Avoiding Nonspecific Interactions in Studies of the Plasma Proteome: Practical Solutions to Prevention of Nonspecific Interactions for Label-Free Detection of Low-Abundance Plasma Proteins

Abstract

The molecular constitution of blood can be highly representative of the physiological state of an individual and offers an ideal target for studies of biomarkers. High-abundance plasma proteins, particularly albumin, dominate the plasma proteome, but it is the low-abundance proteins (such as cytokines) that are commonly associated with many pathophysiological states. Several detection strategies, and particularly those that involve label-free detection, are available for low-abundance protein detection in plasma, but all can be severely compromised by the high-abundance of serum albumin. In the present study, we examine the effect of albumin interference on accurate label-free detection by protein microarrays. Albumin was found to disrupt specific antigen-antibody binding interactions of low-abundance proteins. In clinical analysis, where it is imperative to preserve the integrity of samples, depletion of albumin may further undermine quantitative measurements. We have optimized procedures that permit accurate analysis to be undertaken without the need for prior treatment of samples. The emphasis is placed on disrupting nonspecific interactions including both electrostatic (i.e., Colulombic) and electrodynamic (hydrophobic and other nonpolar based) interactions. These protocols appear to be generic with potential applications in several areas of analytical biotechnology.

Sample Preparation Techniques for the Untargeted LC‐MS‐Based Discovery of Peptides in Complex Biological Matrices

“…However, these separation methods are not protein selective. Another way to reduce a sample's complexity is to specifically remove the most abundant protein(s), by doing (immuno-) affinity capturing [17, 20, 32, 39, 56–61]. …”

“… Figure 1 reflecting the large dynamic range of proteins and peptides in human blood plasma is very illustrative in this respect. Especially-the presence of albumin in a sample has been shown to prevent the successful identification of low-abundance biomarkers in many peptidomic studies [17, 20, 21]. Hence, different methods for capturing, partitioning, fractionating, depleting, or enriching a sample have been developed [22, 23].…”

9G DNAChip Technology: Self-Assembled Monolayer (SAM) of ssDNA for Ultra-Sensitive Detection of Biomarkers

“…Unfortunately, the most widely used techniques for detection of biomarkers OPEN ACCESS such as mass spectrometry [2], 2-D western blotting [3], 2-D gel electrophoresis [4], enzyme-linked immunosorbent assay (ELISA) [5,6] are not suitable for the analysis of large numbers of samples or the multiplexed detection of many targets within an individual sample. The protein microarrays produced by direct immobilization methods are known to suffer from drawbacks like instability of the immobilized proteins, thus resulting in low sensitivity [7,8].…”

Whole blood screening of antibodies using label-free nanoparticle biophotonic array platform

“…In some cases the sample pre-treatment, protein depletion or chaotrope addition, may reduce non-specific contribution to the background (Hifumi et al 2002;Richens et al 2009), this approach is highly problematic as it may affect the specific responses as well. The healthcare objective however would be a point-of-care device which requires no sample preparation and has the potential to assay a number of different blood biomarkers simultaneously.…”