IntroductionRheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation of the joints and the presence of autoantibodies directed against proteins containing the non-standard arginine-derived amino acid citrulline. The protein fibrinogen, which has an essential role in blood clotting, is one of the most prominent citrullinated autoantigens in RA, particularly because it can be found in the inflamed tissue of affected joints. Here, we set out to analyze the presence of citrullinated endogenous peptides in the synovial fluid of RA and arthritic control patients.MethodsEndogenous peptides were isolated from the synovial fluid of RA patients and controls by filtration and solid phase extraction. The peptides were identified and quantified using high-resolution liquid chromatography-mass spectrometry.ResultsOur data reveal that the synovial fluid of RA patients contains soluble endogenous peptides, derived from fibrinogen, containing significant amounts of citrulline residues and, in some cases, also phosphorylated serine. Several citrullinated peptides are found to be more abundantly present in the synovial fluid of RA patients compared to patients suffering from other inflammatory diseases affecting the joints.ConclusionsThe increased presence of citrullinated peptides in RA patients points toward a possible specific role of these peptides in the immune response at the basis of the recognition of citrullinated peptides and proteins by RA patient autoantibodies.
The determination of peptides and proteins in a biological matrix normally includes a sample-preparation step to obtain a sample that can be injected into a separation system in such a way that peptides and proteins of interest can be determined qualitatively and/or quantitatively. This can be a rather challenging, labourious and/or time-consuming process. The extract obtained after sample preparation is further separated using a compatible separation system. Liquid chromatography (LC) is the generally applied technique for this purpose, but capillary zone electrophoresis (CZE) is an alternative, providing fast, versatile and efficient separations. In this review, the recent developments in the combination of sample-preparation procedures with LC and CZE, for the determination of peptides and proteins, will be discussed. Emphasis will be on purification from and determination in complex biological matrices (plasma, cell lysates, etc.) of these compounds and little attention will be paid to the proteomics area. Additional focus will be put on sample-preparation conditions, which can be 'hard' or 'soft', and on selectivity issues. Selectivity issues will be addressed in combination with the used separation technique and a comparison between LC and CZE will be made.
To understand how proteins perform their function, knowledge about their structure and dynamics is essential. Here we use a combination of an efficient chemical lysine acetylation reaction and nanoLC-MALDI tandem mass spectrometry to probe the accessibility of every lysine residue in a protein complex. To demonstrate the applicability of this approach, we studied the interaction between the DNase domain of Colicin E9 (E9) and its immunity protein Im9. Free E9 and E9 in complex with Im9 were rapidly acetylated, followed by proteolytic digestion and analysis by LC-MALDI-TOF/TOF MS/MS. Acetylated peptides could be filtered out of the complex peptide mixtures using selective ion chromatograms of the specific immonium marker ions. Additionally, isobaric acetylated peptides, acetylated at different sites, could be separated by their LC retention times. The combination of LC and MALDI-TOF/TOF MS/MS provided information about the amount of acetylation on each individual lysine even for peptides containing several lysine residues. In general, our data agree well with those derived from the crystal structure of E9 and the E9:Im9 complex. Interestingly, next to in the binding interface expected lysines, K89 and K97, two from the crystal structure data unexpected lysines, K81 and K76, were observed to become less exposed upon Im9 binding. Moreover, K55 and K63, positioned in the predicted DNA binding region, were also found to be less accessible upon Im9 binding. These findings may illustrate some of the described differences in the solution-phase structure of the E9:Im9 complex compared with the crystal structure. (J Am Soc Mass Spectrom 2006, 17, 983-994)
Chemical proteomics is a powerful methodology for identifying the cellular targets of small molecules, however, it is biased towards abundant proteins. Therefore, quantitative strategies are needed to distinguish between specific and nonspecific interactions. Here, we explore the potential of the combination of surface plasmon resonance (SPR) coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) as an alternative approach in chemical proteomics. We coupled cGMP molecules to the SPR chip, and monitored the binding and dissociation of proteins from a human lysate by using sequential elution steps and SPR. The eluted proteins were subsequently identified by LC-MS/MS. Our approach enabled the efficient and selective extraction of low-abundant cyclic-nucleotide-binding proteins such as cGMP-dependent protein kinase, and a quantitative assessment of the less- and nonspecific competitive binding proteins. The data show that SPR-based chemical proteomics is a promising alternative for the efficient specific extraction and quantitative identification of small-molecule-binding proteins from complex mixtures.
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