Julia Dittrich

Apolipoprotein E (apoE) occurs on the majority of plasma lipoproteins and plays a major role in the lipid metabolism in the periphery and in the central nervous system. ApoE is a polymorphic protein with three common isoforms, apoE2, apoE3 and apoE4, derived from respective alleles ε2, ε3 and ε4. The aim of this study was to develop a sample pretreatment protocol combined with rapid mass spectrometry (MS)-based assay for simultaneous apolipoprotein profiling and apoE phenotype identification. This assay was validated in 481 samples from patients with stable atherosclerotic cardiovascular disease (ASCVD) and applied to study association with mild cognitive impairment (MCI) in the LIFE Adult study, including overall 690 study subjects. Simultaneous quantification of 8–12 major apolipoproteins including apoA-I, apoB-100 and apoE could be performed within 6.5 min. Phenotyping determined with the developed MS assay had good agreement with the genotyping by real-time fluorescence PCR (97.5%). ApoE2 isoform was associated with the highest total apoE concentration compared to apoE3 and apoE4 (p < 0.001). In the subgroup of diabetic atherosclerotic cardiovascular disease (ASCVD) patients, apoE2 isoform was related to higher apoC-I levels (apoE2 vs. apoE3, p < 0.05), while in the subgroup of ASCVD patients under statin therapy apoE2 was related to lower apoB-100 levels (apoE2 vs. apoE3/apoE4, p < 0.05). A significant difference in apoE concentration observed between mild cognitive impairment (MCI) subjects and controls was confirmed for each apoE phenotype. In conclusion, this study provides evidence for the successful implementation of an MS-based apoE phenotyping assay, which can be used to assess phenotype effects on plasma lipid and apolipoprotein levels.

A variety of atherosclerosis and cardiovascular disease (ASCVD) phenotypes are tightly linked to changes in the cardiac energy metabolism that can lead to a loss of metabolic flexibility and to unfavorable clinical outcomes. We conducted an association analysis of 31 ASCVD phenotypes and 97 whole blood amino acids, acylcarnitines and derived ratios in the LIFE-Adult (n = 9646) and LIFE-Heart (n = 5860) studies, respectively. In addition to hundreds of significant associations, a total of 62 associations of six phenotypes were found in both studies. Positive associations of various amino acids and a range of acylcarnitines with decreasing cardiovascular health indicate disruptions in mitochondrial, as well as peroxisomal fatty acid oxidation. We complemented our metabolite association analyses with whole blood and peripheral blood mononuclear cell (PBMC) gene-expression analyses of fatty acid oxidation and ketone-body metabolism related genes. This revealed several differential expressions for the heart failure biomarker N-terminal prohormone of brain natriuretic peptide (NT-proBNP) in peripheral blood mononuclear cell (PBMC) gene expression. Finally, we constructed and compared three prediction models of significant stenosis in the LIFE-Heart study using (1) traditional risk factors only, (2) the metabolite panel only and (3) a combined model. Area under the receiver operating characteristic curve (AUC) comparison of these three models shows an improved prediction accuracy for the combined metabolite and classical risk factor model (AUC = 0.78, 95%-CI: 0.76–0.80). In conclusion, we improved our understanding of metabolic implications of ASCVD phenotypes by observing associations with metabolite concentrations and gene expression of the mitochondrial and peroxisomal fatty acid oxidation. Additionally, we demonstrated the predictive potential of the metabolite profile to improve classification of patients with significant stenosis.

