Background: Magnetic bead purification for the analysis of low-abundance proteins in body fluids facilitates the identification of potential new biomarkers by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The aims of our study were to establish a proteome fractionation technique and to validate a standardized blood sampling, processing, and storage procedure for proteomic pattern analysis. Methods: We used magnetic bead separation for proteome profiling of human blood by MALDI-TOF MS (mass range, 1000–10 000 Da) and studied the effects on the quality and reproducibility of the proteome analysis of anticoagulants, blood clotting, time and temperature of sample storage, and the number of freeze–thaw cycles of samples. Results: The proteome pattern of human serum was characterized by ∼350 signals in the mass range of 1000–10 000 Da. The proteome profile showed time-dependent dynamic changes before and after centrifugation of the blood samples. Serum mass patterns differed between native samples and samples frozen once. The best reproducibility of proteomic patterns was with a single thawing of frozen serum samples. Conclusion: Application of the standardized preanalytical blood sampling and storage procedure in combination with magnetic bead-based fractionation decreases variability of proteome patterns in human serum assessed by MALDI-TOF MS.

Background: Peptidome profiling of human urine is a promising tool to identify novel disease-associated biomarkers; however, a wide range of preanalytical variables influence the results of peptidome analysis. Our aim was to develop a standardized protocol for reproducible urine peptidome profiling by means of magnetic bead (MB) separation followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS).

A novel analytical platform based on liquid chromatography and tandem mass spectrometry using atmospheric pressure photoionization was applied for the simultaneous quantification of free and esterified β-sitosterol, campesterol, brassicasterol, and stigmasterol. The total time for sample pretreatment and analysis could be reduced from ∼3 h [gas chromatography-mass spectrometry (GC-MS)] to 15 min. The detection limits of the different phytosterols ranged between 0.25 and 0.68 μg/l. Linear ranges were between 1 and 1,000 μg/l. The within-run and between-run variabilities ranged between 1.4% and 9.9%. The analytical sensitivity was at least 150-fold higher compared with GC-MS.

Purpose: Mass spectrometry–based serum peptidome profiling is a promising tool to identify novel disease-associated biomarkers, but is limited by preanalytic factors and the intricacies of complex data processing. Therefore, we investigated whether standardized sample protocols and new bioinformatic tools combined with external data validation improve the validity of peptidome profiling for the discovery of pancreatic cancer–associated serum markers. Experimental Design: For the discovery study, two sets of sera from patients with pancreatic cancer (n = 40) and healthy controls (n = 40) were obtained from two different clinical centers. For external data validation, we collected an independent set of samples from patients (n = 20) and healthy controls (n = 20). Magnetic beads with different surface functionalities were used for peptidome fractionation followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Data evaluation was carried out by comparing two different bioinformatic strategies. Following proteome database search, the matching candidate peptide was verified by MALDI-TOF MS after specific antibody-based immunoaffinity chromatography and independently confirmed by an ELISA assay. Results: Two significant peaks (m/z 3884; 5959) achieved a sensitivity of 86.3% and a specificity of 97.6% for the discrimination of patients and healthy controls in the external validation set. Adding peak m/z 3884 to conventional clinical tumor markers (CA 19-9 and CEA) improved sensitivity and specificity, as shown by receiver operator characteristics curve analysis (AUROC_combined = 1.00). Mass spectrometry–based m/z 3884 peak identification and following immunologic quantitation revealed platelet factor 4 as the corresponding peptide. Conclusions: MALDI-TOF MS-based serum peptidome profiling allowed the discovery and validation of platelet factor 4 as a new discriminating marker in pancreatic cancer.

Diatoms are major contributors to the aquatic primary productivity and show an efficient acclimation ability to changing light intensities. Here, we investigated the acclimation of Phaeodactylum tricornutum to different light quality with respect to growth rate, photosynthesis rate, macromolecular composition and the metabolic profile by shifting the light quality from red light (RL) to blue light (BL) and vice versa. Our results show that cultures pre-acclimated to BL and RL exhibited similar growth performance, photosynthesis rates and metabolite profiles. However, light shift experiments revealed rapid and severe changes in the metabolite profile within 15 min as the initial reaction of light acclimation. Thus, during the shift from RL to BL, increased concentrations of amino acids and TCA cycle intermediates were observed whereas during the BL to RL shift the levels of amino acids were decreased and intermediates of glycolysis accumulated. Accordingly, on the time scale of hours the RL to BL shift led to a redirection of carbon into the synthesis of proteins, whereas during the BL to RL shift an accumulation of carbohydrates occurred. Thus, a vast metabolic reorganization of the cells was observed as the initial reaction to changes in light quality. The results are discussed with respect to a putative direct regulation of cellular enzymes by light quality and by transcriptional regulation. Interestingly, the short-term changes in the metabolome were accompanied by changes in the degree of reduction of the plastoquinone pool. Surprisingly, the RL to BL shift led to a severe inhibition of growth within the first 48 h which was not observed during the BL to RL shift. Furthermore, during the phase of growth arrest the photosynthetic performance did not change. We propose arguments that the growth arrest could have been caused by the reorganization of intracellular carbon partitioning.