Are Samples in Your Freezer Still Good for Biomarker Discovery

Diagnostic markers can be used for detecting diseases, predicting response to a therapy, making prognosis for a patient, or estimating the outcome of a treatment. Despite great progress in instrumentation and analytical techniques, few new biomarkers have been discovered during the past decade. Some reasons for slow progress are the low throughput of analysis, high false discovery rate, lack of data on verification of the discovered potential biomarkers, and poor stability of many biomolecules. Human blood contains enzymes and proteases, which alter proteins in vivo and in vitro. Studies have suggested that many peptides identified in biological samples are generated by proteolytic cleavage of plasma proteins, which are subsequently altered by peptidases. 3,4 Some of the degradation takes place in vivo, while many of the products could be generated in vitro during blood collection and sample storage. Considering this, peptides identified in the biomarker discovery experiments could be intermediates of sequential reactions, which are unstable over time and could arise from biological variability related to sample collection or handling, or be detected because of technical variability during the analysis. 5,6 It is known that the stability of many proteins differs between serum and plasma samples collected in tubes with different types of anticoagulants. Differences associated with different sample types are associated with inhibition of the endogenous proteases by anticoagulants, which reduce activity of the proteases. Residual plasma protease and peptidase activity results in time-dependent variations and instability of the samples, especially during sample collection and processing. Results of a study by Yi et al 7 suggest that mixing protease inhibitors with blood immediately during blood collection results in enhanced stabilization of plasma proteome. Proteases can be subdivided into 4 classes: serine, cysteine, aspartic, and metalloproteases. Peptidases belong to 1 of 3 classes: endo-, exo-, and carboxypeptidases. In vivo, proteins are either protected from proteolysis in specialized compartments or reside in a protective conformation. When proteins are isolated from cells, there is a potential for their degradation via proteolytic enzymes. Protease inhibitors (PIs) are often used in experimental protocols to protect proteins from degradation. More than 100 naturally occurring PIs have been identified and can be used during the sample collection to prevent protein degradation. Monitoring and controlling preanalytical variations have a major impact on the success of work in biomarker discovery and verification. In this issue of the Journal, Findeisen et al 8 assess the exposure of serum and plasma samples to suboptimal storage conditions. Considering that the outcome of experiments is affected by protein stability, the authors suggest determining the preanalytical quality of serum and plasma by monitoring the time-dependent decay of a synthetic reporter peptide (RP) added to the samples at the time of sample collection. In the study, serum and plasma specimens were spiked with the RP, and proteolytic fragments were monitored at different time points up to 24 hours after blood sample collection. In the proposed method, analysis of the RP and its degradation product was performed by liquid chromatography/mass spectrometry. The sequence of the RP and the monitored fragments were selected with a goal of having properties, allowing time-dependent degradation of the RP with accumulation of stable RP. Quantitative measurement of the RP was shown to enable monitoring of the preanalytical