Characterization of the Small Intestinal Lesion in Celiac Disease by Label-Free Quantitative Mass Spectrometry.

Global characterization of tissue proteomes from small amounts of biopsy material has become feasible because of advances in mass spectrometry and bioinformatics tools. In celiac disease (CD), dietary gluten induces an immune response that is accompanied by pronounced remodeling of the small intestine. Removal of gluten from the diet abrogates the immune response, and the tissue architecture normalizes. In this study, differences in global protein expression of small intestinal biopsy specimens from CD patients were quantified by analyzing formalin-fixed, paraffin-embedded material using liquid chromatography-mass spectrometry and label-free protein quantitation. Protein expression was compared in biopsy specimens collected from the same patients before and after 1-year treatment with gluten-free diet (n = 10) or before and after 3-day gluten provocation (n = 4). Differential expression of proteins in particular from mature enterocytes, neutrophils, and plasma cells could distinguish untreated from treated CD mucosa, and Ig variable region IGHV5-51 expression was found to serve as a CD-specific marker of ongoing immune activation. In patients who had undergone gluten challenge, coordinated up-regulation of wound response proteins, including the CD autoantigen transglutaminase 2, was observed. Our study provides a global and unbiased assessment of antigen-driven changes in protein expression in the celiac intestinal mucosa.

Intrathecal BCR transcriptome in multiple sclerosis versus other neuroinflammation: Equally diverse and compartmentalized, but more mutated, biased and overlapping with the proteome.

The mechanisms driving the intrathecal synthesis of IgG in multiple sclerosis (MS) are unknown. We combined high-throughput sequencing of transcribed immunoglobulin heavy-chain variable (IGHV) genes and mass spectrometry to chart the diversity and compartmentalization of IgG-producing B cells in the cerebrospinal fluid (CSF) of MS patients and controls with other neuroinflammatory diseases. In both groups, a few clones dominated the intrathecal IGHV transcriptome. In most MS patients and some controls, dominant transcripts matched the CSF IgG. The IGHV transcripts in CSF of MS patients frequently carried IGHV4 genes and had more replacement mutations compared to controls. In both groups, dominant IGHV transcripts were identified within clusters of clonally related B cells that had identical or related IGHV transcripts in the blood. These findings suggest more pronounced affinity maturation, but an equal degree of diversity and compartmentalization of the intrathecal B-cell response in MS compared to other neuroinflammatory diseases.

Assessing the citrullinome in rheumatoid arthritis synovial fluid with and without enrichment of citrullinated peptides.

Protein citrullination is a posttranslational modification that has attracted increased attention, especially for its involvement in rheumatoid arthritis (RA). Here, we assess the citrullinome in RA synovial fluid by direct LC-MS/MS analysis and by the use of an enrichment strategy based on citrulline specific biotinylation. RA synovial fluid was depleted for abundant proteins, and total and depleted fractions were analyzed. Frequency of citrullinated peptides and their degree of citrullination could be determined for four known RA autoantigens, as well as a novel in vivo autocitrullination site of peptidylarginine deiminase 4. From the analysis of total and depleted synovial fluid after enrichment we could estimate the numbers of citrullinated peptides to be approximately 3600 and 2100, respectively. However, identification of these biotinylated peptides by MS/MS turned out to be very difficult due to fragmentation of the biotin moiety. By direct MS analysis of the total and depleted synovial fluid without enrichment, 119 and 157 citrullinated peptides were identified, respectively. This indicates that direct analysis allows identification of only a fraction of the citrullinated proteins present in synovial fluid and that specific enrichment is still needed for a comprehensive in-depth elucidation of the citrullinome.

Specific biotinylation and sensitive enrichment of citrullinated peptides.

Protein citrullination is a posttranslational modification where peptidylarginine is enzymatically deiminated to form peptidylcitrulline. Although the role of protein citrullination in both health and disease is being increasingly recognised, techniques available to identify citrullinated proteins and to map their citrullination site(s) are rare and often show poor sensitivity. Here, we present a sensitive technique for specific modification and selective enrichment of citrullinated peptides from complex biological samples. The technique is based on highly specific in-solution biotinylation of citrulline residues followed by selective enrichment of modified peptides using streptavidin beads. We demonstrate that a synthetic citrulline-containing peptide can be selectively enriched when less than 0.5 pmol is spiked into a highly heterogeneous peptide mixture. After enrichment, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of an aliquot of the streptavidin eluate corresponding to theoretically 50 fmol of the spiked-in peptide showed a prominent signal. We further demonstrate the sensitivity of our technique by enrichment of citrullinated peptides from enzymatically deiminated myelin basic protein (MBP), when 10 pmol was spiked into a heterogeneous biological digest. In MALDI-TOF MS analysis, six MBP-derived citrullinated peptides were observed, showing the efficiency of this enrichment strategy. The high sensitivity combined with the remarkable specificity of the described technique makes it a valuable tool for elucidating citrullination in various biological processes.

A technique for the specific enrichment of citrulline-containing peptides.

Protein citrullination results from enzymatic deimination of peptidylarginine and plays an important role in health and disease. Despite increasing scientific interest, the identity and function of citrullinated proteins in vivo remain widely unknown. Thorough proteomic studies could contribute to a better understanding of the role of this posttranslational modification but will require tools for enrichment of citrullinated polypeptides. This study presents a simple technique for a highly specific enrichment of citrullinated peptides that is based on the specific reaction of glyoxal derivatives with the citrulline ureido group under acidic conditions. Beads were functionalized with 4-hydroxyphenylglyoxal attached via a base-labile linker. Incubation of these "citrulline reactive beads" with peptide mixtures at low pH resulted in selective immobilization of citrullinated peptides. Unbound noncitrullinated peptides were removed by extensive washing. Finally, citrullinated peptides carrying a modified ureido group were cleaved off at high pH and were analyzed by mass spectrometry. The procedure was validated by enrichment of synthetic citrulline-containing peptides from a tryptic digest of bovine serum albumin and from an endoproteinase LysC digest of a cytosolic fraction of a cell line. The technique was further applied to enrich citrullinated peptides from a digest of deiminated myelin basic protein.