BD discuss proteomics

By Jizu Yi, Principle Scientist and David Warunek, WW Director, Scientific Affair and David Craft, PhD Group Leader / Proteomics R&D

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.

During the biomarker discovery phase a wide variety of body fluids have been used ranging from blood, plasma, serum, bone marrow, urine, saliva, sputum, synovial fluid, and cerebrospinal fluid (CSF).  Blood has been the biospecimen of choice due to its non-invasive collection, its readily availability, and sufficient quantities.  The use of blood specimens are, however, subject to several challenges which particularly effect 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 five years an immense scientific effort has been placed on biomarker discovery research resulting in a surplus of potential biomarker candidates.  Typically, researchers are talking a broad shot gun 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 have 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 entering into the clinical setting for any specific application.  They now require verification and validation phases.  One of the major hurdles hindering the transition from bench to the clinic is preanalytical variability.  Most notably, time and temperature have significant impact on blood enzymes ability to degrade specific analytes.

There are many preanalytical variables and alternatives that impact virtually every clinical study, and as more studies are done, the more important these aspects are found to be with respect to proteomics and biomarker goals.  Common variables during sampling and analysis include (i) the choice of plasma versus serum samples, (ii) the addition of protease inhibitors or other additives, and (iii) the processing and handling of 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 / plasma sample. To this end, a detailed pre-analytical strategy for sample handling is essential. 

We have 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 in the field of metabolic disorder research especially diabetes drug research. We further developed a cocktail of inhibitors to minimize this variability / instability in the blood collection tube ie P800. The BD P800* tube has a proprietary cocktail which includes a DPP‑IV, esterase and other protease inhibitors that are optimized for blood while yielding high-quality hemolysis-free plasma.

Using time-course mass spectrometry, we have characterized the kinetic digestion of each incretin peptide caused by active plasma endogenous enzymes (figure 1, above).  Mass spectrometry not only enables confident detection of intact peptides it also allows identification of degradation products.  Further analysis of the cleavage products identifies which class of enzyme was responsible for digesting the parent peptide.  We have used this information to add targeted enzyme inhibitors for biomarker preservation.  Once we have identified a solution we then validate the performance of our tube against commercially available ELISA’s.  ELISA allows us to spike plasma within physiological levels however some ELISA’s don’t necessary detect the full intact peptide depending where the antibody epitopes are targeting.  Figure 2 displays ELISA’s for GLP-1 and GIP using commercial kits.  GLP-1 ELISA displays loss in signal for EDTA plasma and consistent signal over 70 hours in P800, complementing the MALDI-MS data.  Total GIP ELISA assay shows no difference between EDTA and P800, not consistent with the MS data.  The GIP assay is not specific to N-terminal degradation and thus is not able to differentiate between intact and DPP-IV cleaved GIP.

The plasma obtained by processing 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 grow the need for stabilizing proteins and peptides will be required through the three phases (discovery, verification, and validation) ultimately improving the success rate of transitioning biomarker candidates from discovery lists to clinical applications.

For Research Use Only – Not for Use in Diagnostic Procedures