Background: Changes in the metabolic fingerprint of blood during child growth and development are a largely under-investigated area of research. The examination of such aspects requires a cohort of healthy children and adolescents who have been subjected to deep phenotyping, including collection of biospecimens for metabolomic analysis. The present study considered whether amino acid (AA) and acylcarnitine (AC) concentrations are associated with age, sex, body mass index (BMI), and puberty during childhood and adolescence. It also investigated whether there are associations between amino acids (AAs) and acylcarnitines (ACs) and laboratory parameters of glucose and lipid metabolism, as well as liver, kidney, and thyroid parameters. Methods: A total of 3989 dried whole blood samples collected from 2191 healthy participants, aged 3 months to 18 years, from the LIFE Child cohort (Leipzig, Germany) were analyzed using liquid chromatography tandem mass spectrometry to detect levels of 23 AAs, 6 ACs, and free carnitine (C0). Age- and sex-related percentiles were estimated for each metabolite. In addition, correlations between laboratory parameters and levels of the selected AAs and ACs were calculated using hierarchical models. Results: Four different age-dependent profile types were identified for AAs and ACs. Investigating the association with puberty, we mainly identified peak metabolite levels at Tanner stages 2 to 3 in girls and stages 3 to 5 in boys. Significant correlations were observed between BMI standard deviation score (BMI-SDS) and certain metabolites, among them, branched-chain (leucine/isoleucine, valine) and aromatic (phenylalanine, tyrosine) amino acids. Most of the metabolites correlated significantly with absolute concentrations of glucose, glycated hemoglobin (HbA1c), triglycerides, cystatin C (CysC), and creatinine. After age adjustment, significant correlations were observed between most metabolites and CysC, as well as HbA1c. Conclusions: During childhood, several AA and AC levels are related to age, sex, BMI, and puberty. Moreover, our data verified known associations but also revealed new correlations between AAs/ACs and specific key markers of metabolic function.

Laborious sample pretreatment of biological samples represents the most limiting factor for the translation of targeted proteomics assays from research to clinical routine. An optimized method for the simultaneous quantitation of 12 major apolipoproteins (apos) combining on‐line SPE and fast LC‐MS/MS analysis in 6.5 min total run time was developed, reducing the manual sample pretreatment time of 3 μL serum or plasma by 60%. Within‐run and between‐day imprecisions below 10 and 15% (n = 10) and high recovery rates (94–131%) were obtained applying the high‐throughput setup. High‐quality porcine trypsin was used, which outperformed cost‐effective bovine trypsin regarding digestion efficiency. Comparisons with immunoassays and another LC‐MS/MS assay demonstrated good correlation (Pearson's R: 0.81–0.98). Further, requirements on sample quality concerning sampling, processing, and long‐term storage up to 1 year were investigated revealing significant influences of the applied sampling material and coagulant on quantitation results. Apo profiles of 1339 subjects of the LIFE‐Adult‐Study were associated with lifestyle and physiological parameters as well as establish parameters of lipid metabolism (e.g., triglycerides, cholesterol). Besides gender effects, most significant impact was seen regarding lipid‐lowering medication. In conclusion, this novel highly standardized, high‐throughput targeted proteomics assay utilizes a fast, simultaneous analysis of 12 apos from least sample amounts.

The increasing number of peptide and protein biomarker candidates requires expeditious and reliable quantification strategies. The utilization of liquid chromatography coupled to quadrupole tandem mass spectrometry (LC-MS/MS) for the absolute quantitation of plasma proteins and peptides facilitates the multiplexed verification of tens to hundreds of biomarkers from smallest sample quantities. Targeted proteomics assays derived from bottom-up proteomics principles rely on the identification and analysis of proteotypic peptides formed in an enzymatic digestion of the target protein. This protocol proposes a procedure for the establishment of a targeted absolute quantitation method for middle- to high-abundant plasma proteins waiving depletion or enrichment steps. Essential topics as proteotypic peptide identification and LC-MS/MS method development as well as sample preparation and calibration strategies are described in detail.

The simultaneous quantification of protein concentrations via proteotypic peptides in human blood by liquid chromatography coupled to quadrupole MS/MS is an important field of bioanalytical research with a high potential for routine diagnostic applications. This review summarizes currently available sample preparation procedures and trends for absolute protein quantification in blood using LC‐MS/MS. It discusses approaches of transferring established qualitative protocols to a quantitative analysis regarding their reliability and reproducibility. Techniques used to enhance method sensitivity such as the depletion of high‐abundant proteins or the immunoaffinity enrichment of proteins and peptides are described. Furthermore, workflows for (i) protein denaturation, (ii) disulfide bridge reduction and (iii) thiol alkylation as well as (iv) enzymatic digestion for absolute protein quantification are presented. The main focus is on the tryptic digestion as a bottleneck of protein quantification via proteotypic peptides. Conclusively, requirements for a high‐throughput application are discussed.