Dataset Information

Variable blood processing procedures contribute to plasma proteomic variability.

ABSTRACT:

Background

Plasma is a potentially rich source of protein biomarkers for disease progression and drug response. Large multi-center studies are often carried out to increase the number of samples analyzed in a given study. This may increase the chances of variation in blood processing and handling, leading to altered proteomic results. This study evaluates the impact of blood processing variation on LC-MS/MS proteomic analysis of plasma.

Methods

Initially two batches of patient plasma samples (120 and 204 samples, respectively) were analyzed using LC-MS/MS shotgun proteomics. Follow-up experiments were designed and carried out on healthy donor blood in order to examine the effects of different centrifugation conditions, length of delay until first centrifugation, storage temperature and anticoagulant type on results from shotgun proteomics.

Results

Variable levels of intracellular proteins were observed in subsets of patient plasma samples from the initial batches analyzed. This observation correlated strongly with the site of collection, implicating variability in blood processing procedures. Results from the healthy donor blood analysis did not demonstrate a significant impact of centrifugation conditions to plasma proteome variation. The time delay until first centrifugation had a major impact on variability, while storage temperature and anticoagulant showed less pronounced but still significant effects. The intracellular proteins associated with study site effect in patient plasma samples were significantly altered by delayed processing also.

Conclusions

Variable blood processing procedures contribute significantly to plasma proteomic variation and may give rise to increased intracellular proteins in plasma. Accounting for these effects can be important both at study design and data analysis stages. This understanding will be valuable to incorporate in the planning of protein-based biomarker discovery efforts in the future.

SUBMITTER: Halvey P

PROVIDER: S-EPMC7816468 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Publications

Variable blood processing procedures contribute to plasma proteomic variability.

Halvey Patrick P Farutin Victor V Koppes Laura L Gunay Nur Sibel NS Pappas Dimitrios A DA Manning Anthony M AM Capila Ishan I

Clinical proteomics 20210119 1

<h4>Background</h4>Plasma is a potentially rich source of protein biomarkers for disease progression and drug response. Large multi-center studies are often carried out to increase the number of samples analyzed in a given study. This may increase the chances of variation in blood processing and handling, leading to altered proteomic results. This study evaluates the impact of blood processing variation on LC-MS/MS proteomic analysis of plasma.<h4>Methods</h4>Initially two batches of patient pla ...[more]

PMID: 33468058

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Project description:Synaptosomes are frequently used research objects in neurobiology studies focusing on synaptic transmission as they mimic several aspects of the physiological synaptic functions. They contain the whole apparatus for neurotransmission, the presynaptic nerve ending with synaptic vesicles, synaptic mitochondria and often a segment of the postsynaptic membrane along with the postsynaptic density is attached to its outer surface. As being artificial functional organelles, synaptosomes are viable for several hours, retain their activity, membrane potential, and capable to store, release, and reuptake neurotransmitters. Synaptosomes are ideal subjects for proteomic analysis. The recently available separation and protein detection techniques can cope with the reduced complexity of the organelle and enable the simultaneous qualitative and quantitative analysis of thousands of proteins shaping the structural and functional characteristics of the synapse. Synaptosomes are formed during the homogenization of nervous tissue in the isoosmotic milieu and can be isolated from the homogenate by various approaches. Each enrichment method has its own benefits and drawbacks and there is not a single method that is optimal for all research purposes. For a proper proteomic experiment, it is desirable to preserve the native synaptic structure during the isolation procedure and keep the degree of contamination from other organelles or cell types as low as possible. In this article, we examined five synaptosome isolation methods from a proteomic point of view by the means of electron microscopy, Western blot, and liquid chromatography-mass spectrometry to compare their efficiency in the isolation of synaptosomes and depletion of contaminating subcellular structures. In our study, the different isolation procedures led to a largely overlapping pool of proteins with a fairly similar distribution of presynaptic, active zone, synaptic vesicle, and postsynaptic proteins; however, discrete differences were noticeable in individual postsynaptic proteins and in the number of identified transmembrane proteins. Much pronounced variance was observed in the degree of contamination with mitochondrial and glial structures. Therefore, we suggest that in selecting the appropriate isolation method for any neuroproteomics experiment carried out on synaptosomes, the degree and sort/source of contamination should be considered as a primary aspect.

Dataset Information

Variable blood processing procedures contribute to plasma proteomic variability.

Background

Methods

Results

Conclusions

Publications

Variable blood processing procedures contribute to plasma proteomic variability.

Similar Datasets

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