Understanding and controlling preanalytical variables: The importance of insuring the accuracy of analysis for various proteomics biomarkers

The field of proteomics has made tremendous strides over the past decade, driven by advancements in mass spectrometry and bioinformatics. The increased sensitivity and throughput of mass spectrometers coupled with high-powered software algorithms have enabled the ability to identify thousands of proteins from very complex mixtures and perform quantitation between different sample types.

A wide variety of body fluids, such as blood, plasma, serum, bone marrow, urine, saliva, sputum, synovial fluid and cerebrospinal fluid, can be used for biomarker discovery activities. Nevertheless, blood has been the biospecimen of choice because it is easily collected and readily availability in sufficient quantities. The use of blood specimens may create several analytical challenges related to proteomic studies, such as the large dynamic range of plasma protein concentration, lipid concentration variability, intrinsic enzymatic activity, and many preanalytical variations in how blood is collected and handled. These challenges limit overall reproducibility, sensitivity and resolution in proteomics biomarker discovery efforts, and are even more critical for translating biomarker discovery into clinical application.

In the past several years, a broad scientific effort has been expended on biomarker discovery research, resulting in an abundance of potential biomarker candidates. Typically, researchers are taking a broad, shotgun approach using mass spectrometry to identify and quantitate potential protein biomarkers from different sample types. This approach has the advantage of quantitatively looking at a large subset of proteins. Once a subset of proteins has been identified as either up or down regulated, the next common approach is to perform either MRM (multiple reaction monitoring), ELISA (enzyme-linked immunosorbent assay), or a hybrid of the two techniques.

Further, these promising new biomarkers require investigation before advancing into the clinical setting for any diagnostic application. They must be subjected to verification and validation phases. One of the major hurdles hindering the transition from laboratory bench to the clinic laboratory is controlling preanalytical variability. It is most important to understand the significant impact of variable time and temperature on blood enzymes which can degrade specific analytes.

There are many preanalytical variables that impact every clinical study, and it is important to understand and control these variables to insure the accuracy of analysis for various proteomics biomarkers. Common variables during blood sample collection, processing and analysis include the choice of processing plasma or serum, (the addition of protease inhibitors or other additives, and the processing and handling conditions for blood specimens.

Only with an understanding of the challenges associated with developing a reproducible proteomics measurement system can one begin to understand the complexity involved in selecting, studying and optimizing a serum or plasma sample. To this end, a detailed preanalytical strategy for sample handling is essential.

Our research has focused on the potential impact sample handling can have on protein and peptide stability and how this variability can be controlled through the use of protease inhibitors. Specifically, we have focused on the stabilization of GLP-1, GIP, glucagon, and ghrelin. These four peptides are of particular interest within the field of metabolic disorder research, especially related to diabetes drug research.

Using time-course mass spectrometry, we have characterized the kinetic digestion of each incretin peptide caused by active plasma endogenous enzymes. We further developed a cocktail of inhibitors to minimize this variability and instability in the blood collection tube. The BD P800* Blood Collection System has a proprietary cocktail which includes multiple protease inhibitors (i.e., DPP‑IV, esterase, etc.) whose performance have been optimized for acting in whole blood while minimizing hemolysis. The plasma obtained from the P800 tube can be used immediately, transported or stored frozen. Stabilization of plasma peptides, such as GLP-1, GIP, glucogan, and ghrelin, enable them to be used in pharmacokinetic and pharmacodynamic studies.

As the field of biomarker research continues to expand, there will be a growing need for preanalytical system that effectively stabilizing proteins and peptides and ultimately enable the transition of these biomarker candidates from the research environment to the clinical laboratory.

Biography

David Craft earned his PhD degree in chemistry from the University of Alberta in Canada, where he studied under the mentorship of Professor Liang Li. He has been working in the field of proteomics for over 12 years, developing mass spectrometry applications towards the detection of biological molecules.

* For research use only – not for use in diagnostic procedures.