Plasma degradome affected by variable storage of human blood

Abstract

BackgroundThe successful application of—omics technologies in the discovery of novel biomarkers and targets of therapeutic interventions is facilitated by large collections of well curated clinical samples stored in bio banks. Mining the plasma proteome holds promise to improve our understanding of disease mechanisms and may represent a source of biomarkers. However, a major confounding factor for defining disease-specific proteomic signatures in plasma is the variation in handling and processing of clinical samples leading to protein degradation. To address this, we defined a plasma proteolytic signature (degradome) reflecting pre-analytical variability in blood samples that remained at ambient temperature for different time periods after collection and prior to processing.MethodsWe obtained EDTA blood samples from five healthy volunteers (n = 5), and blood tubes remained at ambient temperature for 30 min, 8, 24 and 48 h prior to centrifugation and isolation of plasma. Naturally occurred peptides derived from plasma samples were compared by label-free quantitative LC–MS/MS. To profile protein degradation, we analysed pooled plasma samples at T = 30 min and 48 h using PROTOMAP analysis. The proteolytic pattern of selected protein candidates was further validated by immunoblotting.ResultsA total of 820 plasma proteins were surveyed by PROTOMAP, and for 4 % of these, marked degradation was observed. We show distinct proteolysis patterns for talin-1, coagulation factor XI, complement protein C1r, C3, C4 and thrombospondin, and several proteins including S100A8, A9, annexin A1, profiling-1 and platelet glycoprotein V are enriched after 48 h blood storage at ambient temperature. In particular, thrombospondin protein levels increased after 8 h and proteolytic fragments appeared after 24 h storage time.ConclusionsThe overall impact of blood storage at ambient temperature for variable times on the plasma proteome and degradome is relatively minor, but in some cases can cause a potential bias in identifying and assigning relevant proteomic markers. The observed effects on the plasma proteome and degradome are predominantly triggered by limited leucocyte and platelet cell activation due to blood handling and storage. The baseline plasma degradome signature presented here can help filtering candidate protein markers relevant for clinical biomarker studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-016-9126-9) contains supplementary material, which is available to authorized users.

Quantification of protein markers monitoring the pre‐analytical effect of blood storage time before plasma isolation using 15N metabolically labeled recombinant proteins

“…We measured the amounts of 15 plasma proteins at a single LC‐MRM‐MS run to investigate the pre‐analytical effects of storage time and temperature on blood samples. The proteins were chosen from a dataset of PROTOPMAP analysis with one exception . Seven proteins were chosen from the top 20 presumed to be strongly associated with pre‐analytical effects.…”

“…In this method, analytes are quantified by using a known amount of corresponding stable isotope‐labeled proteins. A cytosolic protein, PFN1, was reported to increase in the plasma prepared from blood stored for a long time . This implies that PFN1 leaked from the blood cells into the soluble fraction during blood storage before plasma isolation.…”

“…For developing protein‐based clinical assay, the pre‐analytical effects coming from blood sampling and handling should be carefully considered. Mass spectrometry (MS)‐based omics approach has been well suited for detecting proteins or metabolites under the pre‐analytical effects . A recent study identified 20 plasma proteins that showed significant abundance changes in a profiling LC‐MS/MS analysis .…”

“…8,9 A recent study identified 20 plasma proteins that showed significant abundance changes in a profiling LC-MS/MS analysis. 8 Systematic monitoring of such proteins may provide a valuable information about how much plasma sample is affected. In this study, we developed and validated an assay for the absolute quantification of profilin-1 (PFN1) and thymosin beta-4 (TMSB4X) and relative quantification of other 13 proteins in human plasma using multiple reaction monitoring mass spectrometry (MRM-MS).…”

Degradomics in Biomarker Discovery

“…Among the identified biomarker candidates, the proteolytic processing of the transcriptional regulator STAT1 was found to be highly upregulated in HCC tissue, indicating its pathological relevance and diagnostic potential. Interestingly, PROTOMAP was also used to evaluate the effect of storage temperature on the stability of clinical blood samples . The results showed that blood storage at room temperature caused the degradation of about 4% out of 820 identified plasma proteins, which could cause potential bias in identifying and assigning potential markers.…”