Anti-citrullinated protein antibodies acquire a pro-inflammatory Fc glycosylation phenotype prior to the onset of rheumatoid arthritis.

OBJECTIVE: To determine whether anticitrullinated protein antibodies (ACPA) exhibit specific changes in Fc glycosylation prior to the onset of arthritis. METHODS: Serum samples of patients with ACPA-positive arthralgia (n=183) were collected at baseline and at various time points of follow-up. 105 patients developed arthritis after a median of 12 months (IQR 6-24) and were classified as having either rheumatoid arthritis (RA, n=48) or undifferentiated arthritis (UA, n=57) based on the 1987 American College of Rheumatology (ACR) criteria. ACPA and total serum IgG were isolated by affinity purification and cleaved by trypsin. ACPA-IgG1 Fc-glycopeptides were subsequently analysed by nano-liquid chromatography mass spectrometry and compared to those of total IgG1. RESULTS: At baseline, ACPA-IgG1 and total IgG1 from arthralgia patients displayed similar Fc glycosylation patterns. By contrast, at the onset of arthritis, ACPA exhibited a decrease in galactose residues in RA patients, but not in UA patients. This decrease occurred around 3 months prior to diagnosis and was paralleled by an increase in systemic inflammation (erythrocyte sedimentation rate). Galactosylation of total IgG1 was also decreased in RA, but this did not precede the onset of arthritis. Interestingly, we additionally noted a higher degree of ACPA-IgG1 Fc core fucosylation at baseline as compared with total IgG1, which further increased prior to diagnosis. CONCLUSIONS: ACPA display significant changes in Fc galactosylation and fucosylation prior to the onset of RA. These changes towards a more pro-inflammatory phenotype could be involved in driving the disease process.

Determination of trehalose-6-phosphate in Arabidopsis seedlings by successive extractions followed by anion exchange chromatography-mass spectrometry.

A method for the detection of trehalose-6-phosphate (T6P) in tissue of the model plant Arabidopsis thaliana is presented. Liquid-liquid extraction (LLE) and mixed mode solid-phase extraction (SPE) were used for sample pretreatment followed by anion exchange chromatography (AEC) coupled with electrospray ionization mass spectrometry (MS) for highly selective quantitative analysis. LLE of plant material was performed with chloroform/acetonitrile/water (3:7:16, v/v/v) followed by SPE with Oasis MAX material, which significantly reduced the complexity of the extracts. On-line coupling of MS with gradient AEC using a sodium hydroxide eluent was accomplished with a postcolumn ion suppressor. The method allows specific quantification of T6P with good linearity for spiked plant extracts, from 80 nM to 1.3 microM (r(2)>0.98). The limit of detection in plant extracts was 40 nM. The recovery of the method was above 80% for relevant T6P levels. The method was applied to the determination of T6P in seedlings from four mutant A. thaliana lines (TRR1-4) resisting growth arrest caused by external supply of trehalose. Results reveal that T6P accumulation differed substantially in the four mutant lines and wild type (WT). It is concluded that the mutants circumvent the growth arrest observed in WT seedlings on 100mM trehalose by different mechanisms.