Changes in mean contribution of the glycan group GP1 following zebularine treatment and recovery.

<p>The observed changes were normalized to control values, which were set to 100%. The error bars represent standard deviations obtained on the basis of 4 independent experiments.</p

Mannosidase treatment of glycans released from enzymatically untreated HeLa cells.

<p>Column height of each individual glycan group is normalized to the highest one, set to 100 (GP7). Gray column (GP11) represents an internal standard (2-aminobenzamide labeled dextran peak) added in order to equalize and normalize peak signals, correctly interpret background signals and verify the composition and percentages of oligomannose structures within these peaks.</p

Changes in mean contributions of glycan groups GP1, GP5 and GP10 following TSA treatment and recovery.

<p>The observed changes were normalized to control values, which were set to 100%. The error bars represent standard deviations obtained on the basis of 4 independent experiments.</p

Changes in mean contributions of glycan groups GP1, GP5 and GP7 following Na-butyrate treatment and recovery.

<p>The observed changes were normalized to control values, which were set to 100%. The error bars represent standard deviations obtained on the basis of 3 independent experiments.</p

Composition of membrane and cell lysate <i>N</i>-glycomes.

<p><b>A.</b> Glycans released from either HeLa cells embedded in polyacrylamide gels (H) or HeLa cell lysates (HL) were labeled with 2-AB and separated by HPLC. The histogram shows the partition (percentage + SD) of individual glycan fractions (GP1 – GP12) of HPLC separated glycome. <b>B.</b> Coefficients of variation (%CV) of individual glycan peaks in HPLC separated glycome of HeLa cells embedded in polyacrylamide gels (H) and HeLa cell lysates (HL) from 6 identical experiments.</p

Enzymatically untreated HeLa cells embedded in polyacrylamide gels release glycan structures.

<p>Confocal images of HeLa cells embedded in polyacrylamide gel. The peripheral cellular glycans are stained with Ricinus Communis Agglutinin I. Scale bar is 5 µm.</p

LaCyTools: A Targeted Liquid Chromatography–Mass Spectrometry Data Processing Package for Relative Quantitation of Glycopeptides

Bottom-up glycoproteomics by liquid chromatography–mass spectrometry (LC–MS) is an established approach for assessing glycosylation in a protein- and site-specific manner. Consequently, tools are needed to automatically align, calibrate, and integrate LC–MS glycoproteomics data. We developed a modular software package designed to tackle the individual aspects of an LC–MS experiment, called LaCyTools. Targeted alignment is performed using user defined <i>m</i>/<i>z</i> and retention time (<i>t</i>_r) combinations. Subsequently, sum spectra are created for each user defined analyte group. Quantitation is performed on the sum spectra, where each user defined analyte can have its own <i>t</i>_r, minimum, and maximum charge states. Consequently, LaCyTools deals with multiple charge states, which gives an output per charge state if desired, and offers various analyte and spectra quality criteria. We compared throughput and performance of LaCyTools to combinations of available tools that deal with individual processing steps. LaCyTools yielded relative quantitation of equal precision (relative standard deviation <0.5%) and higher trueness due to the use of MS peak area instead of MS peak intensity. In conclusion, LaCyTools is an accurate automated data processing tool for high-throughput analysis of LC–MS glycoproteomics data. Released under the Apache 2.0 license, it is freely available on GitHub (https://github.com/Tarskin/LaCyTools)

DataSheet_1_Effects of low-calorie and different weight-maintenance diets on IgG glycome composition.docx

Obesity-induced inflammation activates the adaptive immune system by altering immunoglobulin G (IgG) glycosylation in a way to produce more proinflammatory antibodies. The IgG glycome has already been well studied, and its alterations are correlated with a high body mass index (BMI) and central adiposity. Still, the IgG N-glycome susceptibility to different dietary regimes for weight control after the initial weight loss has not been studied. To explore changes in IgG glycosylation induced by weight loss and subsequent weight-maintenance diets, we analyzed 1,850 IgG glycomes from subjects in a dietary intervention Diogenes study. In this study, participants followed a low-calorie diet (LCD) providing 800 kcal/d for 8 weeks, followed by one of five weight-maintenance diets over a 6-month period. The most significant alteration of the IgG N-glycome was present 8 weeks after the subjects underwent an LCD, a statistically significant decrease of agalactosylated and the increase of sialylated N glycans. In the follow-up period, the increase in glycans with bisecting GlcNAc and the decrease in sialylated glycans were observed. Those changes were present regardless of the diet type, and we did not observe significant changes between different diets. However, it should be noted that in all five diet groups, there were individuals who prominently altered their IgG glycome composition in either proinflammatory or anti-inflammatory directions.</p

High-Throughput IgG Fc N‑Glycosylation Profiling by Mass Spectrometry of Glycopeptides

Age and sex dependence of subclass specific immunoglobulin G (IgG) Fc <i>N</i>-glycosylation was evaluated for 1709 individuals from two isolated human populations. IgGs were obtained from plasma by affinity purification using 96-well protein G monolithic plates and digested with trypsin. Fc <i>N</i>-glycopeptides were purified and analyzed by negative-mode MALDI-TOF-MS with 4-chloro-α-cyanocinnamic acid (Cl-CCA) matrix. Age-associated glycosylation changes were more pronounced in younger individuals (<57 years) than in older individuals (>57 years) and in females than in males. Galactosylation and sialylation decreased with increasing age and showed significant sex dependence. Interestingly, the most prominent drop in the levels of galactosylated and sialylated glycoforms in females was observed around the age of 45 to 60 years when females usually enter menopause. The incidence of bisecting <i>N</i>-acetylglucosamine increased in younger individuals and reached a plateau at older age. Furthermore, we compared the results to the total IgG <i>N</i>-glycosylation of the same populations recently analyzed by hydrophilic interaction liquid chromatography (HILIC). Significant differences were observed in the levels of galactosylation, bisecting <i>N</i>-acetylglucosamine and particularly sialylation, which were shown to be higher in HILIC analysis. Age and sex association of glycosylation features was, to a large extent, comparable between MALDI-TOF-MS and HILIC IgG glycosylation profiling

Test of sample preparation.

<p>The activity of the PainOmics samples was analysed by centre (points represent the average activity for each patient (<i>n</i> = 4). These data indicate the homogeneity of serum sample preparation for our analyses. The presence of several outliers from different centres suggest some variation exists between patient samples for this marker.</p