All wells were prewashed using 5 200?L water, followed by equilibration using 3 200?L acetonitrile/water (8020, v/v)

All wells were prewashed using 5 200?L water, followed by equilibration using 3 200?L acetonitrile/water (8020, v/v). changes during acute systemic inflammation associates with increased mortality risk and indicates new avenues for the development of personalized diagnostic and therapeutic approach. N-glycosylation of human proteins is an essential posttranslational modification1 generated by a complex biosynthetic pathway comprising hundreds of glycosyltransferases, glycosidases, transcriptional factors, ion channels and other proteins2. This process results in the creation of branched oligosaccharide chains, called glycans, which become integral a part of proteins and significantly contribute to their structure and function3. Structural details of the attached glycans are of great physiological significance and many pathological conditions are associated with various types of glycan changes4,5. Glycosylation appears to be particularly important in the immune system6 and immunoglobulin G (IgG) is one of the best-studied glycoproteins. Each CH2 domain name of the Fc region of IgG heavy chains WAY-316606 carries a covalently attached bi-antennary em N /em -glycan at the highly conserved asparagine 297 residue7. These glycans are essential for all those pro-inflammatory activities of antibodies by maintaining the heavy chains in an open conformation required for FcR interactions and even a small change in their composition has dramatic consequences for effector function of IgG8,9. The addition of fucose to H3F3A WAY-316606 the core of these glycans radically reduces IgG binding to FcRIIIa10,11 preventing initiation of antibody dependent cellular cytotoxicity (ADCC)12,13 and destruction of target cells. On the other hand, the addition of sialic acid converts IgG from pro-inflammatory to anti-inflammatory agent14,15. Instead of binding to FcRs, sialylated Fcs initiate an anti-inflammatory cascade involving the lectin receptor DC-SIGN16,17. This leads to upregulated surface expression of the inhibitory FcRIIb on inflammatory cells, thus attenuating autoantibody-initiated WAY-316606 inflammation. Sialylation of IgG was also found to be essential for the function of intravenous immunoglobulin (IVIG)18, a therapeutic preparation of highly purified polyclonal IgG antibodies widely used for the treatment of a number of autoimmune diseases. The importance of individual variation in glycosylation in acute inflammation has not been extensively studied, primarily due to the absence of reliable quantitative methods for high-throughput glycomics. We have recently developed methods for quantitative analysis of plasma19 and IgG20 glycomes and here we have applied them to follow the dynamics of glycosylation changes during early course of systemic inflammation caused by cardiac surgery. Acute systemic inflammation is a part of many pathological events and patient’s inflammatory response often determines outcome of a disease. Controlling the inflammatory cascade still represents a great challenge, due to its complexity and individual physiological differences. Cardiac surgery is usually a procedure that provokes a vigorous inflammatory response, which has important clinical implications21,22,23. This inflammatory response is usually caused by blood contact with foreign surfaces and the activation of complement. Besides the activation of the complement system, increasing production of cytokines, oxygen radicals, release of endothelin and the expression of adhesion molecules on leukocytes and the endothelium represent main molecular mechanisms of such inflammation. Cardiovascular surgery with cardiopulmonary bypass (CPB) has improved in past decades, but inflammatory activation in this setting is still unpredictable and is associated with several postoperative complications24. It is generally accepted that CPB initiates a whole-body inflammatory reaction. Factors influencing incidence, severity, and clinical outcome of the inflammatory response, and in particular WAY-316606 the reasons why certain patients develop life-threatening perioperative complications, are currently not well comprehended. EUROSCORE (European System for Cardiac Operative Risk Evaluation) is usually a risk model which allows the calculation of the risk of death after a heart operation. WAY-316606 The model asks for 17 items of information about the patient, the state of the heart and the proposed operation25, and uses logistic regression to calculate the risk of death26. This model has been adopted worldwide, becoming the most widely used risk index for cardiac surgery27, and its use is believed to have contributed substantially to the improvement in the results of heart medical procedures seen at the beginning of the millennium. Due to enhanced production of cytokines in cardiac patients compared to other surgical procedures and modulatory effects of IVIG around the cytokine network, the supplemental application of immunoglobulins represents a potential therapeutic concept in cardiac post-operative high risk patients21. From the results of different studies it seems that immunoglobulins are unlikely to improve outcome in the entire group of patients, but only in specific subgroups. There is a possibility that dynamics and potential individuality of glycosylation changes analyzed in this work should be considered when the immunomodulation therapies are subjected. Results Glycosylation of plasma proteins Total plasma em N /em -glycome has been analysed in 107 individuals (Table 1).