Detection of Trace Proteins in Multicomponent Mixtures Using Displacement Chromatography

Abstract

Model protein feed mixtures containing three abundant and seven trace proteins at various concentrations were identified and employed in a series of displacement experiments. Reversed-phase liquid chromatography (RPLC) and matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry were used to evaluate the compositions of both the feed mixtures and effluent fractions from the displacement experiments. The results demonstrated that trace proteins were focused at the boundaries between the abundant solutes where they were enriched and concentrated. For many of the multicomponent feed mixtures, mass spectrometry analyses of the displacement column effluent fractions resulted in the identification of trace proteins that were not detectable in the feed. In addition, the use of minimal or no salt in the carrier solutions enabled the analysis of displacement fractions by direct infusion mass spectrometry. These results are significant in that they indicate that while the presence of abundant proteins can often be problematic for the detection of trace components, displacement chromatography may be able to employ these abundant proteins to focus trace proteins in the displacement train, thus facilitating detection.

Combinatorial peptides: A library that continuously probes low‐abundance proteins

“…Examples have been published on hydrophobic resins [28] as well as on anion- [29] and cation-exchangers [30]. It has been demonstrated as being very effective for the enrichment of certain low-abundance proteins in lectin affinity chromatography, [31] and in the detection of protein impurity traces [32] as well as in the removal of host cell proteins from purified antibodies [33].…”

High-throughput immunosensor chip coupled with a fluorescent DNA dendrimer for ultrasensitive detection of cardiac troponin T

“…7 Immunosensor chip-based optical imaging platforms show potential in the specic and high-throughput detection of biomarkers, which employs principles of immunoreaction to recognize the target and utilizes spatial resolution strategy to distinguish the optical signals from different sensing sites on the array simultaneously. [8][9][10] Compared with traditional analytical methods such as enzyme-linked immunosorbent assay and chromatography, [11][12][13] it possesses several advantages including easy operation, intuitive testing and the capacity of performing parallel real-time detection for a large number of samples. In our previous work, immunosensor chip always contained 48 sensing sites (diameter of each site: 4 mm), 14,15 on which a maximum of 48 samples could be detected in a single analysis process.…”

Sample displacement chromatography as a method for purification of proteins and peptides from complex mixtures

“…After their removal, residual proteins can be separated either by SDS-PAGE or 2D electrophoresis [14], or by use of a single chromatographic step, or a combination of different chromatographic and electrophoretic methods [15, 16]. However, the need to improve sample preparation methods still remains [16], and the use of additional fractionation methods such as displacement chromatography for both separation of proteins components prior to proteolytic digestion [17, 18], and digested peptides before prior to mass spectrometric analysis [18–20] has recently been discussed.…”