where tyrosine (Y) was isotopically labelled was used to quantify monoclonal IgG1 that had been treated with the enzyme Endo-F2, i.e., yielding tryptic glycopeptide EEQYN(GlcNAc)STYR. The next step was to quantify single subclasses within polyclonal human IgG samples. Although ion abundances in the MALDI spectra often showed higher signals for IgG2 than IgG1, depending on the spotting solvent used, determination of amounts using the newly developed quantitative method allowed to obtain accurate concentrations where IgG1 species were predominant. It was observed that simultaneous analysis of IgG1 and IgG2 yielded non-quantitative results and that more success was obtained when subclasses were quantified one by one. More experiments served to assess the respective extraction and ionization efficiencies of EEQYNSTYR/EEQFNSTFR and EEQYN(GlcNAc)STYR/EEQFN(GlcNAc)STFR mixtures under different solvent and concentration conditions. Graphical Abstract ᅟ.

Rabbit antithymocyte globulin-induced serum sickness disease and human kidney graft survival.

BACKGROUND: Rabbit-generated antithymocyte globulins (ATGs), which target human T cells, are widely used as immunosuppressive agents during treatment of kidney allograft recipients. However, ATGs can induce immune complex diseases, including serum sickness disease (SSD). Rabbit and human IgGs have various antigenic differences, including expression of the sialic acid Neu5Gc and α-1-3-Gal (Gal), which are not synthesized by human beings. Moreover, anti-Neu5Gc antibodies have been shown to preexist and be elicited by immunization in human subjects. This study aimed to assess the effect of SSD on long-term kidney allograft outcome and to compare the immunization status of grafted patients presenting with SSD following ATG induction treatment. METHODS: We analyzed data from a cohort of 889 first kidney graft recipients with ATG induction (86 with SSD [SSD(+)] and 803 without SSD [SSD(-)]) from the Données Informatisées et Validées en Transplantation data bank. Two subgroups of SSD(+) and SSD(-) patients that had received ATG induction treatment were then assessed for total anti-ATG, anti-Neu5Gc, and anti-Gal antibodies using ELISA assays on sera before and after transplantation. RESULTS: SSD was significantly associated with long-term graft loss (>10 years, P = 0.02). Moreover, SSD(+) patients exhibited significantly elevated titers of anti-ATG (P = 0.043) and anti-Neu5Gc (P = 0.007) IgGs in late post-graft samples compared with SSD(-) recipients. CONCLUSION: In conclusion, our data indicate that SSD is a major contributing factor of late graft loss following ATG induction and that anti-Neu5Gc antibodies increase over time in SSD(+) patients. FUNDING: This study was funded by Société d'Accélération du Transfert de Technologies Ouest Valorisation, the European FP7 "Translink" research program, the French National Agency of Research, Labex Transplantex, the Natural Science and Engineering Research Council of Canada, and the Canadian Foundation for Innovation.