Published: December 24, 2010 r2010 American Chemical Society 1667 dx.doi.org/10.1021/pr1009959 | J. Proteome Res. 2011, 10, 1667–1674 ARTICLE pubs.acs.org/jpr Plasma protein N-glycan profiles are associated with calendar age, familial longevity and health L. Renee Ruhaak,* ,† Hae-Won Uh, ‡ Marian Beekman, § Cornelis H. Hokke, † Rudi G. J. Westendorp, || Jeanine Houwing-Duistermaat, ‡ Manfred Wuhrer, † Andr  e M. Deelder, † and P. Eline Slagboom §,^ † Department of Parasitology, Biomolecular Mass Spectrometry Unit, Leiden University Medical Center, Leiden, The Netherlands ‡ Department of Medical Statistics and Bioinformatics, section Medical Statistics, Leiden University Medical Center, Leiden, The Netherlands § Department of Medical Statistics and Bioinformatics, section Molecular Epidemiology, Leiden University Medical Center, Leiden, The Netherlands ) Department of Gerontology and Geriatrics, Leiden University Medical Center, Leiden, The Netherlands ^ Netherlands Consortium of Healthy Aging, The Netherlands ABSTRACT: The development of medical interventions for the preservation of disease-free longevity would be facilitated by markers that predict healthy aging. Altered protein N-gly- cosylation patterns have been found with increasing age and several disease states. Here we investigate whether glycans derived from the total glycoprotein pool in plasma mark familial longevity and distinguish healthy from unhealthy aging. Total plasma N-glycan profiles of 2396 middle aged participants in the Leiden Longevity Study (LLS) were obtained by glycan release, labeling, and subsequent HPLC analysis with fluorescence detection. After normalization and batch correction, several regression strategies were applied to evaluate associations between glycan patterns, familial long- evity, and healthy aging. Two N-glycan features (LC-7 and LC-8) were identified to be more abundant in plasma of the offspring of long-lived individuals as compared to controls. These results were not confounded by the altered lipid status or glucose homeostasis of the offspring. Furthermore, a decrease in levels of LC-8 was associated with the occurrence of myocardial infarction (p = 0.049, coefficient = -0.065), indicating that plasma glycosylation patterns do not only mark familial longevity but may also reflect healthy aging. In conclusion, we describe two glycan features, of which increased levels mark familial longevity and decreased levels of one of these features mark the presence of cardiovascular disease. KEYWORDS: human plasma, N-glycosylation, longevity, aging ’ INTRODUCTION Glycosylation is the enzymatic addition of oligosaccharides (also known as glycans) to proteins and lipids. In N-glycosyla- tion, the glycans are attached to the asparagine residues in the protein. N-Glycans have important functions in many biological processes such as protein folding, 1 protein clearance, 2 cell adhesion, 2-4 receptor binding, and receptor activation. 5,6 Pro- tein N-glycosylation may be very diverse and is a dynamic equilibrium: in a given physiological state, the glycan signature is highly reproducible; 7,8 however, when the physiological state changes, for example, due to aging or disease, the glycosylation machinery of affected cells in an organism may be altered, and the glycan pattern can change dramatically. 7 Therefore, protein N-glycosylation patterns may represent an important group of potential biomarkers of health and disease. 9 Since the biological variation in plasma N-glycan is rather large, 10 larger sample sizes are required for biological interpretation. The analysis methods required for the evaluation in larger sample sets have only recently been developed. Total plasma N-glycosylation patterns were found to be associated with calendar age in a study population of 100 Belgian individuals. It has previously been reported that elderly indivi- duals above 50 years of age showed increased levels of non- galactosylated glycans, whereas the levels of galactosylated glycan structures decreased with increasing calendar age. 11 Even in an exceptionally high-age group, these associations between glyco- sylation and calendar age have been observed. 11 In a more recent study, 12 comprising a larger sample set, changes in levels of glycan features have also been observed with increasing age and were sex specific. In general, females showed more profound associations between glycan patterns and age than males, while Received: October 1, 2010