Biological Variation of the Platelet Proteome in the Elderly Population and Its Implication for Biomarker Research

Abstract

Knowledge about the extent of total variation experienced between samples from different individuals is of great importance for the design of not only proteomics but every clinical study. This variation defines the smallest statistically significant detectable signal difference when comparing two groups of individuals. We isolated platelets from 20 healthy human volunteers aged 56 -100 years because this age group is most commonly encountered in the clinics. We determined the technical and total variation experienced in a proteome analysis using two-dimensional DIGE with IPGs in the pI ranges 4 -7 and 6 -9. Only spots that were reproducibly detectable in at least 90% of all gels (n ‫؍‬ 908) were included in the study. All spots had a similar technical variation with a median coefficient of variation (cv) of about 7%. In contrast, spots showed a more diverse total variation between individuals with a surprisingly low median cv of only 18%. Because most known biomarkers show an effect size in a 1-2-fold range of their cv, any future clinical proteomics study with platelets will require an analytical method that is able to detect such small quantitative differences. In addition, we calculated the minimal number of samples (sample size) needed to detect given protein expression differences with statistical significance. Molecular & Cellular Proteomics 7:193-203, 2008.Biomarkers are used to differentiate between different biological states and to monitor disease progress or the success of medical treatment. To fulfill these tasks, there are not only strict requirements for each biomarker candidate in terms of selectivity and specificity but also for the precision of the analytical process. Therefore knowledge on the extent of variation between the individuals within each group is crucial for the study design as well as the selection of the method of measurement. The total variation experienced between individuals within one group is the result of the biological variation caused by factors like sex, age, genetic background, lifestyle, or health status and the technical variation introduced by the applied sample handling and the method of measurement itself. This study investigated the extent of variation in human blood platelets. Platelets are responsible for the maintenance of vascular integrity and are also involved in inflammation and wound healing (1). They are anucleate cytoplasmic fragments released from megakaryocytes in the bone marrow. During platelet biogenesis, organelles, especially the mitochondria and the ␣-and dense granules, are actively transported into the platelets. Furthermore platelets receive a certain set of mRNAs during their biogenesis from the megakaryocytes and are still capable of protein synthesis and processing (2). Several proteomics studies have focused on different aspects in platelet research using 2D 1 electrophoresis followed by mass spectrometry. These studies provided 2D maps of the total platelet proteome (3, 4), the platelet secretome (5), and the processes during platelet activ...

What Can Proteomics Tell Us About Platelets?

“…From our quantitative MS data, we could furthermore deduce that, using our reproducible isolation procedure, ≈80% of the platelet proteome showed only minor inter-and intradonor variation and were stable (1) between 4 healthy donors and (2) between blood donations of 1 donor over time (≈3 weeks). This was in good agreement with 2-DE derived data from Winkler et al, 46 who found a coefficient of variation of 18% based on 500 spots reproducibly found in platelets from healthy donors. In our study, the calculated amount of the 2 most prominent plasma proteins, serum albumin and transthyretin, allowed measurement of the degree of plasma contamination, which is inevitable because of the sponge-like structure of the platelet open canalicular system.…”

“…These require state-of-the-art equipment and either the usage of costly stable isotope labeling techniques which allow multiplexing of ≤10 samples to save time 50,51 or the successive analysis of single samples by label-free quantification strategies to characterize larger cohorts (Text Box 1). In the past few years, 2-DE was preferably used for disease-driven platelet proteomics, 46,52,53 but, as mentioned above, this technique has limitations when compared with gel-free LC-MS-based proteomics (Table I in the online Data Supplement).…”

“…In one study, proteins from resting and collagen receptor-stimulated platelets were incubated with cAMP/cGMP-coupled beads, and the pull-down eluates were analyzed by LC-MS/MS. 115 Owing to the enrichment provided by specific probes, activity-based protein profiling 116-121 B, These studies are mainly (40%) based on 2-dimensional gel electrophoresis (2-DE)/ difference gel electrophoresis (DIGE), 46,52,53,80,82,83,85,87,88,90,91,[93][94][95][96]98,102,106,110,112,113 whereas gelfree methods (23%) are still under-represented. 21,42,89,97,[114][115][116][117][118]121 Combination refers to studies using 2-DE/DIGE in combination with other approaches.…”

Variability of healthy human proteome

“…В работе по изучению вариабельности протеома тромбоцитов, выполненной Winkler с соавт., было также продемонстрировано, что отличия между индивидуумами значительно выше изменчивости, наблюдаемой при анализе нескольких образцов одного и того же испытуемого, полученных в разное время [47].…”

“…По данным Winkler с соавт., коэффициенты вариации 10 мажорных белков плазмы крови находятся в диапазоне от 9 до 41%. Так, коэффициент вариации альбумина составляет 9%, иммуноглобулина G -20%, трансферрина -14%, гаптоглобина -41%, комплемента С3с -17%, a-1 кислого гликопротеина -21%, a 2 -макроглобулина -20% [47]. Анализ низкомолекулярного субпротеома здоровых лиц также позволил выявить компоненты, характеризующиеся высокой и низкой индивидуальной вариабельностью.…”